µ-Conotoxins are small, potent, peptide voltage-gated sodium (Na_V) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype Na_V1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human Na_V1.7 (hNa_V1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different Na_V channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNa_V channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNa_V1.7. Analysis of other µ-conotoxins at hNa_V1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNa_V1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNa_V1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNa_V1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNa_V1.7 when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNa_V1.7.

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition

PC3 cells are an epithelial prostate tumour cell line. A range of Wnts triggers a delayed Ca2+ rise [1] in these and other cancer cell lines. Recorded in patch clamp the cells have low membrane potential [2] but exhibit an outward current activated apparently at (non-physiological) positive potentials [3]. The main candidate channel carrying this K+ current is the BK(Ca) α subunit although a KCNN4 channel is also reported [4]. Without baseline subtraction, a small ‘leak’ current is sometimes apparent in recordings. All of these currents
are modulated by Wnts as described below.

Cardiac ion channel activity is important to generate cardiac action potentials in appropriate timing and duration. Drug-induced impairment of those ion channels cause abnormal cardiac activity such as QT interval prolongation, ventricular arrhythmia and, in most serious cases, sudden death. These effects are one of the leading causes for drug withdrawal from the market or denied regulatory approval of new therapeutic candidates. Although ICH guideline E14/S7B Q&A published in February focused on IKr (hERG) as well as INa (NaV1.5) and ICa,L (CaV1.2), the other cardiac ion channel targets, including many potassium channels such as IKur (KV1.5), Ito1 (KV4.3), IKs (KVLQT1/minK) and IK1 (Kir2.1) are also important to understand the drug effect on the generation of action potentials in cardiomyocytes. To establish the screening assay for those cardiac potassium channels, we have established the whole cell experiment method using QPatch automated patch clamp system.

Voltage protocols customized for each ion channel were applied to measure biophysical properties and current stability. The currents elicited by the designated voltage protocols were measured in each ion channel, and the half-inactivated voltages (V1/2) were calculated using Boltzmann fitting. The average inactivation V1/2 for KV4.3 and KV1.5 was -39.5 mV and -9.5 mV, respectively. The reference compounds for KV4.3 (Flecainide IC50=11.6µM), KV1.5 (4-Aminopyridine IC50=125.1µM), and KVLQT1/minK (Chromanol 293B IC50=17.4µM, XE-991 IC50=1.7µM) were also assessed. KVLQT1/minK is known to have severe rundown of the currents. The smallest rundown condition was observed in the modified intracellular solution and extracellular solutions as well as shorten voltage protocols. The remaining currents after vehicle solution application for 6 times (approximately 500 seconds) in the best successful combination were 67% in average of groups of experiments.

The optimized assays on QPatch enable measuring the biophysical properties of those four ion channels and assessing the pharmacological effect of reference compound for each of them. QPatch allows the assessment of a novel compounds proarrhythmic risk on those cardiac ion channels other than hERG, NaV1.5 and CaV1.2.

A substantial body of in vitro and in vivo evidence suggests that de novo expression of voltage-gated sodium channels (VGSCs) occurs in several carcinomas and drives the metastatic process (Djamgoz et al. 2019). It is well known that chemotherapy can lead to peripheral neuropathy, and this could be associated with an alteration in VGSC expression/activity. We questioned whether blocking VGSC activity would affect the cytotoxic impact of paclitaxel (PTX) upon human strongly metastatic MDA-MB-231 cells.

The results demonstrate that QPatch 16 can provide high-quality whole-cell current data from fast voltage-activated Na+ channels, and that protocols can be defined that allow analysis of drug potency (IC50), inactivation kinetic properties, as well as state- and frequency-dependency of drug action.

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors.

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC
pharmacological manipulation.

• High fidelity voltage and current clamp recordings of Cor.4U in physiological solution
• Demonstration of the presence of I_Na, I_Ca as well as I_Kr
• The use of fluoride in the internal solution resulted in lower
  I_Ca and shortened action potential durations
• Paced and spontaneous action potentials
• Pharmacological agents show expected effects on action
  potentials in Cor.4U

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

As revising an ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation by Human Pharmaceuticals) was suggested, Comprehensive In
vitro Proarrhythmia Assay (CiPA) activities are being implemented. The suggested S7B revision includes an assay of cardiac ion channels other than hERG, and CiPA-recommended voltage protocols for patch clamping was recently announced. We, Group 5 of the JSPS iSmart (investigation of in silico/in vitro model for arrhythmogenic risk prediction) play a role of wet-data collection. This time we have investigated the applicability of those protocols

Voltage-gated sodium (Na_V) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The Na_V1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding Na_V1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of Na_V1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of Na_V1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of Na_V channels, but little is known about their activity at Na_V1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of Na_V1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of Na_V1.1 (EC₅₀ of 17 and 32 nM, respectively), and they present dual α/β activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αβ fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for Na_V1.1, their unusual dual mode of action provides an alternative approach to the development of selective Na_V1.1 modulators for the treatment of DS.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.

This report summarizes the experiments that were done running fast ligand-gated assays on QPatch 48X. QPatch 48X in multi-hole mode patches 10 cells per well instead of just one which has been shown to increase the overall success rate due to the fact that cells with less or no expression of the ion channel of interest will be patched in parallel with other cells that has better expression. The currents from all 10 holes are thus added together and therefore gives rise to increased current amplitudes from each well. The 10 holes obviously take up more space in the flow channel in the QPlate chip and we were curious to know if these changes had any effect on the measured signal from the ten cells. We set out to test different ligand-gated ion channels that are well known and already validated on QPatch HT to determine if the signal onset was comparable to QPatch 48X in multi-hole mode. Furthermore, we investigated the effect of known compounds: agonists, antagonist and modulators to see if any changes in data quality could be detected.

KV7 (KCNQ) channels are voltage-gated K+ channels with major roles in neurons, muscle cells and epithelia. The biophysical properties of KV7 channels make them essential in controlling the activity of
excitable cells1. They produce currents that are voltage-activated, slowly activating and non-inactivating, such as neuronal M current and cardiac IKs2. These channels often work as ‘excitability brakes’3 and are
targeted by various hormones and modulators to regulate cellular activity outputs. Genetic deficiencies in KCNQ genes result in human excitability disorders, including epilepsy, arrhythmias and deafness4, making
KV7 channels an attractive target for pharmaceutical research.

The aim of the present work was to demonstrate using the Sophion Qube 384-well APC platform for robust and reproducible KV7 currents recordings, suitable for fast and reliable drug testing.

KV3.x receptors have recently emerged as potential targets for treatment of a variety of CNS disorders, including epilepsy, ataxias, hearing disorders, schizophrenia and cognitive impairments. KV3.1–KV3.4 protein isoforms contribute to the high-frequency firing of neurons such as auditory brain stem neurons, fast-spiking cortical and hippocampal GABAergic interneurons, and Purkinje cells of the cerebellum, and play an important role in the regulation of intrinsic excitability and neurotransmitter release at presynaptic terminals of many neurons.

B’SYS has generated a panel of cell lines stably transfected with KV3.1a/b, KV3.2, KV3.3 and KV3.4 subunits. Using automated (QpatchTM ) as well as manual patch-clamp recordings, the aim of this validation study was to characterize these cell lines pharmacologically using the selective serotonin
reuptake inhibitor paroxetine. In addition, the effect of KV modulator 1 was investigated on KV3.1b.

Dorsal root ganglion (DRG) neurons transfer sensory signals from the peripheral to the central nervous system. Understanding the electrophysiological properties of DRG neurons has a significant application for pain research and potential drug development. Given the limited availability of human DRG neurons, the development of human-induced pluripotent stem cell (hiPSC)-derived sensory neurons (SNs) that contain DRG neuron electrophysiological properties provides a promising way to perform in vitro pain research. In addition, the latest expansion of automated patch clamp (APC) into a 384-well format provides the possibility of a high throughput screening (HTS) for thousands of compounds. In this study, RealDRG™ hiPSC-derived SNs were produced at massive scale and consistency to meet the demands of high-throughput APC studies. These SNs bear similarities to human DRG from a whole-transcriptome perspective and have been previously shown to possess functional voltage and ligand-gated channels important for nociception via manual patch clamp. To show the utility of nociceptors in HTS APC, we investigated the electrophysiological properties of hiPSC derived SNs on the Qube 384 APC system over time spans of 9-, 14-, 21-, and 28-day cultures. Using an optimized cell dissociation protocol to obtain healthy cell membranes for patch-clamp, we obtained a whole-cell success rate of 40.69-53.19%. Among these cells, 90.54-97.22% expressed KV currents, and 59.76-77.48% expressed Nav currents. For the Nav current group, 30.7-37.5% of cells carried a detectable TTX-resistant component. Furthermore, under current clamp mode, action potential firings were recorded from 60.12-66.76% of the cells that passed the success filter criteria.

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings, offering the potential to overcome the time-consuming, low-throughput bottleneck, arising from manual patch clamp investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer to the ion channel research community, and this review highlights areas of best practice for both academia and industry. The impact of APC platforms and the prospects of ion channel research and improved therapeutics for chronic pain will be evaluated.

In ion channel safety assessment and drug discovery it is often important to determine the mode of action of a drug candidate, which requires high-quality recordings. We successfully use the QPatch X in multi-hole mode in a screening scenario on Na_v1.5. This correlates with data from Langendorff perfused rabbit hearts, and an in vivo ECG assay using anesthetized rabbit, and effectively discriminates different modes of action of compounds on NaV1.5.
The protocol for QPatch X both tests the decay of the sodium current (30 Hz pulsetrain), and the recovery of the current. The QPatch assay allows combination of these elements into a single protocol, thereby shortening experiment time and costs.
The results show that: 1) Flecainide is a use-dependent blocker of open channels, as demonstrated by a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation in the QPatch experiments. Flecainide produces ECG changes (marked PR and QRS prolongation, and slight QTc prolongation), and lethal arrhythmia (VT ~Vf) in both Langendorff hearts and anesthetized rabbits at low doses. 2) Lidocaine is a state-dependent blocker of inactivated channels, inducing fast decay of I_Na (30 Hz pulsetrain) and fast recovery from inactivation. Lidocaine produces slight ECG changes (PR and QRS prolongation), but no arrhythmia in Langendorff-hearts or anesthetized rabbits. 3) Quinidine demonstrates a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation. Quinidine shows moderate PR and QRS prolongation, and severe morphological changes in ECGs.
The present results suggest that: 1) The QPatch X offers a time- and cost-effective screening scenario to select safe compounds with Na_v1.5 blocking activity and no proarrhythmic activity, and 2) the pro-arrhythmic activity of the Na_v1.5 blockers flecainide and quinidine might be attributable to their marked delay of recovery from inactivation.

Molecular dynamics was used to optimize the droperidol− hERG complex obtained from docking. To accommodate the inhibitor, residues T623, S624, V625, G648, Y652, and F656 did not move significantly during the simulation, while F627 moved significantly. Binding sites in cryo-EM structures and in structures obtained from molecular dynamics simulations were characterized using solvent mapping and Atlas ligands, which were negative images of the binding site, were generated. Atlas ligands were found to be useful for identifying human ether-á-á-go-go-related potassium channel (hERG) inhibitors by aligning compounds to them or by guiding the docking of compounds in the binding site. A molecular dynamics optimized structure of hERG led to improved predictions using either compound alignment to the Atlas ligand or docking. The structure was also found to be suitable to define a strategy for lowering inhibition based on the proposed binding mode of compounds in the channel.

: The use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP). Methods: A retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Cav1.2, and Nav1.5 (peak/late), together with hERG binding. Results: Assay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC50 values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for ∼10% compounds and trended with passive cellular permeability. hERG and Cav1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Cav1.2. For clinical compounds where hERG and Cav1.2 activity was more balanced, an analysis of TdP risk versus hERG/Cav1.2 ratio demonstrated low TdP probability when the hERG/Cav1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Nav1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787). Discussion: The data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

Introduction: The use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP).

Methods: A retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Ca_v1.2, and Na_v1.5 (peak/late), together with hERG binding.

Results: Assay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC₅₀ values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for ∼10% compounds and trended with passive cellular permeability. hERG and Ca_v1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Ca_v1.2. For clinical compounds where hERG and Ca_v1.2 activity was more balanced, an analysis of TdP risk versus hERG/Ca_v1.2 ratio demonstrated low TdP probability when the hERG/Ca_v1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Na_v1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787).

Discussion: The data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the ‘gold standard’, for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch-clamp method, considered the ‘gold standard’, for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

Voltage-gated sodium (Na_V) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of Na_V channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the Na_V channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40–120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4–24-fold higher potency compared to those of the monovalent peptides for the human Na_V1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of Na_V channels.

Professor Ellen Lumpkin, University of California, Berkeley – talk from ICMS 2019, the United States.

Title: TTX-sensitive sodium channels mediate action potential firing in menthol-sensitive neurons of the dorsal root ganglion

CHO cells expressing the cold-sensitive ion channel TRPM8 were tested on the QPatch, and HEK cells expressing TRPM8 were tested on QPatch HTX. Two agonists of TRPM8 were used for EC₅₀ determinations: menthol and icilin. All data presented was obtained with QPatch and analyzed with QPatch Assay Software

Professor Insuk So, Seoul National University, Korea – talk from ICMS 2019, the United Kingdom

Title: TRPC1 as a negative regulator for TRPC4 and TRPC5 channels

True giga seal patch clamping can be performed in parallel with QPatch multi-hole technology. This new multi-hole functionality has been tested on three different transient receptor potential ion channels (TRPA1, TRPV1 & TRPM8). All three targets were tested in both single-hole mode and in multi-hole mode with different agonists and antagonists. The advantages of testing compounds in multihole mode will be a minimization of biological variance and an increase in current amplitude, since the current response from several cells are summarized. The disadvantage is lack of serial resistance compensation and in some cases large leak currents

The synthesis and pharmacological activity of a new series of 5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine derivatives as potent sigma-1 receptor (σ1R) ligands are reported. A lead optimization program aimed at improving the aqueous solubility of parent racemic nonpolar derivatives led to the identification of several σ1R antagonists with a good absorption, distribution, metabolism, and excretion in vitro profile, no off-target affinities, and characterized by a low basic pKa (around 5) that correlates with high exposure levels in rodents. Two compounds displaying a differential brain-to-plasma ratio distribution profile, 12lR and 12qS, exhibited a good analgesic profile and were selected as preclinical candidates for the treatment of pain.

Professor Sylvie Ducki – Institut de Chimie de Clermont-Ferrand – talk from ICMS 2019.

Title: TREK-1 activation to solve the opioid crisis?

Oseltamivir has been shown to prolong the atrial conduction time and effective refractory period, and to suppress the onset of burst pacing-induced atrial fibrillation in vitro. To better predict its potential clinical benefit as an anti-atrial fibrillatory drug, we performed translational studies by assessing in vivo anti-atrial fibrillatory effect along with in vivo and in vitro electropharmacological analyses. Oseltamivir in intravenous doses of 3 (n = 6) and 30 mg/kg (n = 7) was administered in conscious state to the persistent atrial fibrillation model dogs to confirm its anti-atrial fibrillatory action. The model was prepared by tachypacing to the atria of chronic atrioventricular block dogs for > 6 weeks. Next, oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously administered to the halothane-anesthetized intact dogs to analyze its in vivo electrophysiological actions (n = 4). Finally, its in vitro effects of 10-1,000 μM on I_K,ACh, I_Kur, I_Kr, I_Na and I_CaL were analyzed by using cell lines stably expressing Kir3.1/3.4, K_V1.5, hERG, Na_V1.5 or Ca_V1.2, respectively (n = 3 for I_K,ACh and I_Kr or n = 6 for I_Kr, I_Na and I_CaL). Oseltamivir in doses of 3 and 30 mg/kg terminated the atrial fibrillation in 1 out of 6 and in 6 out of 7 atrial fibrillation model dogs, respectively without inducing any lethal ventricular arrhythmia. Its 3 and 30 mg/kg delayed inter-atrial conduction in a frequency-dependent manner, whereas they prolonged atrial effective refractory period in a reverse frequency-dependent manner in the intact dogs. The current assay indicated that IC₅₀ values for I_K,ACh and I_Kr were 160 and 231 μM, respectively, but 1,000 µM inhibited I_Na, I_CaL and I_Kur by 22, 19 and 13%, respectively. The extent of I_Na blockade was enhanced at faster beating rate and more depolarized resting membrane potential. Oseltamivir effectively terminated the persistent atrial fibrillation, which may be largely due to the prolongation of the atrial effective refractory period and inter-atrial conduction time induced by I_K,ACh and I_Kr inhibitions along with I_Na suppression. Thus, oseltamivir can exert a powerful anti-atrial fibrillatory action through its ideal multi-channel blocking property; and oseltamivir would become a promising seed compound for developing efficacious and safe anti-atrial fibrillatory drugs.

Oseltamivir has been shown to prolong the atrial conduction time and effective refractory period and to suppress the onset of burst pacing-induced atrial fibrillation in vitro. To better predict its potential clinical benefit as an anti-atrial fibrillatory drug, we performed translational studies by assessing in vivo anti-atrial fibrillatory effect along with in vivo and in vitro electropharmacological analyses. Oseltamivir in intravenous doses of 3 (n = 6) and 30 mg/kg (n = 7) was administered in a conscious state to the persistent atrial fibrillation model dogs to confirm its anti-atrial fibrillatory action. The model was prepared by tachypacing to the atria of chronic atrioventricular block dogs for > 6 weeks. Next, oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously administered to the halothane-anesthetized intact dogs to analyze its in vivo electrophysiological actions (n = 4). Finally, its in vitro effects of 10–1,000 μM on I_K,ACh, I_Kur, I_Kr, I_Na and I_CaL were analyzed by using cell lines stably expressing Kir3.1/3.4, K_V1.5, hERG, Na_V1.5 or Ca_V1.2, respectively (n = 3 for I_K,ACh and I_Kr or n = 6 for I_Kr, I_Na and I_CaL). Oseltamivir in doses of 3 and 30 mg/kg terminated the atrial fibrillation in 1 out of 6 and in 6 out of 7 atrial fibrillation model dogs, respectively without inducing any lethal ventricular arrhythmia. Its 3 and 30 mg/kg delayed inter-atrial conduction in a frequency-dependent manner, whereas they prolonged atrial effective refractory period in a reverse frequency-dependent manner in the intact dogs. The current assay indicated that IC₅₀ values for I_K,ACh and I_Kr were 160 and 231 μM, respectively, but 1,000 µM inhibited I_Na, I_CaL and I_Kur by 22, 19 and 13%, respectively. The extent of I_Na blockade was enhanced at a faster beating rate and more depolarized resting membrane potential. Oseltamivir effectively terminated the persistent atrial fibrillation, which may be largely due to the prolongation of the atrial effective refractory period and inter-atrial conduction time induced by I_K,ACh and I_Kr inhibitions along with I_Na suppression. Thus, oseltamivir can exert a powerful anti-atrial fibrillatory action through its ideal multi-channel blocking property; and oseltamivir would become a promising seed compound for developing efficacious and safe anti-atrial fibrillatory drugs.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, Ca_V2.2 and Na_V1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPR^Tetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPR^Tetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both Ca_V2.2 and Na_V1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

The adaptive voltage protocol on the Sophion Qube has been used to successfully generate pharmacology for a range of small molecules and toxins, with potency values in line with the literature. Differences in specific IC50 values between this data and published data may be explained by differences in voltage protocols, particularly in the test pulse potential and length of the inactivation period, or use of different cell types. Multi-parameter analysis of Nav1.X current traces allows mechanism of action data, including state-dependence, to be generated from single-well recordings. Additionally, the expected single amino acid substitution in the voltage-sensing domain (3,6). All data presented was obtained from a single Qube experiment for each cell line tested, demonstrating the high quantity and quality of meaningful data which can be obtained using automated patch clamp technology.

Aspidasept (Pep19-2.5) and its derivative Pep19-4LF (&ldquo;Aspidasept II&rdquo;) are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into multiple cell types, including neurons and cardiomyocytes. This allows establishing novel, highly predictive human disease models in vitro. Ion channels represent a class of highly attractive therapeutic targets in the nervous and the cardiovascular system, rendering electrophysiological studies of hiPSCs important for their usage in drug discovery. However, such studies have traditionally been limited by the labor-intensive and low-throughput nature of the manual patch-clamp technique. In the present study, we used our automated, 384 channel patch clamp system Qube to develop robust assays for the study of hiPSC – derived neurons.

Three different hiPSC – based neuronal disease models were investigated. These hiPSC-derived neurons were generated from patients presenting either spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). In a first step, voltage – clamp was used to isolate and characterize individual endogenous currents and compare these to currents recorded from cells derived from healthy individuals. Both the SMA and ALS disease model exhibited elevated voltage-gated sodium channel (Na_v) currents. We confirmed that the isogenic control cells of both models reverse the observed increase in Na_v current. Finally, we tested a set of reference compounds, targeting various ion channels, on all three disease models and evaluated the assay with regard to stability and reproducibility.

Our recordings demonstrate the feasibility of assaying hiPSC-derived neurons on the APC platforms Qube. Altogether, these results can facilitate evaluating the use of hiPSC for early drug development and in extension personal medicine.

Dr Daniel Sauter, Sophion Bioscience, Inc. – talk from ICMS 2019, the United States

Title: Towards more physiological assays: iPSC-derived neurons tested on the 384 channel automated patch clamp platform Qube

Professor Uwe Rudolph – McLean Hospital & Harvard University, USA

Title: Towards a circuit-based pharmacology of GABA-A receptors

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad-spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

Rationale: Enhancing non–CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.

Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.

Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.

Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca²⁺-activated Cl⁻ channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.

Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

TMEM16A (ANO1) is a Ca²⁺-activated Cl- channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as a target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating synergistically depends on voltage and cytosolic Ca²⁺ (Scudieri et al. 2012).
A robust and high-throughput electrophysiology assay for TMEM16A was long awaited, but progress was hampered by the fact that many automated patch clamp devices rely on the use of fluoride (F^–)in the internal solution to promote seal formation. However CaF₂ has very low solubility and the resulting precipitation limits the ability to control precisely the concentration of Ca⁺ in the internal solution.
We recently developed a novel approach to test the pharmacological inhibition of TMEM16A on Qube with following characteristics:

• Consistently high success rates (>80%)
• Low run down
• High degree of pharmacological reproducibility (consistent IC50 values of multiple runs)

TMEM16A (ANO1) is a Ca2+-activated Cl-channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating ynergistically depends on voltage and cytosolic Ca2+ (Scudieri et al. 2012). A robust TMEM16A assay was long awaited but progress was hampered as many automated patch clamp devices rely on the use of fluoride in the internal solution. Fluoride exhibits a very low solubility with calcium which means that calcium that is added to the internal solution to activate the channel has a strong tendency to precipitate, making it impossible to correctly adjust the calcium concentration.

Transient receptor potential melastatin-8 (TRPM8) represents an emerging target in prostate cancer, although its mechanism of action remains unclear. Here, we have characterized and investigated the effects of TRPM8 modulators in prostate cancer aggressiveness disclosing the molecular mechanism underlying their biological activity. Patch-clamp and calcium fluorometric assays were used to characterize the synthesized compounds. Androgen-stimulated prostate cancer-derived cells were challenged with the compounds and the DNA synthesis was investigated in a preliminary screening. The most effective compounds were then employed to inhibit the pro-metastatic behavior of in various PC-derived cells, at different degree of malignancy. The effect of the compounds was then assayed in prostate cancer cell-derived 3D model and the molecular targets of selected compounds were lastly identified using transcriptional and non-transcriptional reporter assays. TRPM8 antagonists inhibit the androgen-dependent prostate cancer cell proliferation, migration and invasiveness. They are highly effective in reverting the androgen-induced increase in prostate cancer cell spheroid size. The compounds also revert the proliferation of castrate-resistant prostate cancer cells, provided they express the androgen receptor. In contrast, no effects were recorded in prostate cancer cells devoid of the receptor. Selected antagonists interfere in non-genomic androgen action and abolish the androgen-induced androgen receptor/TRPM8 complex assembly as well as the increase in intracellular calcium levels in prostate cancer cells. Our results shed light in the processes controlling prostate cancer progression and make the transient receptor potential melastatin-8 as a ‘druggable’ target in the androgen receptor-expressing prostate cancers.

Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel Na_V1.1 is highly expressed in PV-positive interneurons, but only at low levels in principal cells. Positive modulators of Na_V1.1 channels are for this reason considered potential candidates for the treatment of cognitive disorders. Here we examined the effect of the novel positive modulator of voltage-gated sodium channels Lu AE98134. We found that Lu AE98134 facilitated the sodium current mediated by Na_V1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6^+/− mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found that in the presence of Lu AE98134, the firing frequency was increased while the spike duration and the threshold were decreased. Compounds with similar mode of action appear as promising candidates for restoring cognitive deficits present in schizophrenia.

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Ca2+-activated K+ channels (KCa) are integral players in setting the membrane potential and regulating Ca2+ homeostasis. The human genome consists of a total of 8 KCa channels that differ in their unitary conductance, genetic relation to each other and mechanism of Ca2+ binding. The channels are subdivided in two groups according to the aforementioned characteristics. The first group was initially characterized by their structural similarity and comprises of KCa1.1 (BK), KCa4.1, KCa4.2 and KCa5.1. Later studies revealed that these channels have in fact a different activation mode and only KCa1.1 (BK) is a truly Ca2+-activated channel aside from its voltage-dependence. The second group is characterized by their unitary conductance and encompasses three small conductance (SK) channels KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3
(SK3), and an intermediate-conductance channel KCa3.1 that is also known as SK4, IKCa1, IK-1 or KCa4 encoded by KCNN4 (Wei et al., 2005). KCa3.1 was cloned independently by three groups in 1997, therefore, the channels biophysical and pharmacological properties are already well-established (Wulff & Castle, 2010).

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Patients with schizophrenia show alterations in cortical network function and processing of sensory information, which can be monitored by electroencephalography (EEG), particularly auditory event-related potentials (AERP) and auditory steady-state response (ASSR). There is growing evidence that N-methyl-D-aspartate (NMDA) receptor dysfunction contributes to the pathophysiology of schizophrenia. Hence, NMDA receptor antagonists like MK-801 or ketamine can induce symptoms in animals and humans, which resemble those of patients with schizophrenia including cortical network dysfunction. Inhibition of the glycine transporter-1 (GlyT1) is an approach to facilitate NMDA receptor function via increasing its co-agonist glycine. In this study, we tested the ability of the novel GlyT1 inhibitor BI425809 to reverse MK-801 induced deficits on AERPs, ASSR and basal gamma oscillation.

We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel Na_V1.6. In an attempt to decouple channel activation from channel binding, we also synthesized the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin’s excitatory and depressant activities, effectively resulting in a Na_V1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is antinociceptive in mouse models of Na_V1.6-mediated pain.

Treatment and control of schistosomiasis depend on a single drug, praziquantel, but this is not ideal for several reasons including lack of potency against the juvenile stage of the parasite, dose size, and risk of resistance. We have optimised the properties of a series of compounds we discovered through high throughput screening and have designed candidates for clinical development. The best compounds demonstrate clearance of both juvenile and adult S. mansoni worms in a mouse model of infection from a single oral dose of < 10 mg/kg. Several compounds in the series are predicted to treat schistosomiasis in humans across a range of species with a single oral dose of less than 5 mg/kg.

Background Evidence has emerged that small-conductance Ca²⁺-activated K⁺ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited by ventricular adverse effects and efficacy loss as AF progresses.

Methods and Results A total of 43 pigs were used for the studies. AF reversion in conscious long-term tachypaced pigs: Pigs were subjected to atrial tachypacing (7 Hz) until they developed sustained AF that could not be reverted by vernakalant 4 mg/kg (18.8±3.3 days of atrial tachypacing). When the SK channel inhibitor AP14145 was tested in these animals, vernakalant-resistant AF was reverted to sinus rhythm, and reinduction of AF by burst pacing (50 Hz) was prevented in 8 of 8 pigs. Effects on refractory period and AF duration in open chest pigs: The effects of AP14145 and vernakalant on the effective refractory periods and acute burst pacing-induced AF were examined in anaesthetized open chest pigs. Both vernakalant and AP14145 significantly prolonged atrial refractoriness and reduced AF duration without affecting the ventricular refractoriness or blood pressure in pigs subjected to 7 days atrial tachypacing, as well as in sham-operated control pigs.

Conclusions SK currents play a role in porcine atrial repolarization, and pharmacological inhibition of these with AP14145 demonstrates antiarrhythmic effects in a vernakalant-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs.

The human ether-à-go-go related gene (hERG) function is important for cardiac repolarization and inhibition of the channel can prolong the cardiac action potential, which gives increased risk for ventricular arrhythmias including torsade des points (TdP). Therefore, in vitro evaluations of the compound effects is performed on the hERG channel routinely in drug development projects to detect potential arrhythmic side-effects.
Usually these compound measurements are carried out at ambient temperatures. Previously it has been shown that the potency for many compounds has been underestimated when compared to near physiological temperature tests. Therefore, a temperature controlled measuring environment is beneficial when testing compounds for the aims as mentioned here.
Until recently, the only possibility to test compound potency under voltage control conditions has been the manual patch clamp technique. Now automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 8°C and above.
Here we used an automated patch clamp system, Qube, to study the effect of temperature on concentration response relationships on a panel of compounds known to block the hERG channel. Qube has a temperature controlled test environment and in these studies, we show that temperature merits being taken into consideration when evaluating for hERG pharmacology.

To better understand how compounds function in warm-blooded animals such as humans, it is key to be able to test compound potency at physiological temperature. Our automated patch clamp systems provide real time measurement of temperature, and thus the effect of temperature change. Temperature regulation is done directly at the measurement site, and not by thermal control of the whole system. Four different compounds were tested in a dose-response format on Qube 384 at differing temperatures. All four compounds showed increased effect at elevated temperature compared to room temperature. Qube is suitable for screening compounds at temperatures from 8° C to 40° C (or 8° C above dew point).

Fibrosis is a disease that damages organs and even causes death. Because of the complicated pathogenesis, the development of drugs for fibrosis is challenging. In the lysophosphatidic acid receptor type 1 (LPA1) signaling pathway, LPA1 and its downstream Rho-associated coiled-coil forming protein kinase (ROCK) are related to the process of fibrosis. Targeting LPA1 signaling pathway is a potential strategy for the treatment of fibrosis. Area covered: This review describes the process of fibrosis mediated by the LPA1 signaling pathway and then summarizes LPA1 antagonist patents reported since 2010 and ROCK inhibitor patents since 2017 according to their scaffolds based on the Cortellis Drug Discovery Intelligence database. Information on LPA1 antagonists entering clinical trials is integrated. Expert opinion: Over the past decade, a large number of antagonists targeting the LPA1 signaling pathway have been patented for fibrosis therapy. A limited number of compounds have entered clinical trials. Different companies and research groups have used different scaffolds when designing com- pounds for fibrosis therapy. Therefore, LPA1 and ROCK are competitive targets for the development of new therapies for fibrosis to provide a potential treatment method for fibrosis in the future.

Professor Saverio Gentile, University of Illinois Chicaga – talk from the ICMS 2021 Virtual Meeting.

Title: Targeting potassium channels in cancer from cell signaling to precision medicine

About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKV) channel hKV7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hKV7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hKV7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. Methods: Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. Results: Fourteen resin acid derivatives were tested on hKV7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hKV7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hKV7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle–relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hKV11.1, hNaV1.5, or hCaV1.2, or on the amplitude of hKV11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. Significance: The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin – the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

T-type calcium channel (Cav3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved Cav3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual Cav3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two Cav3.2 (2, IC50 = 18.24 µM; 3, IC50 = 6.59 µM) and Cav3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring Cav3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of Cav3.1 activated current (2, IC50 = 5.60 µM; 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2′(3′)-O-(benzoylbenzoyl)adenosine-5′-triphosphate (BzATP)-induced IL-1β release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.

γ-Lactam is an important structural motif in a large number of biologically active natural products and synthetic small pharmaceutical molecules. However, there is currently no effective approach to construct γ-lactam ring directly from natural rigid polycyclic amides. Herein, we report a facile methodology for synthesis of a new group of olean-28,13β-lactams (10a–j) from their corresponding amides, promoted by an easily available reagent 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), through an intramolecular dehydrogenative C–N coupling reaction via a radical ion mechanism. Biological evaluation indicated that the most active lactam 10h displayed potent antiproliferative activity against human cancer cells but 13.84- to 16.92-fold less inhibitory activity on noncancer cells in vitro. In addition, 10h significantly inhibited the growth of implanted prostate cancer in vivo. Furthermore, 10h induced cell cycle arrest and apoptosis and down-regulated the AKT/mTOR signaling in DU-145 cells. Finally, 10h was more stable in rat plasma and human liver microsomes than CDDO-Me and had little hERG channel inhibitory activity. Collectively, 10h may be a potential antiprostate cancer agent for further investigation.

Over expression of c-Met tyrosine kinase is known to promote tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein a series of novel 6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine derivatives were designed, synthesised and evaluated for their c-Met kinase inhibition. Compounds 17e, 17f, 18a, and 18b were further examined for their anti-proliferative activities against four typical cancer cell lines (PC-3, Panc-1, HepG2, and Caki-1). The promising compound 17f was identified as a multi-target receptor tyrosine kinase inhibitor, which also displayed favourable pharmacokinetic properties in rats, had an acceptable safety profile in preclinical studies, and significant anti-tumour activity in the Caki-1 tumour xenograft model.

Herein we describe a focused set of new arylpiperazine derivatives as potential broad‐spectrum antipsychotics. The general structure contains a quinolinone‐like moiety, an arylpiperazine moiety, and a five‐atom linker. Among them, 7‐(5‐(4‐(benzo[d]isothiazol‐4‐yl)piperazin‐1‐yl)pentyl)quinolin‐2(1H)‐one (S6) shows a promising preclinical profile. Compound S6, characterized by partial D₂R agonism, 5‐HT_1AR agonism, 5‐HT_2AR antagonism, and blockade of SERT activities, was found to decrease psychosis‐ and depressive‐like symptoms in rodents. The polypharmacological profile of S6 could provide opportunities for the treatment of various other central nervous system disorders such as anxiety, depression, and psychoses associated with dementia. Furthermore, S6 demonstrated acceptable safety, toxicology, and pharmacokinetic profiles, and has been selected as a preclinical candidate for further evaluation in schizophrenia.

In the present study, a series of multi-target N-substituted cyclic imide derivatives which possessed potent dopamine D2, serotonin 5-HT1A and 5-HT2A receptors properties were synthesized and evaluated as potential antipsychotics. Among these compounds, (3aR,4R,7S,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione hydrochloride (3d) held a promising pharmacological profile. 3d not only showed potent and balanced in vitro activities on D2/5-HT1A/5-HT2A receptors, but also endowed with low to moderate activities on 5-HT2C, H1, α1A, M3 receptors and hERG channel, suggesting a low liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In animal behavioral studies, 3d reduced phencyclidine-induced hyperlocomotion with a high threshold for catalepsy induction. Compound 3d was selected as a potential antipsychotic candidate for further development.

Acute ischemic stroke is a leading cause of disability and death. The development of neuroprotectants is an emerging strategy for the treatment of ischemic stroke. In this work, we designed and synthesized a series of 1,3,5-triaryl substituent triazole derivatives by introducing a phenolic group and phenyl ring to 3,5-diaryl substituents oxadiazole. Structure-activity relationship (SAR) analysis showed that compounds with alkyl groups or with substituents at the 3-position possessed better protective effects. Among the derivatives, 3,5-dimethyl substituted compound 24 exhibited the best neuroprotective effect with weak cytotoxicity. Compound 24 possessed a high plasma protein binding rate, moderate hERG inhibition, low acute toxicity, and suitable pharmacokinetic properties. In vivo experiments demonstrated that compound 24 exerted a protective effect by reducing cerebral infarction size, improving neurological behavior, and restoring redox balance in middle cerebral artery occlusion rats. Further investigation indicated that compound 24 exerted a protective effect against sodium nitroprusside (SNP) induced cell damage by scavenging intracellular reactive oxygen species and restoring mitochondrial membrane potential. Moreover, compound 24 induced the nuclear translocation of nuclear factor erythroid 2-related factor (Nrf2) and promoted the generation of antioxidative proteins, including Heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase (NQO1), and glutamate-cysteine ligase catalytic (GCLC). Surface plasmon resonance (SPR) experiments indicated that compound 24 might activate the Nrf2 signaling pathway by interacting with the Keap1 Kelch domain. Taken together, these facts indicate that compound 24 might have potential in the treatment of ischemic stroke.

The development of inhibitors targeting the PI3K-Akt-mTOR signaling pathway has been greatly hindered by the on-target AEs, such as hyperglycemia and hepatotoxicities. In this study, a series of diaryl urea derivatives has been designed and synthesized based on clinical candidate gedatolisib (6aa), and most of the newly synthesized derivatives showed kinase inhibitory and antiproliferative activities within nanomolar and submicromolar level, respectively. The terminal l-prolineamide substituted derivative 6 ab showed 8.6-fold more potent PI3Kα inhibitory activity (0.7 nM) and 4.6-fold more potent antiproliferative effect against HCT116 cell lines (0.11 μM) compared with control 6aa. The potential antitumor mechanism and efficacy of 6 ab in HCT116 xenograft models have also been evaluated, and found 6 ab showed comparable in vivo antitumor activity with 6aa. The safety investigations revealed that compound 6 ab exhibited more safer profiles in the selectivity of liver cells (selectivity index: >6.6 vs 1.85) and blood glucose regulation than 6aa. In addition, the in vitro stability assays also indicated our developed compound 6 ab possessed good metabolic stabilities.

Ion channels are responsible for the permeation of ions across the membrane and their central role in cellular physiology is well established. Historically, the direct study of ion channels has been considered technically challenging. As such, a significant barrier to drug discovery for ion channels has been the low throughput of high quality electrophysiological data. The emergence of automated high throughput platforms for studying ion channel kinetics and pharmacology has lowered this barrier. Ion channels are now recognized as increasingly important drug targets and a diverse range of ion channels are implicated in a variety of drug discovery and cardiac safety assessment programs. The TRP (Transient Receptor Potential) superfamily of ion channels play a crucial role in a broad range of sensory functions including vision, taste, olfaction, hearing, touch, pain and thermosensation. Many of the TRP channels are polymodal in their activation and deactivation mechanisms and even with conventional patch clamp electrophysiology, the TRP channels are considered to be a very complex target class. Here we present an update on the significant progress made on the TRP receptor assays with the available automated patch clamp systems.

We analyzed the P2X4 receptor structure−activity relationship of a known antagonist 5, a 1,5-dihydro-2H-naphtho[1,2-b][1,4]diazepine-2,4(3H)-dione. Following extensive modification of the reported synthetic route, 4-pyridyl 21u (MRS4719) and 6-methyl 22c (MRS4596) analogues were most potent at human (h) P2X4R (IC 50 0.503 and 1.38 μM, respectively, and selective versus hP2X1R, hP2X2/3R, hP2X3R). Thus, the naphthalene 6-, but not 7-position was amenable to substitution, and an N-phenyl ring aza-scan identified 21u with 3-fold higher activity than 5. Compounds 21u and 22c showed neuroprotective and learning-and memory-enhancing activities in a mouse middle cerebral artery occlusion (MCAO) model of ischemic stroke, with potency of 21u > 22c. 21u dose-dependently reduced infarct volume and reduced brain atrophy at 3 and 35 days post-stroke, respectively. Relevant to clinical implication, 21u also reduced ATP-induced [Ca 2+ ] i influx in primary human monocyte-derived macrophages. This study indicates the translational potential of P2X4R antagonists for treating ischemic stroke, including in aging populations.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antituber-cular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC 50 = 0.52 μM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039−0.0078 μg/mL) and exhibited limited hERG inhibition (IC 50 ≥ 25 μM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.

Several types of drug-resistant epilepsies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. Approximately forty different amino acid substitutions have been identified in patients, involving both transmembrane and intracellular domains of the channel subunit, and with one exception all cause gain-of-function in channel gating. Channel inhibition, therefore, is potentially a stratified approach to treat the disorder. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which have been linked to its low potency and lack of specificity; effects on cardiac ion channels being a major concern. Here we describe the use of high-resolution structures of KNa1.1 channels, generated through cryo-electron microscopy, to identify novel inhibitors using computational methods. From an initial analysis of seventeen compounds identified using this approach, we found six that inhibited KNa1.1 channels. Their IC50 values ranged between 0.6 and 7.4 μM, compared to 100 μM with quinidine, and likely inhibited through pore-blocking mechanisms. The compounds delivered varying results when evaluating state-dependence and in preliminary toxicity assays, with some demonstrating negligible cellular toxicity and low levels of hERG block. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibitors, with the view to treating KCNT1-associated epilepsies. Furthermore, this illustrates the potential for utilising ion channel structures generated through cryo-electron microscopy in drug discovery.

LSD1 is identified as an essential drug target, which is closely correlated to the development of several tumor types. In this work, on the basis of comprehensive analysis of the binding site of LSD1 and other FAD-dependent enzymes, a novel series of potent and selective LSD1 inhibitors were designed by incorporation of privileged indoline scaffold strategies. Representative compound 7e (LSD1; IC50 = 24.43 nM, selectivity over LSD2 and MAOs of >200- and 4000-fold) possessed selective antiproliferative activities against MV-4-11 cell lines. Further study indicates that 7e could activate CD86 expression (EC50 = 470 nM) and induce differentiation of AML cell lines. More importantly, compound 7e demonstrated an acceptable oral PK profile and good in vivo antitumor efficacy with a T/C value of 30.89% in an MV-4-11 xenograft mouse model. Collectively, this work provides a promising lead compound for the development of novel LSD1 inhibitors for the treatment of AML.

In the context of drug repositioning and expanding the existing structure-activity relationship around astemizole (AST), a new series of analogues were designed, synthesized, and evaluated for their antiplasmodium activity. Among 46 analogues tested, compounds 21, 30, and 33 displayed high activities against asexual blood stage parasites (PfNF54 IC50 = 0.025-0.043 μM), whereas amide compound 46 additionally showed activity against late-stage gametocytes (stage IV/V; PfLG IC50 = 0.6 ± 0.1 μM) and 860-fold higher selectivity over hERG (46, SI = 43) compared to AST. Several analogues displaying high solubility (Sol > 100 μM) and low cytoxicity in the Chinese hamster ovary (SI > 148) cell line have also been identified.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Na_v1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Na_v1.7. Compound 24 showed a robust PK/PD response in a Na_v1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Professor Derek Bowie, McGill University – talk from ICMS 2019, the United States

Title: Structural underpinnings of ionotropic glutamate receptor activation

Structural optimization of aminopyrimidine-based CXCR4 antagonists is reported. The optimization is guided by molecular docking studies based on available CXCR4-small molecule crystal complex. The optimization identifies a number of compounds with improved receptor binding affinity and functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for CXCR4 binding in a cell-based assay: IC₅₀ = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium increase: IC₅₀ = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated chemotaxis in a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical properties (MW 367, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate inhibition of CYP isozymes and hERG). These results represent a significant improvement over the initial hit from scaffold hybridization and suggest that compound 23 can be used as a starting point to support lead optimization.

Structural optimization of aminopyrimidine-based CXCR4 antagonists is reported. The optimization is guided by molecular docking studies based on available CXCR4-small molecule crystal complex. The optimization identifies a number of compounds with improved receptor binding affinity and functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for CXCR4 binding in a cell based assay: IC₅₀ = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium increase: IC₅₀ = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated chemotaxis in a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical properties (MW 367, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate inhibition of CYP isozymes and hERG). These results represent significant improvement over the initial hit from scaffold hybridization and suggest that compound 23 can be used as a starting point to support lead optimization.

Structural optimization of aminopyrimidine-based CXCR4 antagonists is reported. The optimization is guided by molecular docking studies based on available CXCR4-small molecule crystal complex. The optimization identifies a number of compounds with improved receptor binding affinity and functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for CXCR4 binding in a cell-based assay: IC₅₀ = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium increase: IC₅₀ = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated chemotaxis in a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical properties (MW 367, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate inhibition of CYP isozymes and hERG). These results represent a significant improvement over the initial hit from scaffold hybridization and suggest that compound 23 can be used as a starting point to support lead optimization.

Dr Yonghong Bai, Amgen – talk at ICMS 2019, the United States

Title: Structural basis for pharmacological modulation of the TRPC6 channel elucidated by cryo-EM

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Nav) subtypes, including Nav1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at Nav1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human Nav channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits Nav subtypes with a rank order of potency of Nav1.4 > 1.2 > 1.7 and Isomer 2 in the order of Nav1.4≈1.2 > 1.7, while the native isomer inhibited Nav1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNav1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant Nav subtypes.

High information-content screenings based on whole-cell current patch-clamp recordings have become available for ion channel pharmacological research with the development of the QPatch automated patch-clamp technology. A significant increase in system throughput was recently achieved by a tripling of the number of parallel operating patch-clamp sites (from 16 to 48) by the introduction of QPatch HT. In a series of hERG screening studies we have subsequently aimed at increasing the throughput further by reducing the average experimental time consumption associated with each IC₅₀ determination, and by employing alternative calculational algorithms. We have investigated the effect of the following three procedures on effective QPatch throughput:

1. Application of multiple drugs per cell
2. Reduction of the duration of the experimental
   protocol
3. Estimation of drug potency (IC₅₀) based on a
   single inhibitor concentration

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

The aim of this study is to observe the consistency of a current signal (elicited by an agonist or a voltage change) before and after antagonist or blocker application. This report presents data monitored on the automated QPatch 16 based on whole-cell current recordings from ligand-gated ion channels (LGIC); the glutamate-activated ligand-gated ion channels Glu5R expressed in HEK293 cells from Neurosearch and GluR6 (GRIK2) from Millipore as well as acid-sensing ion channel 3 (ASIC3) expressed in HEK293 cells. Similar experiments were performed on voltage-gated ion channels (VGIC); HEK293-Na_v1.2a and CHO-hERG.

The ATP-gated P2X₂ and P2X₃ receptors are cation-permeable
ligand-gated ion channels that open in response to extracellular adenosine 5′-triphosphate (ATP) binding. They are widely expressed in the body, including the peripheral nervous system, making them an interesting target against pain^1–3.

When P2X₂ and P2X₃ receptors are expressed together, the functional receptors will be trimeric³ and function either as P2X_2/3 heteromers, P2X₂ homomers, or P2X₃ homomers. The ratio between P2X₂ and P2X₃ homomers and P2X_2/3 heteromers vary from cell to cell, so to limit the variation between sites, only multihole QChips with ten patch holes per site are used in the assays.

P2X₂ and P2X₃ receptors display fast and extensive desensitization, making assays vulnerable to rundown due to poor compound washout or insufficient recovery. Here we show an assay with high stability, high success rates, and low rundown.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1–2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Drug discovery on ion channels is a slow and complicated process and demands a high throughput system with high data quality, but also with a flexible design and easy-to-analyze data. Sophion Qube is a next-generation giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds with a single click on a button. Data analysis is as important as data acquisition. In HTS the need for powerful analysis with efficient quality filtering is evident in order to handle the vast amount of data generated on an electrophysiological platform.

Qube is capable of testing up to 16 different cell lines or cell clones in one experiment. This can be utilized to test a different panel of cell lines or for selecting the best suited cell clone before embarking on a HTS campaign. The integrated analysis software, Sophion Analyzer, ensures analysis to keep track of all the results and whenever another QChip is assayed the analysis is done with the same set of user defined criteria.
Here we demonstrate the power of automated analysis by exploring three types of experiments executed on eight different Nav channel subtypes; 1) TTX sensitivity, 2) IV-relationship for activation and inactivation and 3) pulse train suitable for screening for use dependent sodium channels blockers. For every run Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested in parallel on a QChip. Recorded ion channel whole-cell currents were automatically analyzed for IV-relationships for activation and inactivation (V½ and Boltzmann fit and time constants) and concentration-dependent drug effects (Hill fit and IC50) were analyzed using the Sophion Analyzer. If preferred, data can by a click be exported and calculated by other programs as Spotfire, Genedata screener or implemented in in-house software.
For each subtype, the experiments identified the expected pharmacology for use- and state-dependent drugs as well as biophysical properties. The findings determined the differences between the different subtypes as expected and also that post experiment analysis can be performed with minimum of effort when using integrated, automated analysis software.

Professor Jack Mellor, Bristol University – talk from the ICMS 2021 virtual meeting.

Title: SK channel regulation of synaptic plasticity

High throughput in vitro profile of the cardiac Na_V1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. A variety of differing protocols used across industry and academia may contribute to variation in the data.
The objectives of this study were to (1) assess pharmacology and biophysical properties of veratridine- and ATX-II-induced late INa together with that induced by a mutation in the channel (ΔKPQ-Na_V1.5), (2) develop a protocol to allow simultaneous measurement of both peak and late INa under a single protocol using automated QPatch system. The planar patch clamp technique (QPatch) was applied to record the peak and late I_Na from the Na_V1.5 channel (human SCN5A gene) or ΔKPQ-Na_V1.5 mutant channel expressed in mammalian cells.
When measured at the maximal response during the ramp of the voltage protocol, the ΔKPQ-Na_V1.5 mutant produced a small late I_Na (41.9 ± 5.4 pA). Veratridine and ATX-II-induced concentration-dependent increases in the late INa. The amplitude of late I_Na were 1162.2 ± 258.5 pA and 392.4 ± 71.3 pA in the presences of 100 μM veratridine and 100 nM ATX-II, respectively. Veratridine inhibited the peak I_Na
(IC50 = 84.1 ± 10.5 μM) and altered the biophysical properties of the INa. ATX-II showed minimal effects on the peak I_Na and preserved the biophysical properties of the I_Na. In the presence of 100 nM ATX-II, potencies of 25 clinical I_Na blockers on peak and late INa were characterized. In addition, the IC₅₀ values of these clinical INa blockers on peak I_Na correlated well with and without ATX-II. The results also demonstrated that the potency of a compound blocking late I_Na could be either overestimated or underestimated if the late I_Na was measured at the end of the depolarizing pulse versus during the ramp.
In conclusion, in the presence of ATX-II, both peak and late I_Na could be assessed simultaneously under a single protocol. Our results suggest that late I_Na may be best assessed using the maximum response obtained during the ramp after 200 ms depolarizing pulse at 40 mV.

With the ever-increasing emphasis on ion channel profiling in safety screening, there has been a drive towards increased throughput with respect to patch clamp profiling. The advent of automated 384 well plate-based planar patch clamp instruments such as the Qube^® has significantly aided in this effort, allowing for a significant increase in the number of compounds that can be profiled in a time and cost-effective manner.

To further explore the possibility of increasing efficiencies in these systems we examined whether it would be possible to measure multiple currents from separate cell lines in a single well of a 384 well plate using the Qube^® instrument.

Theoretically, the instrument lends itself to this possibility due to the virtue of being able to select plates that contain multiple holes per well. Hence by adding two independent cell lines, each independently stably expressing a recombinant ion channel, it should be possible to patch two or more cell lines per well.

We have compared a variety of conditions in the study in order to generate a robust assay that allows for the simultaneous profiling of both the Na_V1.5 peak and hERG currents. Na_V1.5 and hERG currents were separated temporally by virtue of the design of the voltage protocol. Our results show that the success rates of simultaneously recording both Na_V1.5 and hERG currents in each well were 34.4%, 87.5%, 93.8%, 93.8% and 93.8% in 2, 6, 10, 16 and 36 hole/well QChips, respectively. The potencies of 28 CiPA compounds on Na_V1.5
peak and hERG currents were determined individually in a single cell line format or in a mixed cell format using a 10 hole/well QChip. The correlation of the IC50 for the compounds was excellent for both hERG (R2=0.96) and Na_V1.5 (R2=0.86) when comparing the single cell line format with the mixed cell format.

Our data demonstrate that is possible to generate a robust and reproducible assay using a single voltage protocol and buffer combination that allows for the measurement of the effects of compounds on both hERG and Na_V1.5 currents simultaneously. Such a format can both significantly increase assay throughput and reduce assay costs while maintaining data quality.

High throughput in vitro profiling of the cardiac Na_V1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. This study assessed the pharmacological and biophysical properties of veratridine and ATX-II, as well as the channel mutation (Na_V1.5-∆KPQ) on the late I_Na. We describe a method for simultaneous measurement of both peak and late I_Na.

Kv1.5

Selective block of Na_V1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several Na_V1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC₅₀for channel block. Here, we report a target engagement assay using transgenic mice that have enabled the development of a second generation of selective NaV1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC₅₀. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.

K⁺ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In non-excitable cells, K⁺ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K⁺ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K⁺ equilibrium potential (E_K). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K⁺ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (K_ATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

K⁺ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In non-excitable cells, K⁺ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K⁺ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K⁺ equilibrium potential (E_K). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K⁺ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (K_ATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

To save time and cost for drug discovery, a paradigm shift in cardiotoxicity testing is required. We introduce a novel screening system for drug-induced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. For 12 drugs reported to have varying cardiotoxicity risks, dose-inhibition curves were determined for six ion channels using automated patch clamp systems. By manipulating the channel models implemented in a heart simulator consisting of more than 20 million myocyte models, we simulated a standard electrocardiogram (ECG) under various doses of drugs. When the drug concentrations were increased from therapeutic levels, each drug induced a concentration-dependent characteristic type of ventricular arrhythmia, whereas no arrhythmias were observed at any dose with drugs known to be safe. We have shown that our system combining in vitro and in silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently.

Ion channels play crucial roles in physiology by modulation of cellular functions that include electrical excitability, secretion, cell migration, and gene transcription. They are an important target class for drug discovery and have historically been targeted using small molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative forms of biologics. Antibodies display high specificity, selectivity, and affinity for their target antigen, thus having the potential to target ion channels very precisely. Nonetheless, isolating antibodies to ion channels is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery and due to the complexities of screening for function. In this overview, we focus on an array of screening methods, ranging from direct antibody binding screens to complex electrophysiological assays, and describe how these assays can be used to identify functional monoclonal antibodies. We also provide some insights into specific considerations which are required to enable these screens to be used for antibody drug discovery

This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate V_half , the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online V_half measurement to obtain a concentration-response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol. In our hands, the sample reference demonstrated good state- and use-dependent inhibition of Nav1.7.

This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch‐clamp system. Three protocols along with step‐by‐step details are provided. First, we describe a protocol to estimate V _half, the voltage at which half of the channels are inactivated, using traditional steady‐state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state‐dependent protocol using adaptive online V _half measurement to obtain a concentration-response curve (CRC) on known reference blockers. Last, we introduce a use‐dependent protocol. In our hands, the sample reference demonstrated good state‐ and use‐dependent inhibition of Nav1.7.

Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real-time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.

Cysteine-knot miniproteins (knottins) have potential as therapeutic agents to block ion channels involved in cancer, autoimmunity and pain but suffer from manufacturing difficulties, short half-lives and a lack of specificity. IONTAS have invented a novel molecular format wherein a peripheral CDR loop (e.g. VL CDR2) of an antibody has been removed and replaced by a naturally occurring knottin. In this novel format (termed a KnotBodyTM), the knottin enjoys the extended half-life of an antibody molecule and the peripheral CDRs gain additional diversity within a scaffold which is pre-disposed to blockade of ion channels. This example of successful fusion of one structural domain within another was initially achieved by inserting a trypsin binding knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBodies were selected from this library using phage display technology on the basis of retained trypsin binding and the correct folding of both domains were confirmed using X-ray crystallography. To further demonstrate the merits of this novel format, the modular nature of the KnotBody binding surface was exploited to: (i) improve existing knottin binding by introducing additional VHcontacts; (ii) create a bispecific molecule by introducing a VH chain that binds to a different target; (iii) engineer novel binding specificity on the knottin scaffold by loop diversification; (iv) substitute the selected (EETI-II trypsin binding) knottin with ion channel blocking knottins.

A scaffold-hopping strategy toward Agomelatine based on in silico screening and knowledge analysis was employed to design novel antidepressant agents. A series of 3, 4-dihydroisoquinoline compounds were selected for chemical synthesis and biological assessment. Three compounds (6a-1, 6a-2, 6a-9) demonstrated protective effects on corticosterone-induced lesion of PC12 cells. Compound 6a-1 also displayed low inhibitory effects on the growth of HEK293 and L02 normal cells and it was further evaluated for its potential antidepressant effects in vivo. The forced swim test (FST) results revealed that compound 6a-1 remarkably reduced the immobility time of rats and the open field test (OFT) results indicated a better general locomotor activity of the rats treated with compound 6a-1 than those with Agomelatine or Fluoxetine. Mechanism studies implied that compound 6a-1 can significantly reduce PC12 cell apoptosis by up-regulation of GSH and down-regulation of ROS in corticosterone-induced lesion of PC12 cells. Meanwhile, the down-regulation of calcium ion concentration and up-regulation of BDNF level in PC12 cells may account for the neuroprotective effects. Furthermore, compound 6a-1 can increase cell survival and cell proliferation, promote cell maturation in the rat hippocampus after chronic treatment. The acute toxicity data in vivo indicated compound 6a-1 exhibited less hepatotoxicity than Agomelatine.

Sangivamycin is a nucleoside analog that is well-tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration (IC50) in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin’s potential for clinical administration, pharmacokinetic, ADME (absorption, distribution, metabolism, and excretion), and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 (COVID-19) therapeutic.

Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high-throughput electrophysiology and its applications in drug discovery is provided.

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1–/y knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1–/y KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1–/y KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

The voltage-gated potassium channel K_V2.1 is the main contributor to the delayed rectifier potassium current in neurons and in the cortex (Du et al. 1998, Murakoshi et al. 1999). It has been suggested that K_V2.1 activation confers neuroprotection in a state of neuronal hyperexcitability via hyperpolarizing the membrane potential (Du et al. 2000, Misonou et al. 2005, Misonou et al. 2005a). Therefore, K_V2.1 may be an attractive target in epilepsy research. Also, the ion channel has previously been shown to play a central role in neurodegeneration, ageing and pancreatic β cell signaling.

Professor Len Kaczmarek, Yale University – talk from ICMS 2019, the United States

Title: Regulation of cellular signaling by potassium channels

Recommended cell concentration on both QPatch ll and Qube depends on both assay complexity, assay longevity and especially on cell quality. We generally recommend having a cell density of 3×10⁶ cells/ml on both Qube and QPatch ll. However, the cell consumption for both systems can be reduced significantly, with a factor of at least 6, and still ensure success rates above 90%. We recommend that optimal cell concentrations are evaluated when working with cells that are expensive or in short supply. Furthermore, QPatch ll can now be loaded with as little as 50 μl cells for a single experiment.

Recommended cell concentration on both QPatch ll and Qube depends on both assay complexity, assay longevity and especially on cell quality. We generally recommend having a cell density of 3×106 cells/ml on both Qube and QPatch ll. However, the cell consumption for both systems can be reduced significantly, with a factor of at least 6, and still ensure success rates above 90%. We recommend that optimal cell concentrations are evaluated when working with cells that are expensive or in short supply. Furthermore, QPatch ll can now be loaded with as little as 50 μl cells for a single experiment.

Professor Yasuo Mori – Kyoto University, Japan

Title: Redox biology of TRP channels

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na_Vs) play a critical role in this process. In particular, Na_V subtype 1.7, has emerged as a promising universal target for the treatment of pain.

Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na_V1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a using sortase A mediated ligation, providing avenues for further pre-clinical development.

We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na_Vs) play a critical role in this process. In particular, the Na_V subtype 1.7, has emerged as a promising universal target for the treatment of pain.

Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na_V1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development.

We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane-binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane-binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) is a non-receptor protein tyrosine phosphatase encoded by the Ptpn11 gene, which regulates cell growth, differentiation and apoptosis via modulating various signaling pathways, such as the RAS/ERK signaling pathway, and participates in the PD-1/PD-L1 pathway governing immune surveillance. It has been recognized as a breakthrough antitumor therapeutic target. Besides, numerous studies have shown that SHP2 plays an important role in the regulation of inflammatory diseases. However, inhibitors targeting the active site of SHP2 lack drug-likeness due to their low selectivity and poor bioavailability, thus none have advanced to clinical development. Recently, allosteric inhibitors that stabilize the inactive conformation of SHP2 have achieved breakthrough progress, providing clinical proof for the druggability of SHP2 as an antitumor drug target. This paper reviews the recently reported design and discovery of SHP2 small molecule inhibitors, focused on the structure-activity relationship (SAR) analysis of several representative SHP2 inhibitors, outlining the evolution and therapeutic potential of the small molecule inhibitors targeting SHP2.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide-Kv1.3). Both the apo-Kv1.3 and dalazatide-Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide-Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

It has been shown that the human muscle cell line TE671 has an endogenous nicotinic acetylcholine alpha 1 receptor (nAChRα1) (Morris et al,. 1991). TE671 cells have therefore been used for experiments with nAChRα1 as a target. This brief report describes the experimental set-up of TE671 and nAChRα1 on the QPatch.

Many spider venom-peptides are known to modulate the activity of the voltage-gated sodium channel Na_V1.7, which has emerged as a promising analgesic target. A class of spider venom-peptides (NaSpTx1) in particular have been found to potently inhibit Na_V1.7 (nanomolar IC50), and shown to produce analgesic effects in animals. However, one member of this family, μ TRTX Hhn2b (Hhn2b), does not inhibit mammalian Na_V channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b leading us to identify two non-pharmacophore residues that contribute to hNa_V1.7 inhibition by non-overlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNa_V1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom-peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.

The MaxCyte® STX™ Scalable Transfection System uses a proprietary flow electroporation technology that can transfect up to 1E10 cells with target, reporter and protein expression plasmids, as well as other molecules, in less than 30 minutes. Transfected cells can be assayed immediately or cryopreserved for future use. Here we demonstrate the use of the MaxCyte STX system in coordination with Corning’s ® HYPERFlask® Cell Culture Vessel to provide an efficient and economical solution for culturing large numbers of adherent CHO cells before and after transfection. The HYPERFlask Cell Culture Vessel features Corning’s HYPER (High Yield PERformance) technology, which utilizes a gas permeable film to provide gas exchange between the internal culture environment and the external atmospheric environment. The unique, space-saving, 10-layer film design results in 1720 cm2 cell growth surface area, which is approximately 10 times that of a normal T-175 flask. We also show that cells transfected with a plasmid encoding a Kv1.3-GFP fusion protein exhibited good seal formation and strong potassium currents following large scale electroporation and cryopreservation. Ion channel activity was assayed on the Sophion QPatch automated patch clamp system. High viability and consistent levels of expression were obtained in three independent large scale electroporations, illustrating robustness and reproducibility of the transfection process. These results demonstrate that the MaxCyte STX system offers a time and labor saving alternative to stable cell line generation for ion channel assays.

Using the optical features of Qube Opto 384, it is possible to evaluate both light-activated ion channels and photoactivated ligands. Here we outline a few of the studies we have performed on Qube Opto 384.

Channelrhodopsin 2 (ChR2) could be activated and the channelrhodopsin 2 mediated current could be manipulated in both a light and voltage-dependent manner, with activation times as short as 4 ms. A light-induced chloride current could also be elicited, employing the chloride-conducting channelrhodopsin iC⁺⁺.

Caged γ-aminobutyric acid (Rubi-GABA) could be activated by light and give rise to a GABA_AR mediated current. The GABA response was both concentration and the light intensity dependent. The optical activation of GABA combined with microfluidic channels also enabled ultra-short ligand exposure times.

The Comprehensive in vitro Proarrhythmia Assay (CiPA) proposal provides an attractive perspective for increasing the efficacy of the drug development process. A detailed electrophysiological analysis of Na_v1.5 (peak and late currents), K_v4.3 (I_to), hERG (I_Kr), KvLQT1/minK (I_Ks), Ca_v1.2 and Kir2.1 (IK1) upon addition of a potential drug is a major part of assessing the drug’s proarrhythmic risk. Gaining a better understanding of the complex relationship between QT-elongation and the occurence of Torsades des Pointes (TdP), potentially enables compounds with properties that today are considered as problematic to be further developed. High throughput screening (HTS) supports this quest and is furthermore of great importance for discovering pharmacologically active substances and understanding ion channels.

Qube is a giga-seal automated patch clamp (APC) instrument, providing 384 amplifiers for the consumable QChip 384 with its integrated electrodes. The QChip has built-in microfluidic flow channels that ensure a fast and complete exchange of liquid for reliable measurements on ligand-gated ion channels and sequential additions to the same site. Here, four different cell lines expressing the cardiac ion channels Na_v1.5, Ca_v1.2, K_v1.5 and hERG, were transferred from a cell clone cell transfer plate (ccCTP) onto the same QChip. A range of pharmacological substances and voltage step protocols were applied to address the suitability of Qube for measuring different cell populations in parallel.

The Qube is the new gigaseal-based 384-channel planar patch clamp system developed by Sophion Bioscience A/S. The system is equipped with 384 individual amplifiers providing continuous recordings at a sampling rate up to 50 kHz from all wells simultaneously. The consumable for the Qube is built on Sophion’s tried and tested silicon technology for optimal giga-seal formation and has been designed to have an effi cient liquid fl ow system which is necessary for testing fast ligand-gated ion channels (LGIC) such as the nicotine acetylcholine receptors (nAChR). In this work we present recordings obtained using the Qube system from two LGICs: ASIC1a and nAChRα1.
The acid-sensitive channel (ASIC) belongs to the ENaC/DEG family. ASIC1a is expressed in the brain and in the peripheral nervous system where it is involved in modulating response to pain (1). The nicotinic acetylcholine receptor alpha 1 (nAChRα1) belongs to the Cys-loop family and is expressed both centrally and in the peripheral nervous system. In the periphery nAChRα1 modulate e.g. the synaptic transmission at the neuromuscular junction (2). Here we show activation of these LGICs by their respective ligand (H+ and acethylcholine (ACh), respectively) and pharmacological block by known reference compounds (amiloride and tetracaine, respectively). These data demonstrate: 1) the design of the Qube consumable allows fast liquid exchange for recordings of fast LGICs and 2) the design of the Qube consumable allows multiple liquid additions to the same recording unit.

Drug discovery on ion channel targets has been an under-explored territory because suitable screening tools for patch clamp were missing. This changed during the 2000’s with the introduction of automated patch clamp (APC) devices. Still, the throughput and running cost of APC devices did not allow for the employment of the patch clamp assays in primary screening. Because of this bottleneck, APC has remained a secondary screening tool. We are now presenting data from the first true gigaseal-based 384-channel planar patch clamp system, the Qube, capable of providing the throughput needed for primary screening. The Qube is a collection of years of experience with planar patch clamp devices: High quality, reliability and easy to use concepts are the core in both the tried-and-tested silicon technology of the consumable, and in the technologies used in the instrument. The Qube offers GΩ seals and efficient integrated liquid flow in 384-well format.
We show here that the Qube provides exceptional data quality for electrophysiological assays with fast desensitizing ligand-gated ion channels. The fast liquid exchange permits accurate XC50 estimations. We have tested the acid sensing ion channel (ASIC1) and the nicotinic acetylcholine receptor (nAchRα1) on the Qube in both agonist and antagonist configurations, showcasing the instrument’s broad flexibility in terms of assay design. The results prove that: 1) Repetitive agonist applications result in reproducible current responses. 2) The agonist can be applied and washed off an unlimited number of times as long as the cell is stable. 3) XC50 values correspond to expected values (QPatch and literature). Collectively, our results show that the design of the flow channels on the Qube consumable enables recordings on ligand-gated ion channels with the highest throughput capacity seen so far, without compromising data quality, reproducibility or reliability.

The QPlate is the biochip applied for electrophysiological measurements using the QPatch automated patch clamp (APC) instrument. The unique QPlate design ensures high and consistent experiment quality and secures several vital properties for high-throughput patch-clamp, including:

– Giga-ohm seal formation in physiological solutions

– Minimal compound adsorption

– Fast and efficient (100%) liquid exchange

– Maintenance-free, single-use electrodes

– Several QPlate configurations to accommodate different experiment- and throughput requirements

– A non-compromised testing environment

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC50 values. The microfluidic pathways also allow for very small amounts of compound used for each experiment (∼ 5 µl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

The ongoing threat of seasonal and pandemic influenza to human health requires antivirals that can effectively supplement existing vaccination strategies. The M2 protein of influenza A virus (IAV) is a proton-gated, proton-selective ion channel that is required for virus replication and is an established antiviral target. While licensed adamantane-based M2 antivirals have been historically used, M2 mutations that confer major adamantane resistance are now so prevalent in circulating virus strains that these drugs are no longer recommended. Here we review the current understanding of IAV M2 structure and function, mechanisms of inhibition, the rise of drug resistance mutations, and ongoing efforts to develop new antivirals that target resistant forms of M2.

In this chapter, you will learn about the methods for analyzing a proteome. After looking at the classical method of 2D electrophoresis, we will explore some concepts and challenges of protein microarrays and discuss different detection methods. Moreover, you will get an introduction to the patch-clamp method for studying the physiology of cells and its use in pharmacology.

You will be able to explain how protein chips work and their potential use in medical diagnostics. Furthermore, you will be able to describe how to assess efficacy and side effects of drugs by patch-clamping techniques.

In this chapter, you will learn about the methods for analyzing a proteome. After looking at the classical method of 2D electrophoresis, we will explore some concepts and challenges of protein microarrays and discuss different detection methods. Moreover, you will get an introduction to the patch-clamp method for studying the physiology of cells and its use in pharmacology.

You will be able to explain how protein chips work and their potential use in medical diagnostics. Furthermore, you will be able to describe how to assess the efficacy and side effects of drugs by patch-clamping techniques.

Professor Anders Jensen – Copenhagen University, Denmark

Title: Probing the molecular basis for potency- and efficacy-based subtype-selectivity exhibited by benzodiazepine-site modulators at GABA-A receptors

Recent preclinical studies have shown that activation of the serotonin 5-HT₇ receptor has the potential to treat neurodevelopmental disorders such as Fragile X syndrome, a rare disease characterized by autistic features. With the aim to provide the scientific community with diversified drug-like 5-HT₇ receptor-preferring agonists, we designed a set of new long-chain arylpiperazines by exploiting structural fragments present in clinically approved drugs or in preclinical candidates (privileged scaffolds). The new compounds were synthesized, tested for their affinity at 5-HT₇ and 5-HT_1A receptors, and screened for their in vitro stability to microsomal degradation and toxicity. Selected compounds were characterized as 5-HT₇ receptor-preferring ligands, endowed with high metabolic stability and low toxicity. Compound 7g emerged as a drug-like 5-HT₇ receptor-preferring agonist capable to rescue synaptic plasticity and attenuate stereotyped behaviour in a mouse model of Fragile X syndrome.

Recent preclinical studies have shown that activation of the serotonin 5-HT₇ receptor has the potential to treat neurodevelopmental disorders such as Fragile X syndrome, a rare disease characterized by autistic features. With the aim to provide the scientific community with diversified drug-like 5-HT₇ receptor-preferring agonists, we designed a set of new long-chain arylpiperazines by exploiting structural fragments present in clinically approved drugs or in preclinical candidates (privileged scaffolds). The new compounds were synthesized, tested for their affinity at 5-HT₇ and 5-HT_1A receptors, and screened for their in vitro stability to microsomal degradation and toxicity. Selected compounds were characterized as 5-HT₇ receptor-preferring ligands, endowed with high metabolic stability and low toxicity. Compound 7g emerged as a drug-like 5-HT₇ receptor-preferring agonist capable to rescue synaptic plasticity and attenuate stereotyped behavior in a mouse model of Fragile X syndrome.

Native cells represent a promising alternative to cell lines due to more physiological behaviour.

Primary brain neurons and astrocytes were tested on Qube 384

• Neuronal Na+ and K+ currents were measured in voltage clamp
• Neuronal action potentials were measured in current clamp
• Astrocyte K+-currents were elicited with both ramp- and step-protocols

A series of dihydropyrazole derivatives was developed as potent, selective, and brain-penetrating T-type calcium channel blockers. An optimized derivative, compound 6c, was advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of generalized nonconvulsive, absence-like epilepsy. Compound 6c was not efficacious in the basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of the PR interval in ECG recordings in rodents.

Recent work by FDA and HESI CiPA working groups indicate that in vitro hERG, Nav1.5 and Cav1.2 potency data in addition to dynamic hERG kinetic data is required to accurately predict proarrhythmic risk. Below we explore two key challenges in exclusively using automated patch clamp for risk prediction:

1. Metrion has previously shown the ability to implement the difficult Milnes protocol on QPatch but the challenges of producing full concentration-response formats required for in silico models are unknown
2. Cav1.2 inhibition values for compounds such as verapamil have been
   variable, with literature IC50 values >10 µM compared to the 202 nM IC50 obtained by manual patch clamp in the original CiPA study. The similar potency of verapamil against Cav1.2 and hERG make it a Multiple Ion Channel Effect (MICE) compound and “rescues” it from being classed as a high-risk proarrhythmia compound as shown by Li et al.
3. QPatch dynamic hERG and Cav1.2 data from multiple voltage protocols
   were used to assess proarrhythmic risk using the FDA’s in silico model.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50 = 26 ± 6 μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2–4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer’s disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

Selective inhibitors of voltage-activated K⁺ channels are needed for the treatment of multiple sclerosis. In this work it was discovered that porphyrins bearing 2–4 carbon alkyl ammonium side chains predominantly blocked the K_v1.1 current whilst K_v1.2 was susceptible to a porphyrin bearing polyamine side chains.

Professor David Beech – Leeds University, UK

Title: Piezo1 channel function and small-molecule modulation

Many rapidly firing neurons, including those in the Medial Nucleus of the Trapezoid Body (MNTB) in the auditory brainstem, express “high threshold” voltage-gated K_v3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate K_v3.1 channels. Using CHO cells stably expressing rat K_v3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of K_v3.1 currents towards negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single channel recordings also showed that AUT1 shifted the open probability of K_v3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage-dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of K_v3.1 currents represents a novel avenue for manipulation of neuronal excitability, and has the potential for therapeutic benefit in the treatment of hearing disorders.

Professor Yoshihiro Kubo – National Institute for Physiological Sciences – talk from ICMS 2019

Title: Phosphoinositides modulate the voltage dependence of Two-Pore Channel 3

Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets – including ion channels, G-protein coupled receptors, transporters and enzymes – with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches – supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques – promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches.

The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3–hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.

Studies of GABA_A ion channels using the automated patch clamp platform Qube 384 with focus on:

• Short ligand exposure with repetitive stimulations with EC₅₀
  concentrations of GABA
• Effects of agonists, antagonists and modulators
• Cumulative and non-cumulative concentration-response relationships
• Characterizing the pharmacological properties of four cell lines expressing different GABA_A subtypes

We here report pharmacological data (EC₅₀ and IC₅₀ values, tests for state-dependency of blockers and steady-state inactivation characteristics) from eight different types of voltage- and ligand-gated ion channels using the automated QPatch 16 system. The data obtained were in accordance with published literature values (not shown). We conclude that the QPatch technology enables fast and reliable characterization of electrophysiological ion channel properties.

Human genetic studies have implicated the voltage-gated sodium channel Na_V1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na_V1.7 (IC₅₀ 0.9 nM) with at least 40–1000-fold selectivity over all other Na_Vsubtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na_V1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na_V1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na_V1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na_V1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

The role of voltage-gated sodium (Na_V) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na_V1.8 and Na_V1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na_V1.8 and mouse Na_V1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na_V isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single-step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na_V1.8 and Na_V1.9 and lacked analgesic efficacy in a murine model of inflammatory pain.

The role of voltage-gated sodium (Na_V) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na_V1.8 and Na_V1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na_V1.8 and mouse Na_V1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na_V isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single-step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na_V1.8 and Na_V1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

In isolated human atrial cardiomyocytes, inhibition of K_2P3.1 K⁺ channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K_2P3.1 (KCNK3), together with K_2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K_2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K_2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K_2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K_2P3.1 current is augmented, K_2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

Objective: To screen a library of potential therapeutic compounds for a woman with Lennox-Gastaut syndrome due to a Y302C GABRB3 (c.905A>G) mutation.

Methods: We compared the electrophysiological properties of cells with wild-type or the pathogenic GABRB3 mutation.

Results: Among 1320 compounds, multiple candidates enhanced GABRB3 channel conductance in cell models. Vinpocetine, an alkaloid derived from the periwinkle plant with anti-inflammatory properties and the ability to modulate sodium and channel channels, was the lead candidate based on efficacy and safety profile. Vinpocetine was administered as a dietary supplement over 6 months, reaching a dosage of 20 mg
three times per day, and resulted in a sustained, dose-dependent reduction in spike-wave discharge frequency on electroencephalograms. Improved language and behavior were reported by family, and improvements in global impression of change surveys were observed by therapists blinded to intervention.

Significance: Vinpocetine has potential efficacy in treating patients with this mutation and possibly other GABRB3 mutations or other forms of epilepsy. Additional studies on pharmacokinetics, potential drug interactions, and safety are needed.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells is obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast, the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca²⁺), prevents channel current “run down,” and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch-clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch-clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

In patch clamp recordings cell dialysis of intracellular constituents can be minimized by employing perforated whole-cell formation.
This method allows electrical access to the cell while maintaining the integrity of the majority of cytoplasmic components. Here we report how to perform perforated patch clamp recordings on Qube 384 with or without the use of intracellular (IC) solution exchange (IC exchange).
• Recommendations for perforated patch clamp experiments with or without using IC exchange
• Comparison of four perforating agents (seal resistance, current level and success rate)
The first part of this report consists of a fast guide for using perforated patch on Qube 384, followed by an in-depth run-through of the experiments underlying our recommendations

Many ion channels are recognized as amenable targets for a range of disease states and conditions. However, the process of discovering drugs is highly influenced by the chemical doability, the biological confidence in rationale of the approach and the ‘screenability’. To date, the absence of informative high throughput technologies for ion channel screening has resulted in ion channels remaining a largely unexplored class of drug targets. This, however, is about to change – a large increase in the number of data points per day should be achieved by the introduction of automated ‘high throughput’ patch clamp machines.

The advent of higher throughput patch clamp electrophysiology systems has begun to change the face of ion channel drug discovery. Systems such as IonWorksHT, PatchXpress and QPatch should allow electrophysiology to be re-positioned from an occasional reagent and compound ‘spot-check’ method, to a frontline gene expression analysis tool and drug screening workhorse. This article chronicles the evolution of these systems, their technical capabilities and impact on drug discovery.

The human α7 nicotinic acetylcholine receptor (α7-nAChR) is a neuronal ligand-gated, fast desensitizing, non-selective cation channel. It is pentahomomeric, consisting of five 50 kD α7 subunits, each composed of 502 amino acids. The α7-nAChR is involved in memory and cognition, and it is widely distributed throughout the nervous system, especially in cholinergic neurons projecting to hippocampus and cortex. The α7-nAChR has also been found to be associated with pathophysiological states. Importantly, it is involved in widespread human neuro-degenerative and psychotic disorders, including Alzheimer’s disease and schizophrenia. Therefore the therapeutic potential of α7-nAChR is substantial, and electrophysiological and pharmacological characterization of the receptor has become an increasingly important issue. The fast kinetics (milliseconds) makes precise patch clamp measurements a challenging task. The present study addresses the possibility of efficient and precise characterization of α7-nAChR function and pharmacology by means of the QPatch automated patch clamp system. Compared to conventional whole-cell patch clamp the system enables a highly increased throughput by simultaneous and asynchronous operation of 16 parallel patch clamp experiments.

The incorporation of the fluorine motif is a strategy widely applied in drug design for modulating the activity, physicochemical parameters, and metabolic stability of chemical compounds. In this study, we attempted to reduce the affinity for ether-à-go-go-related gene (hERG) channel by introducing fluorine atoms in a group of 1H-pyrrolo[3,2-c]quinolines that are capable of inhibiting monoamine oxidase type B (MAO-B). A series of structural modifications guided by in vitro evaluation of MAO-B inhibition and antitargeting for hERG channels were performed, which led to the identification of 1-(3-chlorobenzyl)-4-(4,4-difluoropiperidin-1-yl)-1H-pyrrolo[3,2-c]quinoline (26). Compound 26 acted as a reversible MAO-B inhibitor exhibiting selectivity over 45 targets, enzymes, transporters, and ion channels, and showed potent glioprotective properties in cultured astrocytes. In addition, the compound demonstrated good metabolic stability in rat liver microsomes assay, a favorable safety profile, and brain permeability. It also displayed procognitive effects in the novel object recognition test in rats and antidepressant-like activity in forced swim test in mice. The findings of the study suggest that reversible MAO-B inhibitors can have potential therapeutic applications in Alzheimer’s disease.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single-cell transduction, and subcellular pathways.

Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light-activated ion channels and photoactivated ligands.

Cultured cells from mammalian cell lines incubated at 37 °C can be used for automated patch clamp (APC), including compound screening, for only a relatively brief period of time. Usually this time period extends from the time of ~50 % confluence to full confluence. For CHO cells, the preferred cell type for APC, expressing hERG potassium channels the duration of the usable period is 1-2 days. We here report that incubating CHO-hERG cells at a reduced temperature, 30 °C, for 1-5 days prior to APC experiments is advantageous in several ways: (1) it increases the percentage of cells with acceptable hERG currents (> 50 pA), (2) it increases the average whole-cell current significantly, and (3) it
slows cell proliferation and extends the usable period up to at least 5 days. In addition, we have tested whether the altered conditions affect the IC50 obtained for known hERG inhibitors. For all experiments the automated patch clamp system QPatch 16 was used.

Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 μM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

Drug-induced QT interval prolongation, ventricular arrhythmia and sudden death are one of the leading causes for drug withdrawal from the market or denied regulatory approval. Preclinical profiling for off-target cardiac ion channel interactions using The Comprehensive in vitro Proarrhythmia
Assay (CiPA) paradigm has been initiated to overcome drug attrition due to hERG inhibition only. The aim is to improve the prediction of a drug’s proarrhythmic liability for the pharmaceutical industry. This new paradigm includes a panel of in vitro assays that integrates effects of the test compounds on several cardiac ion channels. Voltage-gated ion channels in this panel, particularly hCa_V1.2 and hKCNQ1/hminK, exhibit rapid
activities decrease when contact between membrane and cytosol is disrupted upon patch excision (run-down). The loss of channel activity without pharmacological interaction poses a challenge for developing accurate, precise and robust assay yielding high success rate, minimal current run-down and reliable pharmacological results. In the present study, hCa_V1.2 and hKCNQ1/hminK cardiac ionic currents were validated on QPatch HT patch clamp system with the specific focus on preventing current run-down. The results demonstrate suitability of these assays for screening and profiling of drug effects by significantly reducing run-down (<20%) by optimizing protocol parameters. Reference pharmacology was
assessed for Ca_V1.2 (Nifedipine IC₅₀ =144nM) and hKCNQ1/hminK (Chromanol 293B IC₅₀ =12.3μM). The optimized assays for hCa_V1.2 and hKCNQ1/hminK on the QPatch are able to provide data that is comparable to manual patch clamp, which allows the assessment of a novel compounds proarrhythmic risk.

Optical modulation of ion channels is traditionally studied using a manual patch clamp system combined with a light source. This approach, however, is limited by very low throughput. In the present work, we show data recorded using a 384-well based automated patch clamp system equipped with 384 integrated light sources (Qube Opto 384).

Optogenetics
In this study, we evaluated Channelrhodopsin 2 (ChR2), a light-sensitive non-selective cation channel permeable to Na+, K+ and Ca2+ opened upon illumination (Berndt et. al., 2012). Furthermore, we employed the chloride-conducting channelrhodopsin (iC++, Govorunova et Al. 2015), which was developed from a non-selective cation conducting channelrhodopsin through a mutational approach. Finally, we used photoactivatable adenylyl cyclase, bPAC, to modulate cellular cAMP levels and thereby alter biophysical properties of the HCN2 channel (Stierl et Al., 2011).

Optopharmacology
Compound activation by light enables the pharmacological manipulation of receptors, ion channels and other proteins with a high degree of temporal control. We used caged GABA (Rubi-GABA, Zayat et. al., 2003) to study the light activation of ligands, in combination with the microfluidic flow channel of the QChip 384, to give a higher degree of experimental control.

Optical modulation of ion channels is traditionally studied using a manual patch clamp system combined with a light source. This approach, however, is limited by a very low throughput. In the present work, we show data recorded using a 384-well based automated patch clamp system equipped with 384 integrated light sources (Qube Opto 384).

Optogenetics
In this study we evaluated Channelrhodopsin 2 (ChR2), a light-sensitive non-selective cation channel permeable to Na⁺, K⁺ and Ca²⁺ opened upon illumination (Berndt et al., 2012). Furthermore, we employed the chloride-conducting channelrhodopsin (iC++, Govorunova et al., 2015), which was developed from a non-selective cation conducting channelrhodopsin through a mutational approach.

Optopharmacology
Compound activation by light enables the pharmacological manipulation of receptors, ion channels and other proteins with a high degree of temporal control. We used caged GABA (Rubi-GABA, Zayat et al., 2003) to study the light activation of ligands, in combination with the microfluidic flow channel of the QChip 384, to give a higher degree of experimental control.

There is no refuting that America’s population is growing older: for the first time in US history, by 2034 older adults (defined as >65 years of age) are projected to outnumber children under the age of 18, representing approximately 70 million people or almost 25% of the population (Lloyd-Jones et al., 2010). Described as the “silver tsunami”, this flood of older adults is driven by the baby boomers (people born after World War II, from 1946 to 1964): they are now reaching old age, living longer due to significant advances in healthcare coupled with a record low birth rate, resulting in a skewed elderly population demographic.

Unfortunately, older adults are also becoming increasingly unhealthy. Many often suffer from several chronic disorders requiring the use of multiple medications at a level higher than any other age group, resulting in an increased risk of drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Indeed, because of age-related changes in pharmacokinetics (PK) and pharmacodynamics (PD), older adults are also more vulnerable to drug toxicity. Prescribed drugs certainly improve a range of health outcomes, but also often cause considerable ADRs, leading to devastating consequences for patients, clinicians, and manufacturers. Therefore, safe and effective pharmacotherapy remains one of the greatest growing challenges in geriatric medicine.

In this review we examine the effects of aging and its impact on the increased risk of experiencing ADRs, resulting in devastating consequences for patients and manufacturers. We assess the current regulatory considerations related to the development of drugs for this population and highlight issues, concerns, and propose alternatives to the standard battery of tests focused on assessing cardiovascular (CV) safety in an attempt to develop safer and efficient new drugs for the growing elderly demographic.

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38–0.98 μM) over Stat1 or Stat5 (IC50 > 15.8 μM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo.

In this work, we investigated the in vitro neurotoxicity of Calliophis intestinalis venom using chick biventer cervicis neuromuscular preparations and electrophysiological analysis of voltage-gated sodium (NaV) channels expressed in HEK293 cells. We found that the indirect twitches of the neuromuscular preparations decreased over time when exposed to venom. However, the responses of these preparations to the agonists acetylcholine, carbachol, and potassium chloride were not changed after incubation with the venom. Our electrophysiological experiments show that C. intestinalis venom acts as a NaV channel antagonist—the first known from a vertebrate venom—by decreasing the peak current of NaV1.4 channels without changing the kinetics of activation or inactivation. Our proteomic results accord with earlier analyses and find that the venom contains three-finger toxins, cysteine-rich secretory proteins, kunitz peptides, phospholipase A2s, snake venom metalloproteases, and vespryns. Some of the three-finger toxins are similar to the δ-elapitoxins from the venom of the closely related Calliophis bivirgatus. However, δ-elapitoxins act as NaV channel agonists in C. bivirgatus whereas C. intestinalis venom contains NaV channel antagonists. The toxins and mechanisms responsible for the neuromuscular symptoms remain unclear as does the identity of the NaV channel antagonists. These aspects of this unusual venom require further study.

This study reports the synthesis and evaluation of novel indirect AMP-activated protein kinase (AMPK) activators. The series of compounds selectively inhibited cell growth in several human breast cancer cell lines by activating AMPK. We performed back-up medicinal chemistry synthetic research on ASP4132, a previously reported as a compound for clinical development that acts as an indirect AMPK activator. This led to the successful identification of 4-({4-[5-({1-[(5-ethoxypyrazin-2-yl)methyl]-4-fluoropiperidin-4-yl}methoxy)-3-methylpyridine-2-carbonyl]piperazin-1-yl}methyl)benzonitrile succinate (27b), a potent, highly aqueous soluble and metabolically stable compound in human hepatocytes. Compound 27b also showed weaker human Ether-a-go-go Related Gene (hERG) inhibitory activity than that of compound 13 and ASP4132. Therefore, 27b was a promising AMPK activator and a second-generation clinical candidate for treatment for human cancer.

This study reports the synthesis and evaluation of novel indirect AMP-activated protein kinase (AMPK) activators. The series of compounds selectively inhibited cell growth in several human breast cancer cell lines by activating AMPK. We performed back-up medicinal chemistry synthetic research on ASP4132, a previously reported as a compound for clinical development that acts as an indirect AMPK activator. This led to the successful identification of 4-({4-[5-({1-[(5-ethoxypyrazin-2-yl)methyl]-4-fluoropiperidin-4-yl}methoxy)-3-methylpyridine-2-carbonyl]piperazin-1-yl}methyl)benzonitrile succinate (27b), a potent, highly aqueous soluble and metabolically stable compound in human hepatocytes. Compound 27b also showed weaker human Ether-a-go-go Related Gene (hERG) inhibitory activity than that of compound 13 and ASP4132. Therefore, 27b was a promising AMPK activator and a second-generation clinical candidate for treatment for human cancer.

We have recently developed a series of novel, first-in-class, voltage-gated calcium channel (VGCC) positive allosteric gating modifiers that are selective for the types of VGCCs that regulate transmitter release at neuromuscular synapses (Ca_V2). These small molecules do not cross the blood-brain barrier well and have been shown to have therapeutic potential to treat a variety of neuromuscular diseases characterized by weakness of presynaptic origin (Lambert-Eaton myasthenic syndrome (LEMS) and Spinal Muscular Atrophy (SMA).

In Gram-negative bacterial sepsis, production of excess pro-inflammatory cytokines results in hyperinflammation and tissue injury. Anti-inflammatory cytokines such as IL-10 inhibit inflammation and enhance tissue healing. Here, we report a novel approach to treat septicemia associated with intra-abdominal infection in a murine model by delicately balancing pro- and anti-inflammatory cytokines. A novel oligosaccharide compound AVR-25 selectively binds to the TLR4 protein (IC₅₀ = 0.15 µM) in human peripheral blood monocytes and stimulates IL-10 production. Following the cecal ligation and puncture (CLP) procedure, intravenous dosing of AVR-25 (10 mg/kg, 6–12 h post-CLP) alone and in combination with antibiotic imipenem protected both young adult (10–12 week old) and aged (16–18 month old) mice against polymicrobial infection, organ dysfunction, and death. Proinflammatory cytokines (TNF-α, MIP-1, i-NOS) were decreased significantly and restoration of tissue damage was observed in all organs. A decrease in serum C-reactive protein (CRP) and bacterial colony forming unit (CFU) confirmed improved bacterial clearance. Together, these findings demonstrate the therapeutic ability of AVR-25 to mitigate the storm of inflammation and minimize tissue injury with high potential for adjunctive therapy in intra-abdominal sepsis.

Neutrophil elastase, a powerful physiological defence tool, may serve as drug target for diverse diseases due to its bystander effect on host cells like chronic obstructive pulmonary disease (COPD). Here, we synthesised seven novel benzoxazinone derivatives and identified that these synthetic compounds are human neutrophil elastase inhibitor that was demonstrated by enzyme substrate kinetic assay. One such compound, PD05, emerged as the most potent inhibitor with lower IC50 as compared to control drug sivelestat. While this inhibition is competitive based on substrate dilution assay, PD05 showed a high binding affinity for human neutrophil elastase (Kd = 1.63 nM) with faster association and dissociation rate compared to notable elastase inhibitors like ONO 6818 and AZD9668, and its interaction with human neutrophil elastase was fully reversible.Preclinical pharmacokinetic studies were performed in vitro where protein binding was found to be 72% with a high recovery rate, aqueous solubility of 194.7 μM, low permeability along with a favourable hERG. Experiments with cell line revealed that the molecule successfully prevented elastase induced rounding and retracted cell morphology and cell cytotoxicity. In mouse model PD05 is able to reduce the alveolar collapse induced by neutrophil elastase. In summary, we demonstrate the in situ, in vitro and in vivo anti-elastase potential of the newly synthesised benzoxazinone derivative PD05 and thus this could be promising candidate for further investigation as a drug for the treatment of COPD.

A series of Novel Bacterial Topoisomerase Inhibitors (NBTIs) employing a linker derived from isomannide were synthesized and evaluated. Reduced hERG inhibition was observed compared to structure-matched analogues with different linkers, and compound 6 showed minimal proarrhythmic potential using an in vitro panel of cardiac ion channels. Compound 6 also displayed excellent activity against fluoroquinolone-resistant MRSA (MIC₉₀ = 2 μg/mL) and other Gram-positive pathogens.

A series of Novel Bacterial Topoisomerase Inhibitors (NBTIs) employing a linker derived from isomannide were synthesized and evaluated. Reduced hERG inhibition was observed compared to structure-matched analogues with different linkers, and compound 6 showed minimal proarrhythmic potential using an in vitro panel of cardiac ion channels. Compound 6 also displayed excellent activity against fluoroquinolone-resistant MRSA (MIC₉₀ = 2 μg/mL) and other Gram-positive pathogens.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors permeable to Ca²⁺, Na⁺ and K⁺. To be activated, they need to bind to glutamate (via GluN2 subunits), glycine (via GluN1) and release the Mg²⁺ blockade by
membrane depolarization. The majority of NMDARs are tetrameric complexes, consisting of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. GluN1 is coded by a single gene with at least eight different splice variants; four different GluN2 genes originate GluN2A, GluN2B, GluN2C, and GluN2D subunits. NMDARs containing different GluN2 subunits have different pharmacological and kinetic properties.

NMDA receptor modulators can be studied using a variety of techniques.

NIH/3T3 is a mouse embryonic fibroblast cell line. NIH/3T3 cells are established from NIH Swiss mouse embryos. These cells are highly contact inhibited and are sensitive to sarcoma virus focus formation and leukaemia virus propagation. These cells have now lost their contact inhibition. Contact inhibition is the natural process of arresting cell growth when two or more cells come into contact with each other. The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. It is therefore used for DNA transfection studies.
Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a chloride channel and controls the regulation of other transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens (CBAVD). The ΔF508CFTR mutation of the CFTR gene was stably transfected into the NIH/3T3 cell line and used for functional studies on QPatch.

Solid preclinical evidence links vasopressin to social behavior in animals, so, extensive work has been initiated to find new vasopressin V1a receptor antagonists which can improve deteriorated social behavior in humans and can treat the core symptoms of autistic behavior, as well. Our aim was to identify new chemical entities with antagonizing effects on vasopressin V1a receptors. Continuing our previous work, we found an in vitro and in vivo orally active V1a selective antagonist molecule (40) among [1,2,4]triazolo[4,3-a][1]benzazepines.

Small-conductance (K_Ca2) and intermediate-conductance (K_Ca3.1) calcium-activated K⁺ channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both K_Ca2 and K_Ca3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate K_Ca2.2 and K_Ca2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward K_Ca3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for K_Ca3.1 (EC₅₀ 111 ± 27 nM) over K_Ca2.3 (EC₅₀ 13.7 ± 6.9 μM), and SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for K_Ca3.1 (EC₅₀ 109 nM ± 14 nM) over K_Ca2.3 (EC₅₀ 4.4 ± 1.6 μM). Both compounds are 200- to 400-fold selective over representative KV (K_V1.3, K_V2.1, K_V3.1, and K_V11.1), Na_V (Na_V1.2, Na_V1.4, Na_V1.5, and Na_V1.7), as well as Ca_V1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of KCa3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in K_Ca3.1−/− mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a K_Ca3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of K_Ca3.1 activation.

New cardiac safety testing guidelines are being developed as part of the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, which aims to remove the reliance on screening against the hERG channel by expanding the panel to include other human ventricular ion channels such as Nav1.5, Ca_V1.2, K_V4.3/KChiP2.2, K_ir2.1 and K_V7.1/KCNE1. In addition, the CiPA working groups have recently identified two additional ion channel assay readouts required for in silico models to reliably predict proarrhythmia. The first is a ‘late’ Na_V1.5 assay, as inhibition of persistent inward current can affect repolarisation and mitigate proarrhythmia (e.g. Ranolazine). The second is a kinetic hERG assay that measures drug trapping using the Milnes voltage protocol(1) and improves the prediction of proarrhythmia risk(2). Here we describe validation of these additional CiPA assays on the gigaseal QPatch48 automated patch clamp platform.

New cardiac safety testing guidelines are being finalised as part of the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, which aims to remove the over-reliance on screening against the hERG channel by expanding the panel to include Na_v1.5, Ca_v1.2, K_v­4.3/KChiP2.2, K_ir2.1 and K_v7.1/KCNE1 human cardiac ion channels. In addition, the CiPA working groups have recently identified two additional in vitro assays required for in silico models to reliably predict proarrhythmia. The first is a ‘late’ sodium current assay, as inhibition of persistent inward current can affect repolarisation and mitigate proarrhythmia (e.g. Ranolazine). The second assay determines the degree of drug trapping in the hERG channel using the Milnes voltage protocol (Milnes et al, 2010), which can improve the prediction of proarrhythmia risk (Li et al. 2018).

We have validated these additional cardiac assays on the gigaseal quality QPatch48 automated patch clamp platform. Rather than rely on pharmacological activators such as ATX-II or Veratridine to induce late openings of the Na_v1.5 channel, we created a HEK cell line expressing the LQT3 DKPQ hNav1.5 mutant which promotes activation of persistent sodium currents (Chandra, Starmer & Grant, 1998). Late sodium current was measured using step pulse, step-ramp, ramp, and action potential waveform voltage protocols in proprietary solutions to minimise rundown and increase whole-cell seal duration.

Late hNav1.5 DKPQ cells exhibited Ranolazine-sensitive late sustained inward currents that were ~1-3% magnitude of the peak current (>4 nA) in single hole Qplate recordings. A CHO cell line expressing the hERG channel and published Milnes hERG kinetic protocols were optimised for the QPatch to create a biophysically and pharmacologically validated ‘dynamic’ hERG assay. We compensated for slower activation kinetics at room temperature (compared to manual patch at 37^oC) and optimised liquid addition and voltage protocols to create a stable assay during prolonged vehicle applications. Our QPatch Milnes hERG assay correctly reports the drug trapping profiles of Cisapride (10%) and Dofetilide (95%) as well as several other CiPA toolbox reference compounds.

We developed a late Na_v1.5 DKPQ mutant cell line and a Milnes dynamic hERG kinetic assay on a gigaseal quality screening platform that meet the additional requirements of the CiPA initiative, and can provide high quality HTS automated patch clamp data for more accurate cardiac safety assessment.

The nonclinical safety profile of GS-8873, a hepatitis B virus RNA transcript inhibitor was evaluated in rat and monkey 13-week toxicity studies with 8-week recovery phases. Vehicle or GS-8873 was dosed orally for 13 weeks at 2, 6, 20, and 60 mg/kg/day to Wistar Han rats and at 0.5, 1.5, 3, and 6 mg/kg/day to cynomolgus monkeys. In vitro and in vivo screening results from an analog discovered prior to GS-8873 informed the 13-week toxicology study designs. Neuroelectrophysiology and neurobehavioral evaluations were included at weeks 4 and 13 of the dosing and recovery phases for GS-8873. No adverse neurobehavioral effects were observed. Significant nerve conduction velocity (NCV) decreases and latency increases occurred at the high doses after 4 weeks of dosing. By week 13, dose-responsive NCV reductions and latency increases worsened across all dose groups compared with controls. Some reversal occurred 8 weeks after the last dose administered, but not to vehicle control levels. A minimal, axonal degeneration was observed in rat spinal and peripheral nerves across dose groups compared with controls. No monkey nervous system microscopic findings were observed. No-observed-adverse-effect-levels could not be determined for either species due to the neuroelectrophysiology findings and development was halted in the interest of safety. A retrospective risk assessment approach utilizing benchmark dose (BMD) modeling contributed 13-week NCV BMDL estimates (lower limits of the 95% confidence interval) in lieu of no-observed-adverse-effect-levels. The best-fitted models extrapolated NCV BMDLs for the rat caudal and monkey sural nerve at 0.3 and 0.1 mg/kg/day, respectively.

Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target Na_V1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess Na_V1.6 sodium current, or other indications where Na_V1.6 mediated hyper-excitability contributes to disease (Gardella & Moller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of Na_V1.6 (IC₅₀ 0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756X for Na_V1.1, 134X for Na_V1.2, 276X for Na_V1.7, and >583X for Na_V1.3, Na_V1.4, and Na_V1.5). NBI-921352 is a state-dependent inhibitor, preferentially inhibiting inactivated channels. The state dependence leads to potent stabilization of inactivation, inhibiting Na_V1.6 currents, including resurgent and persistent Na_V1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where Na_V1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse, as well as in wild-type mouse and rat seizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 was well tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A–developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target Na_V1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess Na_V1.6 sodium current, or other indications where Na_V1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of Na_V1.6 (IC₅₀0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for Na_V1.1, 134 X for Na_V1.2, 276 X for Na_V1.7, and >583 X for Na_V1.3, Na_V1.4, and Na_V1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivated channels. The state dependence leads to potent stabilization of inactivation, inhibiting Na_V1.6 currents, including resurgent and persistent Na_V1.6 currents while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action potential firing in glutamatergic excitatory pyramidal neurons while sparing fast-spiking inhibitory interneurons, where Na_V1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse, as well as in wild-type mouse and rat seizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 was well tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

Despite extensive study, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain after corneal refractive surgery provides a model, in humans, of the effect of injury to trigeminal afferent nerves. Axons of trigeminal ganglion neurons that innervate the cornea are transected by laser assisted in situ keratomileusis (LASIK). While most patients do not experience post-operative pain, a small subgroup develop persistent ocular pain. We previously carried out genomic analysis and determined that some patients with persistent pain after axotomy of corneal axons during refractive surgery carry mutations in genes that encode the electrogenisome of trigeminal ganglion neurons – the ensemble of ion channels and receptors that regulate excitability within these cells -including SCN9A, which encodes sodium channel Nav1.7, a threshold channel abundantly expressed in sensory neurons, that has been implicated in a number of pain-related disorders. Here, we describe the biophysical and electrophysiological profiling of the P610T-Nav1.7 mutation found in two male siblings with persistent ocular pain after refractive surgery. Our results indicate that this mutation impairs the slow inactivation of Nav1.7. As expected from this pro-excitatory change in channel function, we also demonstrate that this mutation produces hyperexcitability in trigeminal ganglion neurons. These findings suggest that this gain-of-function mutation in Nav1.7 may contribute to pain after injury to the axons of trigeminal ganglion neurons.

Humans with loss-of-function mutations in the Nav1.7 channel gene (SCN9A) show profound insensitivity to pain, whereas those with gain-of-function mutations can have inherited pain syndromes. Therefore, inhibition of the Nav1.7 channel with a small molecule has been considered a promising approach for the treatment of various human pain conditions. To date, clinical studies conducted using selective Nav1.7 inhibitors have not provided analgesic efficacy sufficient to warrant further investment. Clinical studies to date used multiples of in vitro IC50 values derived from electrophysiological studies to calculate anticipated human doses. To increase the chance of clinical success, we developed rhesus macaque models of action potential propagation, nociception, and olfaction, to measure Nav1.7 target modulation in vivo. The potent and selective Nav1.7 inhibitors SSCI-1 and SSCI-2 dose-dependently blocked C-fiber nociceptor conduction in microneurography studies and inhibited withdrawal responses to noxious heat in rhesus monkeys. Pharmacological Nav1.7 inhibition also reduced odor-induced activation of the olfactory bulb (OB), measured by functional magnetic resonance imaging (fMRI) studies consistent with the anosmia reported in Nav1.7 loss-of-function patients. These data demonstrate that it is possible to measure Nav1.7 target modulation in rhesus macaques and determine the plasma concentration required to produce a predetermined level of inhibition. The calculated plasma concentration for preclinical efficacy could be used to guide human efficacious exposure estimates. Given the translatable nature of the assays used, it is anticipated that they can be also used in phase 1 clinical studies to measure target modulation and aid in the interpretation of phase 1 clinical data.

As an alternative to standard pharmacological procedures for Na_V1.5(Late) assays, we present a more reliable and accurate Na_V1.5(Late) assay on QPatch that removes the requirement for activators like veratridine and ATX-II and delivers improved cardiac safety screening reliability and cost.

• High fidelity QPatch recordings of small amplitude Na_V1.5 (Late) currents from Long QT3 syndrome mutant channels.
• Stable recordings without pharmacological activators allow more reliable drug potency assessment.
• Pharmacologically validated with sodium channel blockers with a preference for the Na_V1.5(Late).

Torsades de pointes (TdP) is a potentially fatal type of a ventricular tachycardia associated with delayed repolarization of the cardiac action potential. The major reason for pharmacologically induced TdP is the blockade of the voltage-gated potassium channel (KV11.1 or hERG current, I_Kr).
Therefore, the main focus of pre-clinical in vitro tests has been set on detection of I_Kr blockade to effectively discard drugs with a propensity to induce TdP. However, not all compounds that block I_Kr will eventually induce tachyarrhythmia and, therefore, a detected block of I_Kr alone is not specifically
predictive for delayed repolarization and TdP. Not all known I_Kr blockers cause significant arrhythmia because effects caused by induced reduction of potassium outward currents may be
counterbalanced by a reduced calcium inward current (ICaL) or late inward sodium current (I_NaL). hERGand L-type calcium currents (Ca_V1.2) can easily be assessed in in-vitro systems using electrophysiology methods (e.g. patch-clamping). Since the physiological late sodium current exhibits only tiny current amplitudes, I_NaL needs to be increased for drug screening by decreasing or slowing the inactivation of Na_V1.5 channels. This can be pharmacologically achieved by adding a sea anemone toxin II (ATX-II), which binds to the extracellular linker of segments S3-S4 of domain IV or by using inactivation modifying mutations.

For this study, a cell line stably expressing a mutated NaV1.5 (CW) channel was generated and validated using known I_NaL blockers. The substitution L409C/A410W was found to lead to an inactivation-deficient mutation. The mutation is localized in D1S6 and presumably prevents access of the intrinsic fast inactivation particle to the inner cavity. During pharmacological validation using manual and automated (QPatch™) patch-clamping, IC₅₀ values differed by less than a factor of two between ATXII stimulated and CW mutated Na_V1.5 channels. Besides shorter duration of I_NaL experiments and larger current amplitudes, also the observed conserved sensitivity to ATXII and overall reduced assay costs are strong arguments to
screen late sodium currents in mutated rather than in pharmacologically stimulated Na_V1.5 channels.

The sodium channel Na_V1.1, highly expressed in fast spiking interneurons (FSIs), initiates the interneuron’s action potentials and promotes γ-oscillations, which are important for memory encoding and other cognitive functions. An impaired function of FSIs is associated with disorders like autism, schizophrenia, Alzheimer’s and others. Potentiators of Na_V1.1 can restore γ-oscillations by recovering reduced FSI function and they have been shown to minimize cognitive dysfunction in transgenic mice with decreased levels of Na_V1.1 in parvalbumin-positive neurons.

An indazole/aza-indazole scaffold was developed as a novel chemotype for JNK3 inhibition. Extensive structure-activity relationship (SAR) studies utilizing various in vitro and in vivo assays led to potent and highly selective JNK3 inhibitors with good oral bioavailability and high brain penetration. One lead compound, 29, was a potent and selective JNK3 inhibitor (IC50 = 0.005 μM) that had significant inhibition (>80% at 1 μM) to only JNK3 and JNK2 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in the human liver microsome (t1/2 = 92 min), and was orally bioavailable and brain penetrant (brain/plasma ratio: 56%). The cocrystal structure of 29 in human JNK3 at a 2.1 Å resolution showed that indazole or aza-indazole-based JNK3 inhibitors demonstrated a type I kinase inhibition/binding.

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (Na_V), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on Na_V1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at Na_V1.8, while the slope factor of the conductance-voltage curves was significantly increased for Na_V1.7 and peak current was significantly increased for Na_V1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of Na_V1.8 and the tetrodotoxin-sensitive isoforms Na_V1.7 and Na_V1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of Na_V isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

High-throughput automated patch clamp (APC) is quickly emerging as the new gold standard technology for screening compounds against ion channel targets. The latest APC platforms, such as the Qube, enable true giga-seal quality data generation, comparable to conventional patch clamp.

Traditional plate-based screening technologies rarely allowed for analysis of multiple parameters to detect potential differences in the mechanism of action of novel compounds. Using multiple data analysis cursors in the Sophion Analyzer software, it’s possible to gain valuable insight into compound activities on a particular ion channel target, which may translate into different results in further in vivo experiments. This also increases the value of APC data, as multiple parameters can be extracted from each well for both data quality control as well as hit detection.

Here we present Qube data for a 10,000 compound pilot screen for a sodium channel activator project, with analysis for multiple hit parameters, demonstrating the ability of the assay to detect novel compounds with different mechanisms of action.

Dr. Bryan Moyer, Amgen – talk from ICMS 2019, the United States

Title: Multi-modality approach to identify Na_V1.7 inhibitors for pain

Professor Fusao Kato, Jikei University – talk from the ICMS 2021 virtual meeting.

Title: Molecular conversion of presynaptic P2X receptor subtype at brainstem synapse

Professor Sara Liin’s talk from the ICMS 2021 virtual meeting.

Title: Modulation of wildtype and mutant cardiac Kv.1/KCNE1 potassium ion channels by lipid molecules

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons and an increase in extracellular ATP concentration under pathological conditions such as tissue damage or visceral distension induces channel opening, membrane depolarization and initiation of pain signaling. Hence these receptors are considered important therapeutic targets for pain management and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against hP2X3. We have found that these antibodies produce distinct functional effects depending on the homomeric or heteromeric composition of the target, its kinetic state and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nM on hP2X3 after short-term exposure (up to 18 minutes), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short-term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to approximately 20 hours resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, CFA and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated K_V1.3 potassium channels in T-lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to K_V1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for K_V1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with ¹⁸F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10–100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated K_V1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to K_V1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for K_V1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with ¹⁸F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10–100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

Professor Nikita Gamper – Leeds University, UK

Title: Modulation of Kv7 potassium channels by intracellular zinc

Associate Professor Bo Hjorth Bentzen – Copenhagen University, DK

Title: Modulation and anti-arrhythmic potential of SK channels

Developing an effective antidote for fentanyl-induced overdose (OD) is an unmet medical need that requires both lipophilicity comparable to fentanyl and fast onset of overdose reversal. We synthesized and evaluated a bioreversible derivative of naloxone (NX-90) in silico, in vitro and in vivo to yield a robust reversal of fentanyl-induced OD in rats. All monitored reflexes along with the heart rate (HR) and respiratory rate (RR) were fully restored faster in the NX-90 groups than in naloxone groups on equimolar bases when given intranasally. In NX-90 treated rats RR over the time of observation (RR AUC) was significantly higher at all respective doses with no re-narcotization observed. Apart from the enhanced pharmacodynamics profile, NX-90 was found to have lower circulating levels of naloxone, clean profile in in vitro selectivity panels, as well as Ames and CYP450 counter screens. Finally, we demonstrated a robust release of the parent naloxone in brain matrix, as well as lower peripheral naloxone levels after NX-90 iv administration. With the demonstrated pharmacological profile superior yet congruent to naloxone we nominated NX-90 for preclinical development as an effective intranasal fentanyl antidote.

Drug-induced block of the cardiac hERG (human Ether-a`-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Na_v1.5 and Ca_v1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Ca_v1.2.

Professor Raimund Dutzler – Zurich University, Switzerland

Title: Mechanism of ion conduction and gating in the calcium-activated chloride channel TMEM16A

Opioid receptor stimulants are analgesics used in patients with and without cancer; however, they often cause constipation, resulting in poor adherence and deterioration of the quality of life. Hence, suitable treatments for constipation are required. In this study, we investigated the pharmacological mechanisms of action of mashiningan (MNG), a Kampo medicine used to treat constipation, and evaluated the effect of MNG on opioid-induced constipation in rats. MNG (100 or 300 mg/kg) was orally administered to normal or codeine phosphate (CPH)-induced constipation in rats, and its effect was evaluated on the basis of fecal counts, characteristics, and weight. Small intestinal fluid secretion was measured after treatment with MNG alone or co-administration with a cystic fibrosis transmembrane conductance regulator (CFTR) -specific inhibitor (CFTRinh-172). The effects of MNG on the CFTR and type-2 chloride channel were determined using patch clamp or short-circuit current experiments, respectively. MNG increased the fecal weight and proportion of soft feces in normal rats. CPH-induced constipation in rats decreased fecal counts and weight, whereas MNG prevented these effects and increased the proportion of soft feces. MNG increased the electronic chloride current, and this effect was inhibited by the CFTRinh-172 in the CFTR assay. Furthermore, MNG increased small intestinal fluid secretion, and this effect was abolished by co-administration with the CFTRinh-172. MNG improved opioid-induced constipation in rats, and this improvement may have been mediated by increasing intestinal fluid secretion via CFTR chloride channel activation. Therefore, MNG is expected as a medicine of the treatment for constipation in patients taking opioids.

Compelling human genetic studies have identified the voltage-gated sodium channel Na_V1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na_V1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa_V1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3–S4 loop of domain II of hNa_V1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na_V1.7 (20-fold) while maintaining >100-fold selectivity over the major off-targets Na_V1.4, Na_V1.5, and Na_V1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na_V1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na_V1.7 in pain.

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analog (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (Na_V1.7), a channel targeted in pain management. Here, we show that replacements of the positively charged residues that contribute to the activity of the peptide can improve gHwTx-IV’s potency and selectivity for Na_V1.7. Using HwTx-IV, gHwTx- IV,
[R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human Na_V1.7 and their affinity for the lipid bilayer. [R26A]gHwTxIV consistently displayed the most improved potency and selectivity for Na_V1.7, examined alongside off-target Na_Vs, compared with HwTxIV and gHwTx-IV. The lipid affinity of each of the three novel analogs was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at Na_V1.7 and affinity for lipid bilayers. In a murine Na_V1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable to that of native HwTx- IV. In summary, the present study reports the development of an HwTx-IV analog with improved in vitro selectivity for the pain target Na_V1.7 and with an in Vivo efficacy similar to that of native HwTx-IV.

Professor Bernard Attali – Tel Aviv University

Title: M-current inhibition in hippocampal neurons triggers intrinsic and synaptic homeostatic responses at different temporal scales

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS) and the binding of GABA to ionotropic GABA receptors (GABA_AR) is a crucial process in the healthy brain. An imbalance of GABA secretion or the malfunction of the receptor is associated with multiple disease areas like anxiety disorders, seizures and schizophrenia. Pharmacological manipulation of the receptor has, therefore, a large therapeutic potential, which is underscored by the amount of available treatment possibilities and the ongoing search for alternatives thereof^1-4.
GABA_A receptors are ligand-gated ion-channels that consist of 5 membrane-spanning subunits^5,6 and are permeable to Cl^– ions. So far, 16 different subunits have been identified in humans (ɑ1-6, ꞵ1-3, γ1-3, δ, ε, γ, θ, π) and their composition within the GABA receptor leads to different pharmacological responses7. Here, we show pharmacological modulation of the GABA_AR using our high-throughput automated patch clamp (APC) systems QPatch and Qube 384. Our study includes a characterization of the heterogeneous GABA_AR population of cultured primary hippocampal astrocytes and an evaluation of the GABA_AR clone ɑ5ꞵ3γ2. In addition, we utilize the well-characterized GABA_AR response to establish a novel method for ligand release, namely the light-stimulated release of ruthenium-bipyridine-triphenylphosphine(RuBi)-caged GABA using light stimulation on the Qube 384 platform.

Studies on GABAA ion channels using the automated patch clamp platform QPatch with the focus on:

• Effects of agonists, antagonists, and modulators
• Concentration-response relationships
• EC₅₀ and IC₅₀ determination
• Characterizing both the pharmacology of a specific isolated GABAA subtype and the physiological GABA response of cultured rat astrocytes

The global increase in multidrug-resistant pathogens has caused severe problems in the treatment of infections. To overcome these difficulties, the advent of a new chemical class of antibacterial drug is eagerly desired. We aimed at creating novel antibacterial agents against bacterial type II topoisomerases, which are well-validated targets. TP0480066 (compound 32) has been identified by using structure-based optimization originated from lead compound 1, which was obtained as a result of our previous lead identification studies. The MIC₉₀ values of TP0480066 against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and genotype penicillin-resistant Streptococcus pneumoniae (GPRS) were 0.25, 0.015, and 0.06 μg/mL, respectively. Hence, TP0480066 can be regarded as a promising antibacterial drug candidate of this chemical class.

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.

It was possible to obtain both manual and automated patch clamp (APC) recordings of the labile and scarce Wnt proteins (350-400 AA, 35-45 kDa) after thorough optimisation of compound handling.

Here we highlight aspects of handling of Wnt proteins optimised for APC testing, which could also be applied to other large molecules (e.g. toxins, peptides, antibodies).

Qube 384 is capable of performing pharmacology measurements with large molecules.

Low volume microfluidic application ensures that expensive and scarce molecules like antibodies and toxins can be efficiently tested.

Key pharmacology metrics defined in these assays are comparable to values reported in the literature.

To determine if engineered IgG anti-α-cobratoxin antibodies would prevent the blocking of the nicotinic acetylcholine receptor, a protocol was set up using QPatch II. Here we show that:

• QPatch II is well suited for efficiently characterizing both peptide toxins and antibody function.
• Current induced by adding acetylcholine could be abolished completely using α-cobratoxin, and that this effect could be reversed by preincubating the toxin with different IgGs before injection.
• Prevention of current inhibition by the IgGs was dose-dependent, and the potency of different IgGs could be determined using this method.
• Increased affinity between the IgGs and α-cobratoxin resulted in higher neutralization potency.

Lacosamide, developed as an anti-epileptic drug, has been used for the treatment of pain. Unlike typical anticonvulsants and local anesthetics which enhance fast-inactivation and bind within the pore of sodium channels, lacosamide enhances slow-inactivation of these channels, suggesting different binding mechanisms and mode of action. It has been reported that lacosamide’s effect on Na_V1.5 is sensitive to a mutation in the local anesthetic binding site and that it binds with slow kinetics to the fast-inactivated state of Na_V1.7. We recently showed that the Na_V1.7-W1538R mutation in the voltage-sensing domain 4 completely abolishes Na_V1.7 inhibition by clinically-achievable concentration of lacosamide. Our molecular docking analysis suggests a role for W1538 and pore residues as high affinity binding sites for lacosamide. Aryl sulfonamide sodium channel blockers are also sensitive to substitutions of the W1538 residue but not of pore residues. To elucidate the mechanism by which lacosamide exerts its effects, we used voltage-clamp recordings and show that lacosamide requires an intact local anesthetic binding site to inhibit Na_V1.7 channels. Additionally, the W1538R mutation does not abrogate local anesthetic lidocaine-induced blockade. We also show that the naturally occurring arginine in Na_V1.3 (Na_V1.3-R1560), which corresponds to Na_V1.7-W1538R, is not sufficient to explain the resistance of Na_V1.3 to clinically-relevant concentrations of lacosamide. However, the Na_V1.7-W1538R mutation conferred sensitivity to the Na_V1.3-selective aryl-sulfonamide blocker ICA-121431. Together, the W1538 residue and an intact local anesthetic site are required for lacosamide’s block of Na_V1.7 at a clinically-achievable concentration. Moreover, the contribution of W1538 to lacosamide inhibitory effects appears to be isoform-specific.

CiPA recommended Ca_V1.2 assay protocols were run on Qube
384 using Ready-To-Use (RTU) Ca_V1.2 cells from NMI TT with
~80% success rate, low run-down and reliable pharmacology.
We demonstrate the quality and versatility of ready-to-use Ca_V1.2 cells, but also that RTU cells can be a valuable addition to the laboratory tool pack to 1) decrease variation from cell culture and 2) decrease dependence on skilled cell culture personnel.

• Ready-to-use cells easily ignites your experiment within
minutes
• Up to 82% success rate
• Reliable pharmacology including safety pharmacology
recommended by CiPA
• Rundown of only +0.2% ± 0.06% per minute

Glycyrrhiza extract has been used for the treatment of oral and gastric ulcers, but the analgesic mechanism remains unknown. In the present study, we investigated the effects of isoliquiritigenin, an active ingredient of Glycyrrhiza, on Na_v channels in vitro and nociceptive behaviors in vivo. In an autopatch-clamp study, isoliquiritigenin inhibited the currents of Na_v1.1, Na_v1.3, Na_v1.6, Na_v1.7, and Na_v1.8 in a channel expression system. In small- and medium-sized cultured trigeminal ganglion neurons, the compound suppressed Na_v currents in many neurons (78%) and K_v currents in all neurons, dose-dependently. In current-clamp mode, isoliquiritigenin blocked action potential generation in many neurons (64%), but it conversely accelerated action potential generation in the remaining neurons. The opposing effects on action potentials were reproduced in a computational simulation of a modified Hodgkin-Huxley-based model, based on the electrophysiological data. In behavioral experiments, local treatment with isoliquiritigenin suppressed nociceptive behaviors in response to oral ulcer development or nociceptive TRP channel agonists in the oral mucosa and hind paw. These results suggest that isoliquiritigenin exerts an analgesic effect predominantly via inhibitory action on Na_v channels on sensory nociceptive fibers. This pharmacological mechanism indicates that isoliquiritigenin is useful for pain relief and provides scientific evidence for Glycyrrhiza at the ingredient level.

A series of isoindolinone compounds have been developed showing good in vitro potency on the K_v1.5 ion channel. By modification of two side chains on the isoindolinone scaffold, metabolically stable compounds with good in vivo PK profile could be obtained leaving the core structure unsubstituted. In this way, low microsomal intrinsic clearance (CLint) could be achieved despite a relatively high logD. The compounds were synthesized using the Ugi reaction, in some cases followed by Suzuki and Diels–Alder reactions, giving a diverse set of compounds in a small number of reaction steps.

Transport proteins, which are embedded in and span most cell membranes, play essential roles in regulating ionic homeostasis in all mammalian cell types. It is well established that the electrolyte compositions of intracellular and extracellular fluids in the human body are very different, with K⁺ (~140 mM) being the dominant cation intracellularly and Na⁺ (~140 mM) and Cl^– (~100 mM) being the dominant ions extracellularly. In fact, most ions have multiple-fold differences in concentration across the plasma membrane and across the membranes of intracellular organelles. Ionic concentration gradients are established and maintained by active, energy-requiring pumps, including the electrogenic Na⁺/K⁺-ATPase in the plasma membrane and SERCA, a Ca²⁺-ATPase that is present in the membrane of the sarcoendoplasmic reticulum. These concentration gradients are possible because lipid-based membranes are impermeable to ions. Ion channels constitute a class of pore-forming proteins that provide regulated permeability pathways that allow the passive flow of ions from one side of the membrane to the other. Ion channels can be grouped into families that are defined by their structural features, tissue localization, and functional and pharmacological characteristics. Typically, they are classified according to their gating mechanism, for example, voltage-gated or ligand-gated and, in some cases, by their permeant ion(s):¹ cation channels are permeable either selectively or nonselectively to K⁺, Na⁺ and Ca²⁺, whereas anion channels are permeable to Cl^–.

In this issue of SLAS Discovery, we present a special collection of manuscripts, including three original research papers and one review, that reflect recent advances and continuing challenges in the development and application of assay technologies to drug discovery for ion channel targets. First, though, we provide our perspectives on the specific challenges and opportunities in this field.

Transport proteins, which are embedded in and span most cell membranes, play essential roles in regulating ionic homeostasis in all mammalian cell types. It is well established that the electrolyte compositions of intracellular and extracellular fluids in the human body are very different, with K⁺ (~140 mM) being the dominant cation intracellularly and Na⁺ (~140 mM) and Cl^– (~100 mM) being the dominant ions extracellularly. In fact, most ions have multiple-fold differences in concentration across the plasma membrane and across the membranes of intracellular organelles. Ionic concentration gradients are established and maintained by active, energy-requiring pumps, including the electrogenic Na⁺/K⁺-ATPase in the plasma membrane and SERCA, a Ca²⁺-ATPase that is present in the membrane of the sarcoendoplasmic reticulum. These concentration gradients are possible because lipid-based membranes are impermeable to ions. Ion channels constitute a class of pore-forming proteins that provide regulated permeability pathways that allow the passive flow of ions from one side of the membrane to the other. Ion channels can be grouped into families that are defined by their structural features, tissue localization, and functional and pharmacological characteristics. Typically, they are classified according to their gating mechanism, for example, voltage-gated or ligand-gated and, in some cases, by their permeant ion(s):¹ cation channels are permeable either selectively or nonselectively to K⁺, Na⁺ and Ca²⁺, whereas anion channels are permeable to Cl^–.

Dr Anna Greka, Broad Institute of MIT – talk from ICMS 2019, the United States

Title: Ion channel targeted therapy for rare disease

For every heartbeat, movement and thought, ion channels have to open and close, and thus, it is not surprising that malfunctioning of these membrane proteins leads to serious diseases. Today, only 7% of all marketed drugs act on ion channels but the systematic exploitation of this important target class has started mainly enabled by novel screening technologies. Thus, the discovery of selective and state-dependent drugs is on the horizon, hopefully leading to effective novel medicines.

Free intracellular calcium ([Ca²⁺]_i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of ([Ca²⁺]_i) is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on ([Ca²⁺]_i) release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of ([Ca²⁺]_i) in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases ([Ca²⁺]_i) was increased by modulator concentrations comparable to those used clinically. The increase in ([Ca²⁺]_i) in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize ([Ca²⁺]_i). We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.

Internal addition of compounds targeting the chloride channel CIC-1. A robust assay with good pharmacology and high Z-score

• A robust assay of ClC-1 with Z’ constantly >0.5
• Biophysical and pharmacological characteristics as expected for ClC-1
• Qube offers the possibility to apply compounds both intra- and extracellularly
• Advanced analysis of compound effects on channel kinetics are available at a few click

In this report, we show a ClC-1 assay on Qube that shows biophysical characteristics as expected for ClC-1. We further introduce Sophion`s Analyzer software that allows rapid analysis of large data sets to answer advanced electrophysiological questions, in the present case: What is the mode of action of a novel, unknown compound?

Dr Paul Miller, University College London, UK – talk from the ICMS 2021 virtual meeting.

Title: Insights into understanding and developing modulators of GABAA receptors

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Na_V1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Na_V1.7 have not yet fully recapitulated the dramatic analgesia observed in Na_V1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Na_V1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Na_V1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Na_V1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and non-psychoactive compounds are Δ9-tetrahydrocannabidiol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated anti-epileptic effects have been from case reports or smaller surveys. The mechanisms for CBD’s anticonvulsant effects are unclear and likely involve non-cannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1-1.7 currents, with an IC50 of 1.9–3.8 μM, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ~3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD’s mode of action likely involves (1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and (2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

The purpose of the current analysis was to investigate the direct inhibitory effects of perampanel and other anti-seizure medications (ASMs) on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), and kainate glutamate receptor subtypes using electrophysiological assessments.

Neuropathic pain is a debilitating, chronic condition with a significant unmet need for effective treatment options. Recent studies have demonstrated that in addition to neurons, non-neuronal cells such as microglia contribute to the initiation and maintenance of allodynia in rodent models of neuropathic pain. The Ca²⁺– activated K⁺channel, K_Ca3.1 is critical for the activation of immune cells, including the CNS-resident microglia. In order to evaluate the role of K_Ca3.1 in the maintenance of mechanical allodynia following peripheral nerve injury, we used senicapoc, a stable and highly potent K_Ca3.1 inhibitor. In primary cultured microglia, senicapoc inhibited microglial nitric oxide and IL-1β release. In vivo, senicapoc showed high CNS penetrance and when administered to rats with peripheral nerve injury, it significantly reversed tactile allodynia similar to the standard of care, gabapentin. In contrast to gabapentin, senicapoc achieved efficacy without any overt impact on locomotor activity. Together, the data demonstrate that the K_Ca3.1 inhibitor senicapoc is effective at reducing mechanical hypersensitivity in a rodent model of peripheral nerve injury.

Background and Purpose: Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor of ∆9-tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10 μM) blocks voltage-gated sodium (Nav) currents in CNS neurons, although the underlying mechanism is not well understood. Genetic and functional studies have validated Nav1.7 channels as an opportune target for analgesic drug development. The effects of CBG on Nav1.7 channels, which may contribute to its analgesic properties, have not been previously investigated. Experimental Approach: To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling. Key Results: CBG is a ~10-fold state-dependent Nav channel inhibitor (KI-KR: ~2–20 μM) with an average Hill-slope of ~2. We determined that, at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation. However, as the concentration is increased, CBG also induces a hyperpolarizing shift in the half-voltage of inactivation. Our modelling and multielectrode array recordings suggest that CBG attenuates DRG excitability. Conclusions and Implications: Inhibition of Nav1.7 channels in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav1.7 channels are inactivated at the resting membrane potential of DRG neurons, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav1.7 channels, compared with other Nav channels (via Gmax block).

Hypokalemia was associated with an increased incidence of VF, an effect that also seen in the presence of dofetilide. In comparison, the structurally and functionally different KCa2 channel inhibitors, ICA, AP14145, and AP30663 protected the heart from hypokalemia induced VF. These results support that KCa2 inhibition may be associated with a better safety and tolerability profile than dofetilide.

Hypokalemia reduces the cardiac repolarization reserve. This prolongs the QT-interval and increases the risk of ventricular arrhythmia; a risk that is exacerbated by administration of classical class 3 anti-arrhythmic agents.

Small conductance Ca²⁺-activated K⁺-channels (K_Ca2) are a promising new atrial selective target for treatment of atrial fibrillation. Under physiological conditions, K_Ca2 plays a minor role in ventricular repolarization. However, this might change under hypokalemia because of concomitant increases in ventriculay -60r intracellur Ca²⁺.

Professor Mustafa Djamgoz, Imperial College London – talk from ICMS 2019

Title: In vivo evidence for expression of voltage-gated sodium channels in cancer and potentiation of metastasis

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of coronavirus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 μM–100 μM range with a 2–4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 μM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 μM and 30 μM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

The ongoing pain associated with arthritis reduces the quality of human life. Injection of complete Freund’s adjuvant (CFA) into the knee of mice induced inflammation and a concomitant decrease in their natural digging behaviour within 24-hours. After inflammation, retrogradely labelled knee-innervating dorsal root ganglion neurons showed a decreased action potential threshold and increased transient receptor potential vanilloid-1 (TRPV1) expression (validated with immunohistochemistry). Subsequently, systemic administration of a TRPV1 antagonist normalized mouse digging behaviour within 30-minutes, likely due to inhibition of pain signalling. To test the translational potential of these results, we recorded from mouse sensory neurons incubated overnight with synovial fluid from humans with osteoarthritis experiencing chronic pain (OA-SF). We find that OA-SF directly sensitizes mouse knee neurons by decreasing the action potential threshold and increasing the resting membrane potential implicating voltage-gated sodium (Na_v) and potassium (K_v) channels. Using Ca²⁺-imaging we also find that OA-SF increases TRPV1 and TRPM8 activity compared to synovial fluid from healthy individuals, highlighting the importance of TRP channels in OA pain. Taken together, our data suggest that inflammatory mediators in OA-SF excite sensory neurons and hence drive knee pain. This in vitro, translational model can be incorporated into clinical trials to unbiasedly assess the efficacy of drugs on sensory neurons and hence identify drivers of OA pain.

The monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against α-cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified α-cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.

The TRPV1 ion channel is a neuronal sensor that plays an important role in nociception and neuropathic as well as inflammatory pain. In clinical trials, hyperthermia and thermo-hypoaesthesia turned out as major side effects of TRPV1 antagonists, preventing successful development of such molecules as analgesics. In vitro studies demonstrated that the TRPV1 ion channel is a polymodal sensor integrating stimuli from molecular modulators with temperature, pH and transmembrane potential. Temperature-dependent gating is suggested to constitute the molecular basis for its role in heat sensation and body temperature regulation. Drug discovery scientists since many years seek to obtain “thermoneutral” TRPV1 inhibitors, blocking the channels sensitivity for painful stimuli while keeping its temperature mode of activation unaffected.

Aiming for a screening rational for the identification of thermoneutral TRPV1 antagonists, we broadly characterized the prototypic small molecule TRPV1 inhibitors GRT12360V and GRTE16523. In vitro, GRT12360V demonstrated pan-modality inhibition on human, cynomolgus and rodent TRPV1, whereas GRTE16523 selectively bypassed the channels temperature mode on human and cynomolgus TRPV1 and revealed partial agonism on rodent channels. Strikingly, in vivo, GRT12360V induced hyperthermia in all species tested whereas GRTE16523 proved thermoneutral in cynomolgus monkeys and induced hypothermia in rodents.

Hence, working out the different in vitro to in vivo correlations of two compounds, we suggest temperature-dependent voltage gating as key parameter when screening for thermoneutral TRPV1 inhibitors. We highlight a species difference of molecular TRPV1 pharmacology between primates and rodents and provide a methodological breakthrough to engineer thermoneutral TRPV1 antagonists with improved therapeutic safety.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

Activation of the voltage-gated Kv7 channels holds therapeutic promise in several neurological and psychiatric disorders, including epilepsy, schizophrenia, and depression. Here, we present a pharmacological characterization of Lu AA41178, a novel, pan-selective Kv7.2–7.5 opener, using both in vitro assays and a broad range of in vivo assays with relevance to epilepsy, schizophrenia, and depression.

Electrophysiological characterization in Xenopus oocytes expressing human Kv7.2-Kv7.5 confirmed Lu AA41178 as a pan-selective opener of Kv7 channels by significantly left-shifting the activation threshold. Additionally, Lu AA41178 was tested in vitro for off-target effects, demonstrating a clean Kv7-selective profile, with no impact on common cardiac ion channels, and no potentiating activity on GABAA channels.

Lu AA41178 was evaluated across preclinical in vivo assays with relevance to neurological and psychiatric disorders. In the maximum electroshock seizure threshold test and PTZ seizure threshold test, Lu AA41178 significantly increased the seizure thresholds in mice, demonstrating anticonvulsant efficacy. Lu AA41178 demonstrated antipsychotic-like activity by reducing amphetamine-induced hyperlocomotion in mice as well as lowering conditioned avoidance responses in rats. In the mouse forced swim test, a model with antidepressant predictivity, Lu AA41178 significantly reduced immobility. Additionally, behavioral effects typically observed with Kv7 openers were also characterized. In vivo assays were accompanied by plasma and brain exposures, revealing minimum effective plasma levels <1000 ng/ml.

Lu AA41178, a potent opener of neuronal Kv7 channels demonstrate efficacy in assays of epilepsy, schizophrenia and depression and might serve as a valuable tool for exploring the role of Kv7 channels in both neurological and psychiatric disorders.

Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer, however, only a relatively 33 small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding 34 tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated 35 with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are 36 characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a 37 new Kv1.3 targeting radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours 38 successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic 39 colon cancer model, we compared tumour retention of [ 18F]AlF-NOTA-KCNA3P with changes in the tumour immune 40 microenvironment determined by flow cytometry. Imaging with [18F]AlF-NOTA-KCNA3P reliably differentiated tumours 41 responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially 42 Kv1.3-expressing CD8+ effector memory T cells.

Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical, [18 F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of [18 F]AlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with [18 F]AlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8+ effector memory T cells.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

Two voltage-dependent potassium channels, K_V1.1 (KCNA1) and K_V1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer K_V1.1-1.2((3)). Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of K_V1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer K_V1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the K_V1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by K_V1.1-1.2((3)) channels by more than 80 % at 10 μM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6 μM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of K_V1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Na_V1.5, and Ca_V1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC₅₀ = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC₅₀ = 10 nM) with reduced hERG activity (14 μM).

Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

Here, we describe the identification of PARP1/2 as direct binding proteins of andrographolide (Andro) using protein microarray, surface plasmon resonance (SPR), and enzyme activity assays. We then evaluated the proliferation inhibition, apoptosis, and cell migration effects of Andro on the MDA-MB-436 cell line in vitro. The final biological evaluation confirmed that Andro was a highly effective single agent in the MDA-MB-436 xenograft model and had a low hERG-mediated cardiac toxicity. Therefore, Andro represents the first natural product, non-amide member of a novel nanomolar-potency PARP1/2 inhibitor family.

As highlighted in the OliveNet™ library, Olea europaea consists of a diverse collection of chemical compounds. We have classified over 600 compounds into 13 main classes and 47 subclasses. Various compounds, including oleuropein and hydroxytyrosol, have been investigated for their potential beneficial effects in multiple human pathologies. However, the vast majority of compounds remain largely unexplored and approximately 50% are currently non-commercially available. Method: Here, we utilise conventional software to characterise the absorption, distribution, metabolism, excretion, and toxicity profile of OliveNet™ compounds. Molecular docking was performed for assessment of P-glycoprotein (P-gp) inhibition and interactions with the human ether-à-go-go-related gene (hERG) channel. Potential hERG ion channel inhibition was calibrated using in vitro patch clamp assays and steered molecular dynamics (SMD) simulations were used to examine membrane permeability of a subset of compounds. Results: Our findings indicated that 313 out of 675 olive compounds were predicted to be absorbed by the gastrointestinal tract. Hydroxytyrosol required the least amount of force to pass through the lipid bilayer compared to elenolic acid diglucoside in SMD simulations. Based on the ADMET and molecular docking data, the hERG inhibitory activities of verbascoside, oleuropein, and hydroxytyrosol were investigated using patch clamp assays and they were identified as non-inhibitors. Conclusions: While the favourable properties of well-known compounds were confirmed, we identified oleuropein aglycone decarboxymethyl dialdehyde acetal form, decarboxymethyl elenolic acid dialdehyde, acetal of decarboxymethyl elenolic acid dialdehyde, methyl malate-β-hydroxytyrosol ester, hydroxytyrosil elenolate, D-(+)-erythro-1-(4-hydroxy-3-methoxy)-214-phenyl-1,2,3-propantriol, (+)-1-acetoxypinoresinol-4″-O-methyl ether, and 3-[1-(hydroxymethyl)-(E)-1-propenyl] glutaric acid as potential candidates for synthesis and further evaluation.

Growing functional and genetic evidence points to an emerging role for two-pore domain potassium channels (K2Ps) in numerous pathophysiologies. K2Ps carry background (or leak) potassium current and primarily function to maintain cellular resting membrane potential. However, K2Ps have proved a challenging superfamily to modulate with small molecules and there is currently a lack of pharmacological tools which target K2Ps. This has limited the investigation of K2Ps precise physiological function and efforts to generate therapeutics which act upon K2Ps. Our initial aim was to develop a screening system which would allow the identification of small molecule K2P activators. We initially showed that the identification of channel activators can be compromised in systems in which the target (K2P) is over-expressed at high levels. To avoid this, we used baculovirus (‘BacMam’) to transduce K2P expression in mammalian cells. ‘BacMam’ allows the precise titration of expression of the gene of interest. This enabled us to develop cell systems in which we were able to intricately and robustly select a level of K2P expression (via functional studies) optimized for the identification of channel activators. An example assay development using the K2P TRAAK is described. TRAAK is known to be expressed in nociceptors and has been shown to be a genetic predictor of post-surgical neuropathic pain. An appropriate level of TRAAK channel function was chosen after ‘BacMam’ optimization and channel function measured via thallium flux. The pharmacology of previously described TRAAK activators is also shown. A representative sub-group of K2Ps (TWIK-1, THIK-1, TASK-3, TASK-2, TREK-2) was screened using the BacMam/ Thallium flux assay against the LifeArc Index set (11k compounds). This allowed us to assess which K2Ps were ‘ligandable’ or ‘activatable’ by small molecules. Multiple novel activators were identified, and development is on-going. We also screened a library of drug-like molecules against the same group of K2Ps and identified Terbinafine as a novel activator of the TASK-3 channel. The selectivity of this molecule across the K2P superfamily was assessed and structure-activity relationship studies showed that relatively small changes to the molecule had significant effects on activity and selectivity.

Voltage-gated sodium channels (Na_v) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Na_v1.1 modulators. Among these compounds, N,N′-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Na_v1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Na_v1.1 channels to design potential drugs for treating CNS diseases.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtypeselective inhibitors of voltage-gated sodium (Na_V) channels, we screened spider venoms for inhibitors of human Na_V1.7 (hNa_V1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na_V1.7 inhibitor, m-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa_V1.7. hNa_V1.6. hNa_V1.2 . hNa_V1.1. hNa_V1.3 channels in fluorescent assays. N_aV1.7 inhibition was diminished (IC₅₀ 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form(IC₅₀ 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mM. Unlike most spider toxins that modulate Na_V channels, Tp1a inhibited hNa_V1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa_V1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa_V1.7 inhibitors for treatment of chronic pain.

Rat basophilic leukaemia (RBL) cells endogenously express calcium-release-activated-calcium (CRAC) channels (1). CRAC channels are activated by depletion of intracellular calcium stores, probably via the involvement of STIM-1 (stromal interaction molecule) sensing the depletion of the stores and travelling to the cell membrane activating the channel (2, 3). Several approaches can be used to deplete calcium stores ultimately leading to activation of I_CRAC and most of these have been tried on QPatch. This document describes the results of these experiments and discusses the best conditions for studying I_CRAC with QPatch.

There is clear evidence that the presence of inflammatory factors and impaired GABA-ergic neurotransmission in depressed patients is associated with poor clinical outcome. We designed hybrid molecules, bearing the GABA molecule assembled with chemical fragments that interact with the serotonin 5-HT₆ receptor. Such a combination aimed to curb neuroinflammation, remodel GABA-ergic signaling, and provide antidepressant-like activity. The most promising hybrid 3B exerted nanomolar affinity for 5-HT₆ receptors and exerted agonistic properties on GABA-A receptors. Developability studies conferred that 3B exerted favorable drug-like properties and optimal brain penetration. In in vivo studies, 3B exerted robust antidepressant-like activity and proved to be highly effective in reducing levels of oxidative stress markers and the pro-inflammatory cytokine IL-6. The interesting pharmacological profile of 3B makes it a promising candidate for further development for depression associated with neuroinflammation.

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca²⁺ concentrations [Ca²⁺]_i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca²⁺]_i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca²⁺]_i quantification as an endpoint was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca²⁺]_i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

• High fidelity voltage and current clamp recordings of human iPS cell-derived cardiomyocytes (Cor.4U^®) on Qube
• Demonstration of the presence of I_Na, I_K and I_Ca
• High throughput current clamp recordings
• Pharmacological characterization of action potentials and isolated currents
• Using Sophion Analyzer software it is possible to easily extract key parameters relevant for studying compound effects on action potentials

HTS campaign of the corporate compound collection resulted in a novel, oxalic acid diamide scaffold of α7 nACh receptor positive allosteric modulators. During the hit expansion, several derivatives, such as 4, 11, 17 demonstrated not only high in vitro potency, but also in vivo efficacy in the mouse place recognition test. The advanced hit molecule 11 was further optimized by the elimination of the putatively mutagenic aromatic-amine building block that resulted in a novel, aminomethylindole compound family. The most balanced physico-chemical and pharmacological profile was found in case of compound 55. Docking study revealed an intersubunit binding site to be the most probable for our compounds. 55 demonstrated favorable cognitive enhancing profile not only in scopolamine-induced amnesia (place recognition test in mice) but also in natural forgetting (novel object recognition test in rats). Compound 55 was, furthermore, active in a cognitive paradigm of high translational value, namely in the rat touch screen visual discrimination test. Therefore, 55 was selected as a lead compound for further optimization. Based on the obtained favorable results, the invented aminomethylindole cluster may provide a viable approach for cognitive enhancement through positive allosteric modulation of α7 nAChRs.

The voltage-gated sodium channel, Na_V1.1, highly expressed in fast-spiking interneurons (FSIs), initiates the interneuron’s action potentials and promotes γ-oscillations. This process is critical for memory encoding and other cognitive functions. An impaired function of FSIs is associated with disorders like autism, schizophrenia, Alzheimer’s disease and others. Potentiators of Na_V1.1 can restore γ-oscillations by recovering reduced FSI function. These compounds were further shown to mitigate cognitive dysfunction in transgenic mice with decreased levels of Na_V1.1 expression in parvalbumin-positive neurons.

Na_V1.1 gating is complex, and activators of the ion channel can modulate various channel properties. Amongst many different screening techniques available, the patch clamp technique is the only one that reveals mode of action information. Recent advances of patch clamp devices have enabled the technology to be employed in large compound library screens. Qube 384 is a fully automated 384-well patch clamp device capable of testing thousands of compounds per day whilst providing true giga-ohm seal quality data. Using the Qube 384 in a drug discovery cascade enables acquisition of mode of action data simultaneous with hit detection during the primary screen, minimizing the need for many follow-up validation studies.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of the host response to pathogens.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens.

The Na_V1.5 channel is critical in maintaining the physiological electrical function in cardiomyocytes, as it is the heart’s main depolarising current. Here we focus on the Na_V1.5 channel in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an important target for cardiac-drug and safety screening.

• The hiPSC-CMs Na_V1.5 compound screening was performed with assay success rate (quality filters) up to 60% and ~ 20 useful experiments per 48 recording site plate.

• The study demonstrates that it is possible to screen compounds on hiPSC-CMs using the QPatch II.

• The experimental success rate of hiPSC-CMs assays was variable depending on the quality and maturation of the cell population.

In summary, it is possible to perform measurements on QPatch II of hiPSC-CMs in physiological ringers with voltage-gated assays yielding up to 50% success rate, depending on the ion channel target. These measurements demonstrate a heterogeneous expression of the cardiac ion channels, which consequently lowers the success rates (up to 20%) of action potential measurements. As 1) the hiPSC-CM quality and maturity increases and 2) cell suspension preparation is further optimized, we expect the throughput of these measurements to increase. Eventually, we envision that APC can be used as a characterization tool to assist the continuous development of hiPSC-CMs as well as for cardiac drug screening and disease modelling.

Voltage-gated sodium (Na_V) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na_V1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na_V1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube® is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na_V1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC₅₀ assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC₅₀ potencies generated on the Qube correlate with data generated on the more established Sophion QPatch® APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na_V1.7 in a robust and reliable APC assay for compound screening.

Introduction
Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types, including neurons and cardiomyocytes, and therefore constitute a novel way to model human diseases for drug testing in vitro1. Ion channels represent attractive therapeutic targets in the nervous and cardiovascular systems2 rendering electrophysiological studies of hiPSCs interesting for their usage in drug discovery. However, such studies have traditionally been limited by the labour-intensive and low throughput nature of patch clamp electrophysiology2.

Here we present the electrophysiological characterization of hiPSC-derived motor neurons using our automated patch clamp (APC) platforms, Qube 384 and QPatch. Our results include a measure of channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. voltage-gated Na+ (NaV) and voltage-gated K+ (KV) channels), identification of ligand-gated receptors and recordings of action potentials using current clamp. The major challenge when investigating neurons using APC platforms is the requirement to dissociate the cells from their neuronal network while maintaining cell viability and membrane integrity3. By optimization of the harvest- and whole-cell protocols we have overcome this obstacle resulting in success rates of up to 60% using our 384-well APC system. Utilizing the high throughput nature of our system we tested in parallel two disease models, using hiPSC neurons derived from Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS) patients, together with control cells from healthy subjects and isogenic control cells. The results show a general overactivity of Na+ channels in both cell lines, which could be rescued by a point mutation in the superoxide dismutase (SOD1) gene in the isogenic ALS cell line.

Our results demonstrate the feasibility of conducting electrophysiological characterization and screening of hiPSC-derived neurons on APC platforms like Qube 384 and QPatch, thus paving the way for high throughput ion channel-targeted screening of drugs for neurological disorders.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC₅₀ values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, in particular in the central nervous and cardiovascular systems. Moreover, assessment of cardiac ion-channel activity of new chemical entities is now an integral component of drug discovery programmes to assess potential for cardiovascular side effects. Despite their attractiveness as drug discovery targets ion channels remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of patch-clamp electrophysiology. This Review provides an update on the current state-of-the-art for the various automated electrophysiology platforms that are now available and critically evaluates their impact in terms of ion-channel screening, lead optimization and the assessment of cardiac ion-channel safety liability.

The patch-clamp technique is the gold-standard methodology for analysis of excitable cells. However, throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for applications like drug screening, has been primarily used to study channels and receptors expressed in heterologous systems. We introduce an approach for automated high-throughput patch-clamping that enhances analysis of excitable cells at the channel and cellular levels. This involves dissociating and isolating neurons from intact tissues and patch-clamping using a robotic instrument, followed by using an open-source Python script for analysis and filtration. As a proof of concept, we apply this approach to investigate the biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables voltage- and current-clamp recordings in the same cell, allowing unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of freshly isolated neurons without requiring culturing on coverslips.

• High data quality based on giga-ohm seal resistances in physiological solutions
• High flexibility of assay design
• Flexible voltage clamp and current clamp
• Enable discovery of new therapeutics for channelopathies
• Measure single dose as well as concentration-dependent effects
• Comparable channels recording between different physiological and disease cell models

• Constantly high success rates (99% in 3 experiments)
• Giga-ohm seal resistances in almost all wells
• Very low data spread
  • All CV values of DMSO control below 10%
  • No false positives with cutoff criteria 3 X SD
  • Z’ constantly greater 0.75
• Discrimination between different modes of inhibition (state-dependency)
• Up to 384 individual IC₅₀ values (3072 data points) determined in just one experiment
• Using Qube in combination with Stacker it would be possible to determine up to 7680 IC₅₀ (60,000 data points) per 24 hours

Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

Engineered human IgG could neutralise venom derived neurotoxins

The potency of IgGs could be distinguished using APC

Broader neutralization capacity could be detected

QPatch II and Qube are well suited for efficiently characterizing both peptide toxins and antibody function

Large molecules: evaluating the neutralization potential of recombinant antibodies against snake neurotoxins

Qube 384 is capable of performing pharmacology measurements with large molecules.

Qube 384 provides high throughput and high-quality recordings, enabling the evaluation of neurotoxins and neutralizing antibodies in an assay with a success rate of 95%.

K_V1.5 channels conduct the ultra-rapid delayed rectifier current (I_Kur) that contributes to action potential repolarization of the heart. Mutations in the encoding gene (KCNA5) have been related to both atrial fibrillation and sudden cardiac death. K_V1.5 is also found in other tissues, such as vascular smooth muscle, CNS, microglia, and Schwann cells (Clarkson et Al., 1998).

The pharmacological blockade of K_V1.5 has been suggested as a prevention and treatment of various arrhythmia (Karczewski et al., 2009) In the present study, we present an assay suitable for a high throughput screening campaign.

The citrus flavanone hesperetin (HSP) has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain unexplored. Here we studied the effects of HSP on the human cardiac voltage-gated Na+ channel (hNaV1.5) and compared it to its effects on a recombinant hNaV1.5 channel baring a mutation (R1623Q) associated with lethal ventricular arrhythmias in the Long QT syndrome type 3 (LQT3).

METHODS: Whole-cell patch clamp experiments were used to record Na+-currents (INa) in HEK293T cells expressing hNaV1.5 wild type (WT) or mutant channels.

RESULTS: HSP blocked thehNaV1.5 channels in voltage-dependent manner with an IC50 ≈ 100 μM. Its inhibition was decreased by disruption of the F1760 residue. HSP preferentially accelerated the inactivation phase of INa and decreased the Na+ net influx into the cell. The effects of HSP on the inactivation phase and voltage-dependent inhibition of INa were more marked in the LQT3 mutant.

CONCLUSIONS: HSP could be used as a template to develop drugs against cardiac arrhythmias in LQT3.

In this study, by using our QPatch II system and the temperature control feature, we report a significant difference in hERG kinetics at 22°C and 35°C. By testing four hERG blockers, we were able to measure channel activity and investigate compound effects at the two temperatures.
IC50 values obtained from the compounds tested on the QPatch II are similar to those obtained on the Qube 384 and agree with literature.

The QPatch 16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing TRPV1 channels.

TRPV1 response is stable on the QPatch platform and it is easy to perform agonist and antagonist studies. The half maxima effective and inhibitory concentrations (EC50 and IC50) found in this study are comparable to literature values.

Performing automated patch clamp experiments with different intracellular solutions is somehow difficult. Using QPatch with its multi well reservoir is it possible to test eight different solutions on the same cell line. In this study the multi well reservoir has been used for experiments on the voltage-dependent large conductance Ca2+-activated K+ channels, often referred to as BK channels. A human embryonic kidney cell line (HEK293) expressing the BK channel was used.
BK channels are large conductance calcium and voltage gated potassium channels, which allow potassium fluxwhen activated with intra cellular calcium and/or membrane potential (Strøbæk et al., 1996).

It is common that potential customers ask Sophion Bioscience to use QPatch to test some of their compounds on specific ion channels before buying a system. In this application report, we present some of the results obtained from a validation study done for a pharmaceutical company. Currents from the N-type
voltage-gated calcium channels (Ca_V2.2) were studied using
QPatch. A blocker of the Ca_V2.2 current, compound A, was
applied in increasing concentrations to study the dose-response
effects and estimate IC₅₀ values. Furthermore, flunarizine which is known to block T-type Ca²⁺/Na⁺ channels and have low potency on N-type Ca channels, was tested.

hKir2.1 exhibit strong inward rectification with major current activity at very negative potentials. The channel has a fundamental role in controlling and maintaining the resting membrane potential. hKir2.1 is encoded by the KCNJ2 gene and mutation in this gene can cause cardiac arrhythmia.

The QPatch16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing Ca_V3.2 channels. Whole cell currents were measured upon application of a voltage-step protocol to obtain I-V curves.

The development of screening assays for the Ca_V1.2 channel has been challenging due to the tendency of the channel to exhibit declining current levels (rundown) during the experiment. Here we report a robust Ca_V1.2 assay yielding high success rates, low rundown and reliable pharmacology.

Pharmacology and current-voltage relationship in accordance with literature values

Success rates of up to 98%

Stable currents with rundown as low as 2% per min

The development of screening assays for the Ca_V1.2 channel has been challenging due to the tendency of Ca_V1.2 expressing cell lines to exhibit declining current levels (rundown) during the experiment^1,2. Here we report a robust Ca_V1.2 assay yielding high success rates, low rundown and reliable pharmacology.

Pharmacology and current-voltage relationship in accordance with literature values

Success rates up to 94%

Stable currents with rundown as low as (1.2 ± 0.9)% per minute

The CiPA protocol yields stable currents with rundown as low as (1 ± 1)% per minute

Dr Hongjie Yuan – Emory University, USA

Title: GRIN mutations in neurological diseases: from molecular mechanism to rescue pharmacology

Background and Purpose

TREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.

Experimental Approach

The effect of GI‐530159 on TREK channels was demonstrated using 86Rb efflux assays, whole‐cell and single‐channel patch‐clamp recordings from recombinant TREK channels. The expression of K2P2.1 (TREK1), K2P10.1 (TREK2) and K2P4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current‐clamp recordings from cultured rat DRG neurons were used to measure the effect of GI‐530159 on neuronal excitability.

Key Results

For recombinant human TREK1 channels, GI‐530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current‐clamp recordings from cultured rat DRG neurones showed that application of GI‐530159 at 1 μM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential.

Conclusions and Implications

This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI‐530159 could aid in the development of novel analgesic agents.

This study presents results produced on the QPatch with GH4-C1 cells expressing the homomeric nicotinic ligand gated α7 ion channel (nAChR α7). nAChR α7 is linked to many normal and pathological conditions. It is important in memory formation, arousal, and attention, nicotine addiction, and nicotine-induced reversal of age-related memory deficits. nAChR α7 activation leads to neuroprotection against amiloid-β-induced neurotoxicity, which suggests that the receptor may play an important role in Alzheimer’s disease. Also their role in hippocampal auditory gating has linked the receptor to schizophrenia. These findings makes this particular channel an interesting target for pharmaceutical
companies. The fast kinetics of the α7 channel makes this a difficult ion channel to work on even in conventional patch clamp rigs. At Sophion we have developed assays that captures this current in a reproducible way enabling automated technology to be used in the search for blockers and positive allosteric modulators of this channel.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites.

Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Pathogenic T cell effector memory (TEM) cells drive many autoimmune disorders and are uniquely dependent on the Kv1.3 channel. A number of venom derived knottin (cysteine-rich mini-protein) inhibitors of Kv1.3 are being developed as potential drug candidates, but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this novel KnotBody^TM format, the knottin benefits from the improved therapeutic functionality of an antibody and the antibody gains additional diversity by the addition of a scaffold which is pre-disposed to blockade of ion channels.

A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBody^TM molecules were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography.

To further demonstrate the benefits of this novel format, the modular nature of the KnotBody^TM binding surface was exploited to: (i) improve existing knottin binding by introducing additional V_H contacts; (ii) create a bispecific molecule by introducing a V_H chain that binds to a different target; (iii) substitute the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with ShK, a Kv1.3 ion channel blocking toxin; (iv) develop a panel of low-nM Kv1.3 inhibitors with selectivity exceeding 3000-fold over the Kv1.1 channel, a closely related Kv family member.

The major inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA) and GABA is exerting its effect by binding to GABA receptors. The central role of GABA in the nervous system is underscored by the devastating consequences of pathophysiological changes in GABA signalling. Conversely, manipulation of GABA receptors can offer relief of a large group of neurological and psychiatric disorders. Pharmacological manipulation of GABAA has a large potential and ligands increasing the current will typically have anxiolytic, anticonvulsant, amnesic, sedative, hypnotic, euphoriant, and muscle relaxant effects.

GABAA receptors are ligand-gated ion channels, permeable to Cl- ions, consisting of 5 membrane-spanning subunits. 16 different subunits are identified in humans (α1-6, β1-3, γ1-3, δ, ε, θ, π) and the cellular GABA response is hence composed by a population of GABA receptors with significant different pharmacology. Here we demonstrate pharmacological GABA receptor evaluation in both a stably-transfected cell line containing only α5β3γ2 receptors and a primary cell culture of rat hippocampal astrocytes with a diverse GABA receptor population.

The major inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA) and GABA is exerting its effect by binding to GABA receptors. The central role of GABA in the nervous system is underscored by the devastating consequences of pathophysiological changes in GABA signalling. Conversely, manipulation of GABA receptors can offer relief of a large group of neurological and psychiatric disorders. Pharmacological manipulation of GABAA has a large potential and ligands increasing the current will typically have anxiolytic, anticonvulsant, amnesic, sedative, hypnotic, euphoriant, and muscle relaxant effects^1–4.
GABAA receptors are ligand-gated ion channels, permeable to Cl- ions, consisting of 5 membrane-spanning subunits^5,6. 16 different subunits are identified in humans (α1-6, β1-3, γ1-3, δ, ε, θ, π) and the cellular GABA response is hence composed by a population of GABA receptors with significant different pharmacology⁷. Here we demonstrate pharmacological GABA receptor evaluation in both a stably-transfected cell line containing only α5β3γ2 receptors and a primary cell culture of rat hippocampal astrocytes with a diverse GABA receptor population.

Over the last six decades, voltage-gated sodium (Nav) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the activity of Nav channels, proper determination of their properties still presents serious challenges, depending upon the experimental conditions and the desired subtype of channel to be characterized. The aim of this unit is to review the characteristics of Nav channels, their properties, the cells in which they can be studied, and the currently available techniques. Topics covered include the determination of Nav-channel biophysical properties as well as the use of toxins to discriminate between subtypes using electrophysiological or optical methods. Perspectives on the development of high-throughput screening assays with their advantages and limitations are also discussed to allow a better understanding of the challenges encountered in voltage-gated sodium channel preclinical drug discovery.

Voltage-gated sodium channels are transmembrane proteins that are responsible for the rapid depolarization that elicits the upstroke of action potentials in neurons and cardiac cells. Molecular cloning techniques have identified nine related voltage-gated sodium channels subtypes with distinct biophysical properties, interacting proteins, and cellular patterns of expression that are involved in electrical signaling. The goal of many research groups is to target specific sodium channels subtypes to develop selective inhibitors that could, in theory, produce safer and more efficacious drugs. To address compound sodium subtype profiling, we used a two-pulse
state-dependent protocol to develop a sub-type specific sodium channel (hNav1.1 to hNav1.8) panel on the QPatch HT patch clamp system. Additionally, we tested three stimulus protocols on hNav1.5 with known sodium inhibitors: 1) CiPA protocol for cardiac safety testing (tonic), 2) two-pulse state-dependent protocol (inactivation-dependent), 3) three-pulse protocol (tonic, inactivation-dependent, and V1/2 dependent inactivation). The CiPA protocol identified state-independent compounds (e.g. Flecainide) within three-fold of published
literature values but didn’t identify state-dependent compounds such as Lidocaine. The two-pulse protocol identified both state-dependent and independent compounds at pulse 2 with IC50 values within 4-fold published literature values. Data from the first two pulses of the three-pulse protocol had comparable pharmacology to the two-pulse protocol. The functional activity from V_1/2 inactivation (a delayed 3rd pulse) was less predictive of state-dependent inhibition than the two-pulse protocol but may reflect the in vivo inhibition. The hNav1.x panel using the two-pulse protocol can be useful to profile target identification, selectivity and state-dependence of novel compounds. (QPatch Automated Patch Clamp System is manufactured by Sophion Bioscience).

Professor Mala Shah, University College London – talk from ICMS 2019.

Title: Function and modulation of axonal Kv7 channels in hippocampal neurons

The Na_V 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theatre with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and
conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence.

Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theatre with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence.

Series resistance compensation for fast activating ion channels can help remove voltage clamp errors and increase clamp accuracy. All Sophions patch clamp amplifiers utilize a patented technique enabling up to 100% RS compensation. In addition, Sophion amplifiers are equipped with automatic clip detection to avoid loss of cells due to fatal oscillations, thereby increasing data throughput while maintaining high quality recordings. Here we show that voltage clamp errors can be up to 20-30 mV when uncompensated and in general that errors occur if not using RS compensation. However, we also show that the increase of accuracy due to use of RS compensation comes with a price of a lower success rate, and that the settings are critical to maximize success rates while avoiding or decreasing errors. Last we demonstrate the importance of automated clip detection.

• The nicotinic acetylcholine receptor alpha1 (nAChR α1) is a fast desensitizing channel that makes the development of a stable high throughput assay on automated patch clamp systems challenging.
• The solution stacking feature on Qube reduces ligand exposure times below 1 s. Using this feature, it is now possible to evoke stable acetylcholine signals for agonist concentrations at least up to EC₅₀.
• An assay for nAChR α1 endogenously expressed in TE671 cells with 5 µM acetylcholine showed the following characteristics:
  • Overall success rate of 89%
  • Overall whole-cell seal resistance = 1.2 ± 0.4 GΩ (SD)
  • Coefficient of variance for rows and columns in DMSO control ≤12%
  • IC₅₀(max/min)(tetracaine) of a non-cumulative concentration response experiment = 1.5
  • Z’ greater than 0.70
• The assay stability is also owed to unique architecture of the QChip where microfluidic channels ensure rapid and complete solution exchange of compound and agonist.

Background: Dying tumor cells release intracellular potassium (K+ ), raising extracellular K+ ([K+ ]e) in the tumor microenvironment (TME) to 40–50 mM (high-[K+]e). Here, we investigated the effect of high-[K+]e on T cell functions.

Materials and Methods: Functional impacts of high-[K+]e on human T cells were determined by cellular, molecular, and imaging assays.

Results: Exposure to high-[K+]e suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K+]e inhibited T cell cytokine roduction and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K+]e caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of KCa3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K+]e, a novel KCa3.1 activator, SKA346, rescued T cells from high-[K+]e-mediated suppression.

Conclusion: High-[K+ ]e represents a so far overlooked secondary checkpoint in cancer. KCa3.1 activators could overcome such ‘‘ionic-checkpoint’’-mediated immuno-suppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

There is increasing interest for cardiomyocytes as models for studying cardiac cellular physiology and preclinical drug safety testing. Stem cell-derived cardiomyocytes have the potential for such a model and have the possibility for modeling human diseases. The present investigation is the first to describe current properties from stem cell-derived cardiomyocytes using multi-hole recordings with planar automated patch clamp technology. In our study pluripotent stem cell-derived cardiomyocytes were biophysically and pharmacologically characterized. The cells are differentiated in large numbers and cryo-preserved, which make them suitable for automated patch clamping and facilitate their use in drug screening. We tested the cells in two different recording modes; single-hole and multi-hole, respectively. For multi-hole recordings up to ten cells are patched at the same time and the total current is measured per site. This recording mode can be useful for small currents (e.g. endogenous) and typically increases the success rate for useful data. For all experiments the whole-cell configuration was used and three different types of currents were studied; Na⁺, Ca2⁺ and K⁺. Using specific voltage protocols biophysical characteristics of each current was described and compared from single-hole and multi-hole experiments. We showed that currents recorded from these pluripotent stem cell-derived cardiomyocytes are similar to human cardiomyocytes and the response to known pharmacology is as expected. The V0.5 values, I-V relationships, current kinetics and IC₅₀ values determined for known blockers (TTX, nifedipine and cisapride) were comparable for the two recording modes. Clearly the success rate for usable data per measurement plate was significantly increased with the multi-hole technology. This is the first time current properties of stem cell-derived cardiomyocytes have been described from multi-hole recordings with planar automated patch clamp. Our study has shown that automated patch clamp is ready for stem cell-derived exploration.

The QPatch HTX is the newest member of the of the QPatch family of high-end fully automated patch clamp systems from Sophion Biosciences. The QPatch HTX builds on the proven technology from the QPatch 16 and QPatch HT, where cells are patched on a silicon chip with integrated microfluidic flow channels made in glass. The chip, called a QPlate, allows true gigaseals to be formed and high quality data to be recorded. For QPatch HTX modifications has been made to the QPlate and to the amplifier. To diminish problems with low-expressing cell lines, the QPlate HTX contains multiple holes per recording site and to accommodate the increased current amplitudes, the amplifier has been built with a broader current range.
In this study we present data obtained during the assay validation and optimization of a high throughput screening assay using a HEK293 cell line expressing the voltage gated sodium channel Na_v1.2a. This stable cell line is characterized by a very low functional expression of the Na_v1.2a channel with approximately 30% expressing cells in the population resulting in a relatively low success rate in data recording on QPatch HT. We found that with the QPlate HTX the success rate could be dramatically improved and upon assay optimization on QPatch HTX the effective success rate approaches 100%. We present data to compare throughput and assay quality between the QPatch HTX in multi-hole mode and traditional single cell patch clamp on QPatch HT.
Furthermore we have evaluated the compound profiling properties of the QPatch HTX by characterizing known blockers against the Na_v1.2a channel – a challenging channel for electrophysiological recording due the extremely low time constants of the response elicited upon voltage stimulation of the channel. Surprisingly, our biophysical and pharmacological data suggest that the data quality obtained on the QPatch HTX system is overall comparable to equivalent data obtained on the QPatch HT system.

Small-conductance Ca²⁺-activated K⁺ (SK) channels mediate afterhyperpolarization in neurons and dampen the firing frequency of action potentials. Calmodulin is constitutively associated with the SK2 channels and serves as the Ca²⁺ sensor for the SK-calmodulin complex. The SK2 channel subtype plays a key role in the regulation of excitability of Purkinje cells in the cerebellum. Given their importance in Purkinje cells, SK2 channels are a promising drug target for ataxia, a movement disorder.

Automated patch clamp (APC) machines, such as QPatch, have been used in the pharmaceutical industry to study drug interaction with varieties of ion channels. Over the past decade, technology has contributed significantly to pharmaceutical research and drug discovery. Here, we report to use QPatch as a tool for testing and evaluating the positive allosteric modulators of SK2 channels. A stable cell line of the rat SK2 channel tagged with GFP was established through transfection of HEK293 cells followed by puromycin selection and enrichment using repeated GFP fluorescence-activated cell sorting.

Positive allosteric modulators were tested under whole-cell voltage-clamp configuration with automated QPatch. The compounds that positively modulate the SK2 channels in the automated QPatch was further tested with the inside-out patch manual recordings. The results from automated whole-cell recordings and inside-out patch manual recordings were consistent.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two Nav1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which has previously been shown to increase affinity and inhibitory potency on hNav1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

The human ether-à-go-go related gene (hERG) carries one of the major repolarizing currents in the heart myocytes and block of this channel by certain drugs may lead to prolongation of the cardiac action potential and the potentially lethal arrhythmia torsade de pointes (TdP). Therefore current guidelines are that potency against hERG should be assessed in preclinical drug development. However, not all compounds inhibiting hERG are proarrhythmogenic and more integrated effects of the rug must be taken into consideration.

The comprehensive in vitro proarrhythmic assay (CiPA) initiative have introduced a new paradigm for assessing
proarrhythmic risk based on a thorough understanding of the underlying cellular mechanisms leading to a more predictive estimation of the risk carried by the specific compound.

One pillar of the CiPA paradigm is to measure the major
ionic currents involved in cardiac depolarization and repolarization using the high throughput automated patch clamp (APC) systems. Until recently this has meant that the measurements were carried out at ambient temperature a but recently automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 10 to 40°C.

Here we estimate potencies and binding kinetics to the hERG channel which are important parameters for the outcome of the risk assessment for a panel of clinical drugs using a Qube APC system equipped with temperature control.

The synthesis and pharmacological activity of a new series of pyrazoles that led to the identification of 1-(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethanone (9k, EST64454) as a σ₁ receptor (σ₁R) antagonist clinical candidate for the treatment of pain are reported. The compound 9k is easily obtained through a five-step synthesis suitable for the production scale and shows an outstanding aqueous solubility, which together with its high permeability in Caco-2 cells will allow its classification as a BCS class I compound. It also shows high metabolic stability in all species, linked to an adequate pharmacokinetic profile in rodents, and antinociceptive properties in the capsaicin and partial sciatic nerve ligation models in mice.

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066–660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066–660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially.
To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, K_v1.5, KvLQT1/minK and Na_v1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for K_v1.5 and Na_v1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially. To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, Kv1.5, KvLQT1/minK and Nav1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for Kv1.5 and Nav1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

The prominent role of voltage-gated sodium channel 1.7 (Na_v1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtypeselective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, since they naturally evolved to be potent ion channel blockers. Here we report the engineering of highly potent and selective inhibitors of Na_v1.7 channel based on tarantula Ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to Na_v1.7 channel. The resulting microproteins are highly potent (IC₅₀ to Na_v1.7 of 2.5 nM) and selective. We achieved 80-fold and 20-fold selectivity over the closely related Na_v1.2 and Na_v1.6 channels, respectively, and the IC₅₀ on skeletal (Na_v1.4) and cardiac (Na_v1.5) sodium channels is above 3000nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

A large number of mammalian cell lines are commercially available to be used as expression systems for membrane or cytoplasmal proteins. A number of voltage and ligand gated ion channels of potential interest for the pharmaceutical industry are endogenously expressed in several CNS and non-CNS cell lines including TTX-sensitive Na+ channels, Ca2+-release activated Ca2+ (CRAC) channels, inward rectifier K+ channels, acid-sensing ion channels (ASIC) and muscarinic alpha-adrenergic receptors mAChR). We have explored the applicability of five commonly employed cell lines from American Type Culture Collection (ATTC) for use with Sophions QPatchTM automated patch clamp systems (QPatch 16 and QPatch HT) and characterized the ion channel types that they endogenously express. Specificly we have explored:

• Suitability for automated patch clamp studies
  (‘patchability’)
• Background ionic currents that may interfere
  with currents of experimentally expressed ion
  channels
• Possible use for characterizing ion channels of
  interest without the need to experimentally
  introduce their genes (expression)

The tests have led to the development of a number of simple standard operation procedures (SOPs) for employment of the cell lines in QPatch characterizations of ion channels.

Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl− secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases.

Ligand‐gated ion channels (LGICs) are a group of diverse ion channels that are gated by ligands and play important roles in normal physiological and pathological conditions. Many of them are drug targets that have been pursued, are being pursued, and will likely be pursued in the future by pharmaceutical companies and academic groups for a variety of diseases. One of those LGICs is the GABA_A receptor, a hetero-oligomeric chloride channel that can be blocked and modulated at various sites. In order to study the receptor’s functional response to compounds, the manual patch‐clamp method provides a detailed but low‐throughput electrophysiological characterization. QPatch II, a next‐generation automated patch clamp machine that was recently developed by Sophion Bioscience, provides an automated electrophysiological study of ion channels. In this article, we use the GABA_A receptor as an example for studying LGICs and describe two detailed protocols for using QPatch II to carry out pharmacological studies on the receptor.

Pluripotent stem cell-derived cardiomyocytes, iCells® Cardiomyocytes, were biophysically and pharmacologically characterized with planer automated patch clamp using the QPatch. iCells Cardiomyocytes are differentiated from human induced pluripotent stem cells. They are differentiated in large numbers and cryo-preserved, which make them highly suitable for automated patch clamping and facilitates their use in drug screening. Here we present the results obtained during assay optimization of the cell culture, assay set-up on the QPatch as well as biophysical and pharmacological validation of the cardiac currents. Implementing optimal cell culture routines, the iCells Cardiomyocytes demonstrated spontaneous rhythmical contractions indicating functional properties of adult cardiomyocytes. We tested the cells in two different QPatch recording modes; single-hole recordings and multi-hole recordings where up to ten cells are patched at the same time and the total current is measured per site. Three different types of currents were studied including sodium, calcium and potassium. By using specific buffer solutions and changing the voltage protocols it was possible to characterize calcium, and sodium currents from the same cell. Our study showed that iCells Cardiomyocytes can successfully be applied to the QPatch. The recorded currents are similar to human cardiomyocytes and the response to known pharmacology is as expected. Using the QPatch we believe that iCells Cardiomyocytes have great potential for safety screening and other cardiovascular investigations early in the drug discovery process.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types, including neurons and cardiomyocytes, and therefore constitute a novel way to model human diseases¹. However, hiPSC-based applications are often challenged by the variability among differentiation batches, even when using iPSC lines originating from a single donor, and by heterogeneity among specific cell types derived from iPSCs². Here we demonstrate the application of high throughput electrophysiological measurements using our automated patch clamp (APC) platform, Qube 384, for characterization of iPSC-derived cortical neurons.

Generic cortical neurons were differentiated from iPSCs using small molecules under dual-SMAD inhibition conditions³. The cells were handled and differentiated in parallel, and a total of three independent differentiation batches were carried out. We conducted an electrophysiological analysis of the endogenous ion channel expression in the three different batches of iPSC neurons. In agreement with sequencing data, the measurements revealed a good reproducibility between the three iPSC batches with 60 % – 70 % of the investigated cells expressing voltage-gated Na⁺ (Na_v) channels and 60 % – 80 % expressing voltage-gated K⁺ (Kv) channels. Furthermore, filtering the measurements for the ion channel of interest, allowed a thorough biophysical and pharmacological investigation of the individual ion channels.

Finally, we compared iPSC-derived neurons from a patient (proband) carrying a missense mutation in cyclin-dependent kinase-like 5 (CDKL5) (c.871T>C, p.Cys291Arg), who was clinically diagnosed to present with classic CDKL5 Deficiency Disorder (CDD), with cells derived from an isogenic control. Using immunostaining, whole transcriptome analysis, and electrophysiological measurements we found that proband neurons were larger and had a more immature morphology, suggesting that they do not differentiate as efficiently as the control neurons.

Rat basophilic leukaemia (RBL) cells endogenously express calcium-release-activated-calcium (CRAC) channels (1). CRAC channels are activated by depletion of intracellular calcium
stores via the involvement of STIM-1 (stromal interaction molecule) sensing the depletion of the stores and travelling to the cell membrane activating the channel (2, 3). Several approaches can be used to deplete calcium stores, ultimately leading to activation of ICRAC. The present work was to develop assays using different depletion strategies on the
QPatch automated patch clamp platform.

Human-induced pluripotent stem cells (hiPSCs) were developed a decade ago and hold great promise for disease modelling, drug discovery and personalized medicine, especially in cardiac and neurological diseases1. Dysfunctional ion channels are one of the major targets in these diseases and thus, as the iPSC technology improves, a requirement arises for instrumentation that can characterize electrophysiological properties of iPSCs in a high-throughput fashion. Here, we demonstrate the use of the automated patch clamp (APC) platforms Qube 384 and QPatch in three characterization studies of hiPSC motor neurons derived from patients and healthy individuals. The results include:

APC recording of hiPSC-derived motor neurons with high success rates on Qube (60%) and QPatch (30%)

Biophysical characterization of voltage-gated channels, Na⁺ (Na_V) and K⁺ (K_V), in healthy hiPSCs

Measurements of a ligand-gated channel (GABA_A receptor) in healthy control cells

Screening of hiPSC motor neurons derived from Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) patients

Comparison of hiPSC motor neurons derived from two different healthy individuals

The loss of dopaminergic neurons in the substantia nigra plays an important role in the development of the Parkinson’s disease The symptoms of this disease typically occur after around 80 of these neurons degenerated This cell decay can be caused or promoted by genetic defects or environmental factors
including chemical compounds like pesticides For proper testing of neurotoxic effects on these neurons as well as for the development of neuroprotective drugs, assays based on animal primary cells lack predictivity due to mostly weak correlation between animal and human data. Therefore, models based on
human neuronal cells have a high potential to overcome the limitations of animal models One interesting neuronal cell line is the LUHMES (Lund human mesencephalic) line, which consists of immortalized fetal human mesencephalic cells that can be differentiated into fully postmitotic dopaminergic neurons in 6 days Scholz et al 2011 We here describe functional properties of these cells as a fundament for the
development of LUHMES based pharmacological assays.

The human-induced pluripotent stem-cell (hiPSC) technology was developed in 20071,2 and hiPSC-derived cardiomyocytes (hiPSC-CMs) have since then been recognized as a promising model system for cardiac drug screening and disease modelling3,4. Ion channels represent highly attractive therapeutic targets in the cardiovascular system, rendering electrophysiological studies of hiPSC-CMs important for their usage in drug discovery. However, such studies have traditionally been limited by the labor-intensive and low-throughput nature of patch-clamp electrophysiology. Here we use the automated patch clamp (APC) system QPatch II for high-throughput recordings of voltage-gated ion channels and paced action potentials in physiological solutions.
APC measurements of hiPSC-CMs are inherently challenging due to cell quality (batch-batch variation, differentiation efficiency)5, cardiac maturity (presence of pacemaker current If, and reduced hyperpolarizing current, IK1)6,7 and cell harvest (purity/quality of the single-cell suspension). Improving on all three parameters we were able to record cardiac voltage-gated currents (INa, ICa, IKr, IK1) with up to 50% success rates and paced action potentials with up to 20% success rates. The increased throughput of action potential measurements compared to manual patch clamp (10 cells per measurement plate) allowed us to measure dose-response effects of standard compounds such as nifedipine, E4031 and Bay K8466. In contrast to other APC systems, these measurements were performed in physiological solutions without the use of fluoride or other types of a seal-enhancer.

Inwardly rectifying IK1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding Kir2.1 channels, which primarily conduct ventricular IK1, are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of Kir2.1-mediated IK1 currents is a cardiosafety concern during new drug discovery and development. Kir2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of Kir2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human Kir2.1 channels. We reproduced key electrophysiological and pharmacological features known for native IK1, including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the Kir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of
this assay authorizes medium throughput screening for cardio safety purposes and could help to enrich the currently limited Kir2.1 pharmacology.

Background

Eleclazine (GS-6615) is a sodium channel blocker designed to improve the selectivity for cardiac late Na⁺ current (I_Na) over peak I_Na.

Objectives

The goals of this study were to investigate the inhibition of late INa by eleclazine using a sample of long QT syndrome type 3 (LQT3) and overlap LQT3/Brugada syndrome mutant channels; to compare the apparent binding rates for eleclazine with those for other class 1 antiarrhythmic agents; and to investigate the binding site.

Methods

Wild-type human cardiac voltage-gated sodium channel (hNa_V1.5) and 21 previously reported variants were studied using patch clamp recordings from a heterologous expression system.

Results

Eleclazine inhibited anemone toxin II–enhanced late INa from wild-type hNa_V1.5 with a drug concentration that causes 50% block of 0.62 ± 0.12 μM (84-fold selectivity over peak INa). The drug concentration that causes 50% block of eleclazine to inhibit the enhanced late INa from LQT3 mutant channels ranged from 0.33 to 1.7 μM. At predicted therapeutic concentrations, eleclazine and ranolazine inhibited peak INa to a similar degree as assessed with 4 overlap LQT3/Brugada syndrome mutations. Eleclazine was found to interact with hNa_V1.5 significantly faster than ranolazine and 6 other class 1 antiarrhythmic agents. Engineered mutations (F1760A/Y1767A) located within the local anesthetic binding site decreased the inhibition of late I_Na and peak I_Na by eleclazine.

Conclusion

At predicted therapeutic concentrations, eleclazine elicits potent inhibition of late INa across a cohort of Na_V1.5 mutant channels. These properties are consistent with a class 1b antiarrhythmic agent that associates with unusually rapid binding/unbinding rates.

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

Atrial fibrillation (AF) is the most common type of arrhythmia. Current pharmacological treatment for AF is moderately effective and/or increases the risk of serious ventricular adverse effects. To avoid ventricular adverse effects, a new target has been considered, the small conductance calcium-activated K⁺ channels (K_Ca2.X, SK channels). In the heart, K_Ca2.X channels are functionally more important in atria compared to ventricles, and pharmacological inhibition of the channel confers atrial selective prolongation of the cardiac action potential and converts AF to sinus rhythm in animal models of AF. Whether antiarrhythmic drugs (AADs) recommended for treating AF target K_Ca2.X channels is unknown. To this end, we tested a large number of AADs on the human K_Ca2.2 and K_Ca2.3 channels to assess their effect on this new target using automated whole-cell patch clamp. Of the AADs recommended for treatment of AF only dofetilide and propafenone inhibited hK_Ca2.X channels, with no subtype selectivity. The calculated IC₅₀ were 90±10 µmol/l vs 60±10 µmol/l for dofetilide and 42±4 µmol/l vs 80±20 µmol/l for propafenone (hK_Ca2.3 vs hK_Ca2.2). Whether this inhibition has clinical importance for their antiarrhythmic effect is unlikely, as the calculated IC₅₀ values are very high compared to the effective free therapeutic plasma concentration of the drugs when used for AF treatment, 40,000-fold for dofetilide and 140-fold higher for propafenone.

Society urgently needs new, effective medicines for the treatment of tuberculosis. To kick-start the required hit-to-lead campaigns, the libraries of pharmaceutical companies have recently been evaluated for starting points. The GlaxoSmithKline (GSK) library yielded many high-quality hits, and the associated data were placed in the public domain to stimulate engagement by the wider community. One such series, the spiro compounds, are described here. The compounds were explored by a combination of traditional in-house research and open source methods. The series benefits from a particularly simple structure and a short associated synthetic chemistry route. Many members of the series displayed striking potency and low toxicity, and highly promising in vivo activity in a mouse model was confirmed with one of the analogues. Ultimately the series was discontinued due to concerns over safety, but the associated data remain public domain, empowering others to resume the series if the perceived deficiencies can be overcome.

The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative differentiates torsadogenic risk of 28 drugs affecting ventricular repolarization based on multiple in vitro human-derived ionic currents. However, a standardized prospective assessment of the electrophysiologic effects of these drugs in an integrated in vivo preclinical cardiovascular model is lacking. This study questioned whether QTc interval prolongation in a preclinical in vivo model could detect clinically reported QTc prolongation and assign torsadogenic risk for ten CiPA drugs.

The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative differentiates torsadogenic risk of 28 drugs affecting ventricular repolarization based on multiple in vitro human-derived ionic currents. However, a standardized prospective assessment of the electrophysiologic effects of these drugs in an integrated in vivo preclinical cardiovascular model is lacking. This study questioned whether QTc interval prolongation in a preclinical in vivo model could detect clinically reported QTc prolongation and assign torsadogenic risk for ten CiPA drugs.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor β-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca²⁺-activated-Cl⁻ channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ~580,000 compounds. The anthelmintics niclosamide, nitazoxanide and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting β-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the β-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time, we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

Dr Sung-Young Kim, Daewoong Pharmaceutical Co., Ltd. – talk from ICMS 2019, the United Kingdom

Title: Drug development strategies of Na_V1.7 blocker

Professor Matthew Nolan – Edinburgh University, UK

Title: Diverse computational roles for HCN1 ion channels in behaviour

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K⁺ channels (K_V1.1 and 1.2 a subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of K_V1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or K_V1.1-subunit selective (dendrotoxin K) blockers of K⁺ currents suggest enhanced K_V1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K⁺ currents mediated by recombinant channels comprised of different K_V1.1 and 1.2 stoichiometries revealed that the enrichment of KV1 channels K_V1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the patho-physiology of MS and related disorders.

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K⁺ channels (K_V1.1 and 1.2 α subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of K_V1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or K_V1.1-subunit selective (dendrotoxin K) blockers of K⁺currents suggest enhanced K_V1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K⁺ currents mediated by recombinant channels comprised of different K_V1.1 and 1.2 stoichiometries revealed that the enrichment of K_V1 channels K_V1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the pathophysiology of MS and related disorders.

Voltage-gated sodium (Na_V) channel subtypes, including Na_V1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent Na_V channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human Na_V1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus. Here we present the isolation of native peptide, chemical synthesis, characterisation of human Na_V channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique NaV channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human Na_V1.7 putative pore blockers (IC50 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for Na_V channel pore blocker selectivity and subsequently important for chronic pain drug development.

Endocrine therapies in the treatment of early and metastatic estrogen receptor α positive (ERα+) breast cancer (BC) are greatly limited by de novo and acquired resistance. Selective estrogen receptor degraders (SERDs) like fulvestrant provide new strategies for endocrine therapy combinations due to unique mechanisms. Herein, we disclose our structure-based optimization of LSZ102 by replacing 6-hydroxybenzothiophene with 6H-thieno[2,3-e]indazole. Subsequent acrylic acid degron modifications led us to identify compound 40 as the preferred candidate. In general, compound 40 showed much better pharmacological profiles than the lead LSZ102, exhibiting growth inhibition of wild-type or tamoxifen-resistant MCF-7 cells, potent ERα degradation, together with superior pharmacokinetic properties, directional target tissue distribution including the brain, and robust antitumor efficacy in the mice breast cancer xenograft model. Currently, 40 is being evaluated in preclinical trials.

Activation of the stimulator of interferon genes (STING) pathway to achieve antitumor response is an attractive approach for cancer immunotherapy. In this study, we report the identification of BSP16 (LF250) as a potent, orally available STING agonist. BSP16 strongly activates STING signaling in human and mouse cells and binds STING as a homodimer. A 2.4 Å cocrystal structure revealed that BSP16 could induce the “closed” conformation of STING. In vivo studies revealed that BSP16 is well tolerated, has an excellent pharmacokinetic profile as an oral drug, and induces tumor regression and durable antitumor immunity. The promising bioactivities of BSP16 make it valuable for further development as an antitumor agent.

A series of quinazolinyl-containing benzamide derivatives were designed, synthesized and evaluated for their in vitro histone deacetylase 1 (HDAC1) inhibitory activities. Compounds 11a surpassed the known class I selective HDAC inhibitor MS-275 in both HDAC1 enzymatic inhibitory activity and cellular anti-proliferative activity against a selected set of cancer cell types (Hut78, K562, Hep3B and HCT116 cells) with no observed effects on human normal cells. In particular, compound 11a inhibited HDAC1 over the other tested HDACs isoforms (HDAC2, HDAC6 and HDAC8) with acceptable safety profiles. Moreover, compound 11a displayed favorable oral pharmacokinetic properties and showed significant antitumor activity in the A549 tumor xenograft model in vivo.

A series of pyrimidine-bridged CA-4 derivatives (9a-u) targeting colchicine site were designed, synthesized and evaluated. Among them, the most potent compound 9j showed favorable anti-proliferative activities against a panel of cervical cancer cells (IC50 = 0.09–0.15 μM) and tubulin polymerization inhibitory activity (IC50 = 4.6 μM). Meanwhile, compound 9j exhibited superior anti-proliferative activity against cisplatin-resistant HeLa/DDP and SiHa/DDP cells than CA-4 and cisplatin. Particularly, the combination of 30 mg/kg 9j with 3 mg/kg cisplatin resulted in a 73% tumor suppression rate in HeLa xenograft model and reduced the renal dysfunction and injuries caused by high doses of cisplatin. Moreover, 9j was highly selective over the normal human proximal tubular cells (HK-2 cells, IC50 = 188 μM). Mechanism studies revealed that 9j could disrupt tubulin polymerization and vasculature, arrest the cell cycle at the G2/M phase, induce apoptosis, and suppress clonogenesis and migration in HeLa cells. Further druggability characterization in terms of pharmacokinetic profile, acute toxicity, and hERG inhibition confirmed 9j could serve as a promising and safe combination agent for cervical cancer therapy.

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

The paper focuses on the scaffold hopping-based discovery and characterization of novel nicotinic alpha 7 receptor positive modulator (α7 nAChR PAM) ligands around the reference molecule (A-867744). First, substantial efforts were carried out to assess the importance of the various pharmacophoric elements on the in vitro potency (SAR evaluation) by chemical modifications. Subsequently, several new derivatives with versatile, heteroaromatic central cores were synthesized and characterized. A promising, pyrazole-containing new chemotype with good physicochemical and in vitro parameters was identified. Retrospective analysis based on homology modeling was also carried out. Besides its favorable in vitro characteristics, the most advanced derivative 69 also showed in vivo efficacy in a rodent model of cognition (scopolamine-induced amnesia in the mouse place recognition test) and acceptable pharmacokinetic properties. Based on the in vivo data, the resulting molecule with advanced drug-like characteristics has the possibility to improve cognitive performance in a biologically relevant dose range, further strengthening the view of the supportive role of α7 nACh receptors in the cognitive processes.

α9-Containing nicotinic acetylcholine receptors (nAChRs) are key targets for the treatment of neuropathic pain. α-Conotoxin RgIA4 is a peptide antagonist of human α9α10 nAChRs with high selectivity. However, structural rearrangement reveals a potential liability for clinical applications. We herein report our designer RgIA analogues stabilized by methylene thioacetal as nonopioid analgesic agents. We demonstrate that replacing disulfide loop I [CysI–CysIII] with methylene thioacetal in the RgIA skeleton results in activity loss, whereas substitution of loop II [CysII–CysIV] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC50 of 0.9 nM and much reduced degradation in human serum. In vivo studies showed that RgIA-5524 relieves chemotherapy-induced neuropathic pain in wild type but not α9 knockout mouse models, demonstrating that α9-containing nAChRs are necessary for the therapeutic effects. This work highlights the application of methylene thioacetal as a disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

The cytosolic metalloenzyme leukotriene A₄ hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B₄ (LTB₄). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB₄ generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ₁ receptor (σ₁R) and the μ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ₁R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ₁R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ1 receptor (σ1R) and the μ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ1R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

Late sodium current (late I_Na) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Na_v 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late I_Na, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia–ventricular fibrillation (VT–VF). We will describe structure–activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late I_Na inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S–T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC₅₀ values of 190 and 8000 nM, respectively. Compound 4represents a new class of potent late I_Na inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Inhibition of glucosylceramide synthase (GCS) is a major therapeutic strategy for Gaucher’s disease and has been suggested as a potential target for treating Parkinson’s disease. Herein, we report the discovery of novel brain-penetrant GCS inhibitors. Assessment of the structure-activity relationship revealed a unique pharmacophore in this series. The lipophilic ortho-substituent of aromatic ring A and the appropriate directionality of aromatic ring B were key for potency. Optimization of the absorption, distribution, metabolism, elimination, toxicity (ADMETox) profile resulted in the discovery of T-036, a potent GCS inhibitor in vivo. Pharmacophore-based scaffold hopping was performed to mitigate safety concerns associated with T-036. The ring opening of T-036 resulted in another potent GCS inhibitor with a lower toxicological risk, T-690, which reduced glucosylceramide in a dose-dependent manner in the plasma and cortex of mice. Finally, we discuss the structural aspects of the compounds that impart a unique inhibition mode and lower the cardiovascular risk.

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

To date, the clinical use of the anti-tubercular therapy bedaqui-line has been somewhat limited due to safety concerns. Recent investigations determined that modification of the Band C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, Band C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophen-yl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, Band C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds.

A series of 3-aryl(pyrrolidin-1-yl)butanoic acids were synthesized using a diastereoselective route, via a rhodium catalyzed asymmetric 1,4-addition of arylboronic acids in the presence of (R)-BINAP to a crotonate ester to provide the (S) absolute configuration for the major product. A variety of aryl substituents including morpholine, pyrazole, triazole, imidazole, and cyclic ether were screened in cell adhesion assays for affinity against α_vβ₁, α_vβ₃, α_vβ₅, α_vβ₆, and α_vβ₈ integrins. Numerous analogs with high affinity and selectivity for the α_vβ₆ integrin were identified. The analog (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-((R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid hydrochloride salt was found to have very high affinity for α_vβ₆integrin in a radioligand binding assay (pK_i = 11), a long dissociation half-life (7 h), very high solubility in saline at pH 7 (>71 mg/mL), and pharmacokinetic properties commensurate with inhaled dosing by nebulization. It was selected for further clinical investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis.

(R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders. ■ INTRODUCTION Epilepsy is a common neurological disorder characterized by spontaneous and recurrent seizures, often accompanied by a spectrum of neuropsychiatric symptoms. It is also a very heterogeneous disease with multifactorial and complex etiology. There are many different types of epilepsies, and the disease affects more than 70 million patients globally. 1 Progress in epilepsy research has led to the approval and marketing authorization of more than 30 antiseizure drugs (ASDs) over recent decades. Yet, despite this unquestionable therapeutic success, approximately one-third of epilepsy patients still experience uncontrolled and debilitating seizures. 2 These patients are considered as having the so-called drug-resistant epilepsy, which is defined as the failure of adequate trials of two tolerated and appropriately chosen ASD schedules (whether as monotherapies or in combination) to achieve seizure freedom. 3 The mechanisms underlying drug resistance in epilepsy are complex and still relatively poorly understood, which in combination with the multifactorial etiology and pathophysiology of epilepsy immensely complicate the rational selection of ASDs for optimal therapy. As such, drug-resistant epilepsy creates an urgent unmet medical need and propels ongoing drug discovery and development programs globally. Currently approved ASDs work through a number of mechanisms to restore the disturbed balance between excitatory and inhibitory neurotransmission responsible for seizure generation. 4 Such drugs target mainly ion channels (e.g., sodium or potassium channels), excitatory and inhibitory receptors, or presynaptic neurotransmitter release mechanisms. So far, only a few ASDs target neurotransmitter uptake mechanisms, mainly γ-aminobutyric acid (GABA) uptake (i.e., tiagabine). However, glutamate uptake dysregulation, which is not yet specifically targeted by ASDs, emerges as one of the critical drivers of excitotoxicity and seizures.

A phenotypic high-throughput screen allowed discovery of quinazolinone-2-carboxamide derivatives as a novel antimalarial scaffold. Structure-activity relationship studies led to identification of a potent inhibitor 19f, 95-fold more potent than the original hit compound, active against laboratory-resistant strains of malaria. Profiling of 19f suggested a fast in vitro killing profile. In vivo activity in a murine model of human malaria in a dose-dependent manner constitutes a concomitant benefit.

Psoriasis is a common immune-mediated skin disorder manifesting in abnormal skin plaques, and phosphodiesterase 4 (PDE4) is an effective target for the treatment of inflammatory diseases such as psoriasis. Toddacoumalone is a natural PDE4 inhibitor with moderate potency and imperfect drug-like properties. To discover novel and potent PDE4 inhibitors with considerable druggability, a series of toddacoumalone derivatives were designed and synthesized, leading to the compound (2R,4S)-6-ethyl-2-(2-hydroxyethyl)-2,8-dimethyl-4-(2-methylprop-1-en-1-yl)-2,3,4,6-tetrahydro-5H-pyrano[3,2-c][1,8]naphthyridin-5-one (33a) with high inhibitory potency (IC50 = 3.1 nM), satisfactory selectivity, favorable skin permeability, and a well-characterized binding mechanism. Encouragingly, topical administration of 33a exhibited remarkable therapeutic effects in an imiquimod-induced psoriasis mouse model.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 μM) and achieves “artemisinin-like” kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water–metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Ca_v2.2 (IC₅₀ 2.6 μM) but not Ca_v1.3 or Ca_v3.1 (IC₅₀ > 30 μM) in fluorimetric assays. In patch clamp electrophysiological assays Cd1a inhibited rat Ca_v2.2 with similar potency (IC₅₀ 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn’t act on Ca_v2.2 as a classical gating modifier toxin. The Cd1a binding site on Ca_v2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Ca_v2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Na_v1.1–1.2 and Na_v1.7–1.8 (IC₅₀ 0.1–6.9 μM) but not Na_v1.3–1.6 (IC₅₀ > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Na_v1.7 (IC₅₀ 16 nM) and produced a 29 mV depolarising shift in Na_v1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Na_v1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Ca_v2.2 inactivation gating, associated with the Ca_v2.2 α-subunit pore, while altering the activation gating of Na_v1.7. Cd1a was inactive at some of the Na_v and Ca_v channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

: The chemokine receptor CXCR3 is a seven-trans-membrane G-protein-coupled receptor (GPCR) involved in various pathologies, in particular autoimmune diseases. It is activated by the three chemokine ligands CXCL9, CXCL10, and CXCL11 and enables the recruitment of immune cell subsets leading to damage of inflamed tissues. Starting from a high-throughput screening hit, we describe the iterative optimization of a chemical series culminating in the discovery of the selective CXCR3 antagonist ACT-660602 (9j). The careful structural modifications during the lead optimization phase led to a compound with high biological potency in inhibiting cell migration together with improvements of the metabolic stability and hERG issue. In a LPS-induced lung inflammation model in mice, ACT-660602 led to significantly reduced recruitment of the CXCR3 + CD8 + T cell in the bronchoalveolar lavage compartment when administered orally at a dose of 30 mg/kg.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into the 47S ribosomal RNA (rRNA) precursor. Further processing produces the 28S, 5.8S, and 18S rRNAs that are assembled into mature ribosomes. Many cancers exhibit higher Pol I transcriptional activity, reflecting a need for increased ribosome biogenesis and protein synthesis and making the inhibition of this process an attractive therapeutic strategy. Lead molecule BMH-21 (1) has been established as a Pol I inhibitor by affecting the destruction of RPA194, the Pol I large catalytic subunit. A previous structure-activity relationship (SAR) study uncovered key pharmacophores, but activity was constrained within a tight chemical space. This work details further SAR efforts that have yielded new scaffolds and improved off-target activity while retaining the desired RPA194 degradation potency. Pharmacokinetic profiling was obtained and provides a starting point for further optimization. New compounds present additional opportunities for the development of Pol I inhibitory cancer therapies.

Despite the importance of the hERG channel in drug discovery and the sizable number of antagonist molecules discovered, only a few hERG agonists have been discovered. Here we report a novel hERG agonist; SKF-32802 and a structural analog of the agonist NS3623, SB-335573. These were discovered through a similarity search of published hERG agonists. SKF-32802 incorporates an amide linker rather than NS3623’s urea, resulting in a compound with a different mechanism of action. We find that both compounds decrease the time constant of open channel kinetics, increase the amplitude of the envelope of tails assay, mildly increased the amplitude of the IV curve, bind the hERG channel in either open or closed states, increase the plateau of the voltage dependence of activation and modulate the effects of the hERG antagonist, quinidine. Neither compound affects inactivation nor deactivation kinetics, a property unique among hERG agonists. Additionally, SKF-32802 induces a leftward shift in the voltage dependence of activation. Our structural models show that both compounds make strong bridging interactions with multiple channel subunits and are stabilized by internal hydrogen bonding similar to NS3623, PD-307243 and RPR26024. While SB-335573 binds in a nearly identical fashion as NS3623, SKF-32802 makes an additional hydrogen bond with neighboring threonine 623. In summary, SB-335573 is a type 4 agonist which increases open channel probability while SKF-32802 is a type 3 agonist which induces a leftward shift in the voltage dependence of activation.

The P2X4 receptor is a ligand-gated ion channel that is expressed on a variety of cell types, especially those involved in inflammatory and immune processes. High-throughput screening led to a new class of P2X4 inhibitors with substantial CYP 3A4 induction in human hepatocytes. A structure-guided optimization with respect to decreased pregnane X receptor (PXR) binding was started. It was found that the introduction of larger and more polar substituents on the ether linker led to less PXR binding while maintaining the P2X4 inhibitory potency. This translated into significantly reduced CYP 3A4 induction for compounds 71 and 73. Unfortunately, the in vivo pharmacokinetic (PK) profiles of these compounds were insufficient for the desired profile in humans. However, BAY-1797 (10) was identified and characterized as a potent and selective P2X4 antagonist. This compound is suitable for in vivo studies in rodents, and the anti-inflammatory and anti-nociceptive effects of BAY-1797 were demonstrated in a mouse complete Freund’s adjuvant (CFA) inflammatory pain model.

Background and Purpose
The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug-like NCX 1.1 inhibitor.

Experimental Approach
A flavan series‐based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM‐11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM‐11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae.

Key Results
ORM‐11372 inhibited human NCX 1.1 reverse and forward currents; IC50 values were 5 and 6 nM respectively. ORM‐11372 inhibited human cardiac sodium 1.5 (INa) and hERG KV11.1 currents (IhERG) in a concentration‐dependent manner; IC50 values were 23.2 and 10.0 μM. ORM‐11372 caused no changes in action potential duration; short‐term variability and triangulation were observed for concentrations of up to 10 μM. ORM‐11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose.

Conclusion and Implications
ORM‐11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro‐arrhythmic risk.

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na_V1.7 subtype of voltage-gated sodium (Na_V) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na_V1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na_V1.6, compared to Na_V1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa_V1.1–1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na_V1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity.

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.

Class II antiarrhythmics or β-blockers are antisympathetic nervous system agents that act by blocking β-adrenoceptors. Despite their common clinical use, little is known about the effects of β-blockers on free intracellular calcium (Ca2+ i), an important cytosolic second messenger and a key regulator of cell function. We investigated the role of four chemical analogs, commonly prescribed β-blockers (atenolol, metoprolol, propranolol, and sotalol), on Ca2+ i release and whole-cell currents in mammalian cancer cells (PC3 prostate cancer and MCF7 breast cancer cell lines). We discovered that only propranolol activated free Ca2+ i release with distinct kinetics, whereas atenolol, metoprolol, and sotalol did not. The propranolol-induced Ca2+ i release was significantly inhibited by the chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) and by dantrolene, an inhibitor of the endoplasmic reticulum (ER) ryanodine receptor channels, and it was completely abolished by 2-aminoethoxydiphenyl borate, an inhibitor of the ER inositol-1,4,5-trisphosphate (IP3) receptor channels. Exhaustion of ER stores with 4-chloro-m-cresol, a ryanodine receptor activator, or thapsigargin, a sarco/ER Ca2+ ATPase inhibitor, precluded the propranolol-induced Ca2+ i release. Finally, preincubation of cells with sotalol or timolol, nonselective blockers of β-adrenoceptors, also reduced the Ca2+ i release activated by propranolol. Our results show that different β-blockers have differential effects on whole-cell currents and free Ca2+ i release and that propranolol activates store-operated Ca2+ i release via a mechanism that involves calcium-induced calcium release and putative downstream transducers such as IP3 The differential action of class II antiarrhythmics on Ca2+ i release may have implications on the pharmacology of these drugs.

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity and the binding to voltage-gated sodium channels (VGSCs) and GABA_A receptors. Docking simulations allowed us to determine the mode of interactions between the investigated compounds and binding cavity of the human VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial membrane permeability assay (PAMPA), single-cell gel electrophoresis (SCGE) and cytotoxicity evaluation in HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The combined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising agents in the development of new anticonvulsants.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed

Polypharmacology approaches may help the discovery of pharmacological tools for the study or the potential treatment of complex and multifactorial diseases as well as for addictions and also smoke cessation. In this frame, following our interest in the development of molecules able to modulate either the endocannabinoid or the dopaminergic system, and given the multiple and reciprocal interconnections between them, we decided to merge the pharmacophoric elements of some of our early leads for identifying new molecules as tools able to modulate both systems. We herein describe the synthesis and biological characterization of compounds 5a-j inspired by the structure of our potent and selective fatty acid amide hydrolase (FAAH) inhibitors (3a-c) and ligands of dopamine D2 or D3 receptor subtypes (4a,b). Notably, the majority of the new molecules showed a nanomolar potency of interaction with the targets of interest. The drug-likeliness of the developed compounds (5a-j) was investigated in silico while hERG affinity, selectivity profile (for some proteins of the endocannabinoid system), cytotoxicity profiles (on fibroblast and astrocytes), and mutagenicity (Ames test) were experimentally determined. Metabolic studies also served to complement the preliminary drug-likeliness profiling for compounds 3a and 5c. Interestingly, after assessing the lack of toxicity for the neuroblastoma cell line (IMR 32), we demonstrated a potential anti-inflammatory profile for 3a and 5c in the same cell line.

Polypharmacology approaches may help the discovery of pharmacological tools for the study or the potential treatment of complex and multifactorial diseases as well as for addictions and also smoke cessation. In this frame, following our interest in the development of molecules able to modulate either the endocannabinoid or the dopaminergic system, and given the multiple and reciprocal interconnections between them, we decided to merge the pharmacophoric elements of some of our early leads for identifying new molecules as tools able to modulate both systems. We herein describe the synthesis and biological characterization of compounds 5a-j inspired by the structure of our potent and selective fatty acid amide hydrolase (FAAH) inhibitors (3a-c) and ligands of dopamine D2 or D3 receptor subtypes (4a,b). Notably, the majority of the new molecules showed a nanomolar potency of interaction with the targets of interest. The drug-likeliness of the developed compounds (5a-j) was investigated in silico while hERG affinity, selectivity profile (for some proteins of the endocannabinoid system), cytotoxicity profiles (on fibroblast and astrocytes), and mutagenicity (Ames test) were experimentally determined. Metabolic studies also served to complement the preliminary drug-likeliness profiling for compounds 3a and 5c. Interestingly, after assessing the lack of toxicity for the neuroblastoma cell line (IMR 32), we demonstrated a potential anti-inflammatory profile for 3a and 5c in the same cell line.

hERG (human Ether-a go-go-Related Gene) is a voltage-gated ion channel expressed in cardiac tissue which is of particular concern in off-target pharmacology. Blockade of hERG can lead to prolongation of cardiac repolarisation (long QT syndrome) and ventricular arrhythmia (torsades de pointes). Drug-induced LQTS is the most common reason for drug withdrawal in the last ten years; techniques are therefore required to address this and help to reduce attrition as early as possible in the drug development process.

Second generation automated electrophysiology systems such as IonWorks Barracuda use perforated patch technology to allow c.600 dose-response curves to be generated per day, but produce low fidelity megaOhm recordings with poor success rates. We have developed a more robust, reproducible high throughput whole-cell patch clamp assay methodology using the Sophion Qube 384, which provides higher fidelity
recordings and improved reliability in prediction of activity compared to other automated electrophysiology methods

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC50 = 1.0 nmol/L), but the poor metabolic stability in human liver microsomes (t1/2 = 14.6 min) and insufficient selectivity (SI = 2059) with high cytotoxicity (CC50 = 2.08 μmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH2-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC50 = 1.8–349 nmol/L). The best compound 5t in this collection (EC50 = 1.8 nmol/L, CC50 = 117 μmol/L) was 32-fold in selectivity (SI = 66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t1/2 = 74.52 min), approximately 5-fold higher than JK-4b in human liver microsomes (t1/2 = 14.6 min). Also, 5t possessed good stability in both human and monkey plasma. No significant in vitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

The chloride (Cl^–) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl^– currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

The α7 nicotinic acetylcholine receptor (a7 nAChR) is an important and challenging target for drug discovery in the area of neuropsychiatric disorders. The current screening for chemicals targeting α7 nAChRs is primarily achieved by the use of low throughput assay two-electrode voltage clamp (TEVC) in non-mammalian Xenopus oocytes. Automated patch clamp system has emerged as an attractive approach compared to conventional electrophysiology. To develop a mammalian cell-based functional assay in an automated electrophysiology system, we in this study generated a stable expression of α7 nAChRs in GH3 cells that originated from a rat pituitary tumor cell line and utilized automated QPatch 16 to test a set of tool compounds and chemicals identified as α7 agonists by TEVC. For the improvement of evaluating weak or partial α7 nAChRs agonists, we achieved enhancement of the signal-to-noise ratio by the addition of a positive allosteric modulator PNU-120596, which only activates α7 current in the presence of agonist. This improved assay was further validated by using known α7 partial agonists, such as RG3487, EVP-6124, and A-P90. Using this validated assay, we were able to identify a novel agonist 140507C that partially activates α7 nAChRs. Taken together, our results validate the use of QPatch 16 for evaluation α7 partial agonists, demonstrating its utility as an effective tool for α7 ion channel drug discovery.

The ionotropic glutamate receptor GluK1 (previously known as GluR5) is a kainate subtype of the glutamate receptor family and is comprised of a ligand binding domain and an associated ion channel. The receptor is expressed throughout the central nervous system with particularly strong expression in the dorsal root ganglion and trigeminal ganglion; antagonists for the receptor could be treatments for pain disorders, as well as acute migraine.

Calcium (Ca²⁺) is an important second messenger that is involved in critical processes in cell homeostasis and signal transduction. The ratio between extra- and intracellular Ca²⁺ contributes to setting the plasma membrane potential. Intracellularly, Ca²⁺ is stored in organelles such as Mitochondria and the Endoplasmic reticulum (ER). Depletion of these stores triggers the opening of Ca²⁺ – permeable channels that allow entry of Ca²⁺ into the cell along the electrochemical gradient. This process is called store-operated calcium entry (SOCE). Activation of SOCE happens as a secondary result of ER depletion (ref). ER is being depleted by an inositol-triphosphate (IP3) pathway and phospholipase C cascade, via the IP3 receptor. SOCE can experimentally be activated by elevating intracellular levels of IP3 in the presence of a Ca²⁺ chelator.

The activation of SOCE can directly be measured as a change in clamp current when performing patch-clamp experiments. In this study the automated patch clamp system, Qube 384 was used to record Icrac. Qube 384 is a second-generation automated patch clamp device capable of testing thousands of compounds per day whilst providing true giga-ohm seal quality data. Using the Qube 384 in a drug discovery cascade enables acquisition of mode of action data simultaneous with hit detection during the primary screen, thereby minimizing the need for extended follow-up validations studies.

In this study, we present an assay for STIM1/Orai1. Firstly, we present that Orai1 current can be activated either by exchange of the intracellular solution or alternative by de facto caging an agonist in the IC in the on-cell configuration. Secondly, we pharmacologically validate the assay using the trivalent lanthanide cations blocker lanthanum (La). With this, we demonstrate that Qube 384 also is a versatile patch clamp system, even though it is fully automated and can be running unattended.
Conclusion

Members of the voltage-gated K⁺ channel subfamily (Kv1), involved in regulating transmission between neurons or to muscles, are associated with human diseases and, thus, putative targets for neurotherapeutics. This applies especially to those containing Kv1.1 α subunits which become prevalent in murine demyelinated axons and appear abnormally at inter-nodes, underlying the perturbed propagation of nerve signals. To overcome this dysfunction, akin to the consequential debilitation in multiple sclerosis (MS), small inhibitors were sought that are selective for the culpable hyper-polarising K⁺ currents. Herein, we report a new semi-pod and – compound 3 – that was designed based on the modelling of its interactions with the extracellular pore region in a deduced Kv1.1 channel structure. After synthesis, purification, and structural characterisation, compound 3 was found to potently (IC₅₀ = 8 µM) and selectively block Kv1.1 and 1.6 channels. The tested compound showed no apparent effect on native Nav and Cav channels expressed in F-11 cells. Compound 3 also extensively and selectively inhibited MS-related Kv1.1 homomer but not the brain native Kv1.1- or 1.6-containing channels. These collective findings highlight the therapeutic potential of compound 3 to block currents mediated by Kv1.1 channels enriched in demyelinated central neurons.

Members of the voltage-gated K⁺ channel subfamily (Kv1), involved in regulating transmission between neurons or to muscles, are associated with human diseases and, thus, putative targets for neurotherapeutics. This applies especially to those containing Kv1.1 α subunits which become prevalent in murine demyelinated axons and appear abnormally at inter-nodes, underlying the perturbed propagation of nerve signals. To overcome this dysfunction, akin to the consequential debilitation in multiple sclerosis (MS), small inhibitors were sought that are selective for the culpable hyper-polarising K⁺ currents. Herein, we report a new semi-pod and – compound 3 – that was designed based on the modelling of its interactions with the extracellular pore region in a deduced Kv1.1 channel structure. After synthesis, purification, and structural characterisation, compound 3 was found to potently (IC₅₀ = 8 µM) and selectively block Kv1.1 and 1.6 channels. The tested compound showed no apparent effect on native Nav and Cav channels expressed in F-11 cells. Compound 3 also extensively and selectively inhibited MS-related Kv1.1 homomer but not the brain native Kv1.1- or 1.6-containing channels. These collective findings highlight the therapeutic potential of compound 3 to block currents mediated by Kv1.1 channels enriched in demyelinated central neurons.

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC₅₀s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC₅₀s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC₅₀s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC₅₀s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.

The intermediate-conductance Ca²⁺-activated K+ channel KCa3.1 (also known as KCNN4, IK1, or the Ga´rdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca²⁺ sensitivity of KCa3.1 was studied using varying intracellular Ca²⁺ concentrations. A free Ca²⁺ concentration of 1 lM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca²⁺ concentration (*200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

K2P channels are characterised by their four transmembrane
domain, two-pore topology. Sub-units dimerise to form a
functional channel.

K2P channels carry background (or leak) potassium current in
a variety of cell types and primarily act to maintain resting
membrane potential.

Despite a number of important roles they have proved a
difficult target class to modulate with small molecules.

Thus, there is currently a lack of useful specific
pharmacological tools which target K2Ps. This has limited the
interrogation of the precise physiological function of K2Ps and
efforts to generate K2P targeting therapeutics.

K2Ps have been implicated in a number of human
pathophysiologies including, but not limited to, pain (TREK-1,
TREK-2, TRAAK), migraine (TRESK), Birk-Barel syndrome
(TASK-3) and Balkan endemic nephropathy (TASK-2).

Voltage-gated sodium channels (Na_Vs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium
influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNa_V1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the Na_V1.1–1.7 activator veratridine (EC50 10–29 μM) or the Na_V1.1–1.8 inhibitor tetracaine (IC50’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, the development of the Qube 384 automated patch-clamp system has allowed rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale, making it possible to run HTS campaigns and support SAR with a functional readout.

Here, we have used the Qube platform to develop an assay against the NR1/NR2A receptor, which is part of the N-methyl-D-aspartate (NMDA) glutamate receptor family. For this assay, we utilized stacked liquid addition which enabled us to assess the open state kinetics of the channel and to investigate the effects of antagonists with multiple modes of actions.

We observed stable agonist responses for both NMDA and glycine, with EC₅₀ values comparable to literature values. The assay format was stable over multiple agonist applications and wash periods, which meant it was suitable for compound testing and sensitive enough to detect antagonists with multiple modes of action. D-AP-5, a competitive antagonist, showed stable NR1/NR2A receptor over six consecutive agonist/antagonist applications. The potency of ketamine, a use-dependent inhibitor, increased over the six agonist/antagonist applications.

By utilizing of stacked liquid additions in a 384 automated electrophysiology platform we have created an assay against NMDA receptors which is suitable for compound testing and sensitive enough to detect different modes of actions.

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, with the development of the Qube 384 automated patch-clamp system, the rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale is now possible at a greater throughput than previously possible. Here, we have used the Qube platform to develop assays against two ligand-gated families: 1) the P2X receptor and 2) the GABAA receptor families. The P2X family is comprised of 7 family members, which are cation permeable and gated by the binding of extracellular ATP. We have assessed both agonist and antagonist pharmacology of 4 members of the P2X family, P2X1, P2X2, P2X3 and P2X4, as well as two species homologs, rP2X3 and gpP2X3. The GABAA α1β3γ2 receptor is a chloride permeable ion channel gated by the binding of GABA. We utilized stacked liquid addition to assess the open state kinetics of the channel and to investigate the effects of a positive allosteric modulator on channel function. As such, we have successfully characterized and developed assays for both the P2X receptor and GABAA receptor families and present EC/IC50 data for antagonists and positive allosteric modulators.

TRPML1 is a cation-selective ion channel ubiquitously expressed in lysosomes and late endosomes. TRPML1 is intimately involved in the regulation of lysosomal Ca2+ homeostasis and is consequently a critical component of autophagy and lysosomal biogenesis. Malfunctions of these processes are linked to various neurodegenerative disorders, including Alzheimer`s and Parkinson`s disease. The relevance of TRPML1 in neurodegenerative disorders is further validated by a loss-of-function mutation that was shown to cause the neurodegenerative lysosomal storage disease mucolipidosis type IV (MLIV).

The patch clamp technique remains the gold standard for studying ion channels as recordings directly measure the protein’s activity. Whilst powerful, the conventional patch clamp method requires highly trained scientists and allows only a low throughput. Automated patch clamp systems have evolved to overcome these limitations. These platforms are, therefore, well-suited to test large numbers of compounds quickly to support ion channel drug development programs.

High conductance, calcium-activated potassium (BK) channels (K_Ca1.1) are important in regulating physiologic responses in many types of tissues and, as such, present opportunities for development of new therapeutic agents. Both channel agonists and inhibitors could have therapeutic utility, depending on medical application under consideration. However, characterization of molecular pharmacology of BK channels is incomplete and has been difficult to accomplish because of paucity of chemical leads that are acceptable templates for Medicinal Chemistry investigation. Only through continued prosecution of new high-throughput screening campaigns can this situation be rectified. Examples are presented of BK channel agonist and inhibitor discovery paradigms which will be useful for progressing BK channel future drug discovery strategies.

Electroporation is a technique allowing chemicals, drugs, or DNA to be introduced into the cell by applying an electrical field to the cells, thereby increasing the permeability of the cell membrane. The Neon^® Transfection System is a novel benchtop electroporation device that employs the pipette tip as an electroporation chamber to easily and efficiently transfect mammalian cells.

In this study, native HEK 293 cells were transfected with a plasmid encoding the human glycine receptor α1 (hGlyRα1), to demonstrate how a Qube can be used for:

1. optimization of electroporation transfection

2. pharmacological evaluation of transfected cells with varying transfection levels

The chemokine receptor CXCR3 allows the selective recruitment of innate and adaptive inflammatory immune cells into inflamed tissue. CXCR3 ligands are secreted after exposure to pro-inflammatory cytokines. Upon binding to CXCR3 ligands, CXCR3 expressing T-lymphocytes migrate toward sites of inflammation and can promote tissue damage. Therefore, antagonizing this receptor may provide clinical benefits for patients suffering from autoimmune diseases characterized by high concentrations of CXCR3 ligands. Herein, we report the second part of our CXCR3 discovery program where we explored the benzimidazolo-thiazole core scaffold. The optimization of potency and the mitigation of an hERG liability are described. Further pharmacokinetic considerations led to the identification of the potent CXCR3 antagonist ACT-672125 (29). The compound showed good physicochemical properties and safety profile. In a proof-of-mechanism model of lung inflammation, ACT-672125 inhibited the recruitment of CXCR3 expressing T cells into the inflamed lung in a dose-dependent manner.

RIPK1 plays a key role in the necroptosis pathway that regulates inflammatory signaling and cell death in various diseases, including inflammatory and neurodegenerative diseases. Herein, we report a series of potent RIPK1 inhibitors, represented by compound 70. Compound 70 efficiently blocks necroptosis induced by TNFα in both human and mouse cells (EC50 = 17–30 nM). Biophysical assay demonstrates that compound 70 potently binds to RIPK1 (Kd = 9.2 nM), but not RIPK3 (Kd > 10,000 nM). Importantly, compound 70 exhibits greatly improved metabolic stability in human and rat liver microsomes compared to compound 6 (PK68), a RIPK1 inhibitor reported in our previous work. In addition, compound 70 displays high permeability in Caco-2 cells and excellent in vitro safety profiles in hERG and CYP assays. Moreover, pre-treatment of 70 significantly ameliorates hypothermia and lethal shock in SIRS mice model. Lastly, compound 70 possesses favorable pharmacokinetic parameters with moderate clearance and good oral bioavailability in SD rat. Taken together, our work supports 70 as a potent RIPK1 inhibitor and highlights its potential as a prototypical lead for further development in necroptosis-associated inflammatory disorders.

In this study, the synthesis and biological evaluation of a variety of benzoheterocyclic-containing benzamide derivatives were described. Some of these compounds were proved to inhibiting the activity of histone deacetylase 1 (HDAC1) with IC50 values below the micromolar range, retarding proliferation of several human cancer cells, and surprisingly, not possessing toxicity to human normal cells and hERG K+ ion channels. Among those compounds, 3c was the most potent and efficacious derivative. Compound 3c was orally active and displayed excellent in vivo antitumor activity in a HCT-116 xenograft mice model.

: Due to the poor permeability across Gram-negative bacterial membranes and the troublesome bacterial efflux mechanism, only a few GyrB/ParE inhibitors with potent activity against Gram-negative pathogens have been reported. Among them, pyrimido[4,5-b]indole derivatives represented by GP-1 demonstrated excellent broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria but were limited by hERG inhibition and poor pharmacokinetics profile. To improve their drug-like properties, we designed a series of novel pyrimido[4,5-b]indole derivatives based on the tricyclic scaffold of GP-1 and the C-7 moiety of acorafloxacin. These efforts have culminated in the discovery of a promising compound 18r with reduced hERG liability and an improved PK profile. Compound 18r exhibited superior broad-spectrum in vitro antibacterial activity compared to GP-1, including a variety of clinical multidrug G– pathogens, especially Acinetobacter baumannii, and the in vivo efficacy was also demonstrated in a neutropenic mouse thigh model of infection with multidrug-resistant A. baumannii.

Based on our recent finding that α1 selective GABA-A receptor potentiator—zolpidem—(a hypnotic drug) exerts antipsychotic-like effects in rats, we developed a series of fluorinated imidazo[1,2-a]pyridine derivatives as potential novel antipsychotic agents. The selected compounds displayed high affinity and positive allosteric modulator properties at the GABA-A receptor, enhanced metabolic stability and lack of hepatotoxicity. The most promising compound 2-(2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)-N,N-dimethylethanamide (26) showed antipsychotic-like activity in amphetamine-induced hyperlocomotion test in rats (MED = 1 mg/kg) and was characterized by a longer duration of antipsychotic-like activity as compared to zolpidem. These results are an encouraging example of a compound with non-dopaminergic mechanism of action displaying antipsychotic activity and are a point of entry for the future studies in this field.

Metastatic castration-resistant prostate cancer (mCRPC) with high mortality has seriously threatened men’s health. Bifunctional agents simultaneously degrade and antagonize androgen receptor (AR), display robust AR signaling pathway blockade, and show the therapeutic prospect for mCRPC. Herein, systemic structural modifications on the C-3, C-6, and C-17 positions of galeterone led to the discovery of 67-b with the dual functions of AR antagonism and degradation. In vitro, 67-b exhibited excellent antiproliferative activity and potent AR degradation activity in different PCa cells (LNCaP and 22RV1), as well as outstanding antagonistic activity against wild-type and mutant (W741L, T877A, and F876L) ARs. In vivo, 67-b effectively inhibited the growth of hormone-sensitive organs in the Hershberger assay and exhibited tumor regression in the enzalutamide-resistant (c4-2b−ENZ) xenograft model. These results confirmed 67-b to be a promising AR degrader and antagonist for the treatment of mCRPC patients.

CDK4/6 pathway is an attractive chemotherapeutic target for antitumor drug discovery and development. Herein, we reported the structure-based design and synthesis of a series of novel tetrahydronaphthyridine analogues as selective CDK4/6 inhibitors. Compound 5 was identified as a hit and then systematically structure optimization study was conducted. These efforts led to compound 28, which exhibited excellent in vitro potencies against CDK4/6 enzymatic activity with high selectivity over CDK1, and against Colo-205 cell growth. The compound demonstrated favorable in vitro metabolic and robust mice pharmacokineticproperties. In Colo-205 xenograft models, compound 28 showed potent tumor growth inhibition with acceptable toxic effects, which could serve as a novel anticancer agent for further preclinical study.

Bruton’s tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC50 = 21.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak suppression against normal LO2, HEK293 and THP-1 cell lines with IC50 values over 30 μM. Moreover, B16 showed very weak potential to block the hERG channel (IC50 = 11.10 μM) in comparison to ibrutinib (IC50 = 0.97 μM). Owing to its favorable physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL), pharmacokinetic profiles (F = 49.15%, t1/2 = 7.02 h) and reasonable CYP450 profile, B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis.

A series of novel berberine derivatives, 4,7,12,12a-tetrahydro-5H-thieno[3′,2’:3,4]pyrido[1,2-b]isoquinolines was designed, synthesized, and biologically evaluated for their anti-diabetic activity. Following the evaluation in two types of cells, compounds 4aa, 4bq, and 4bv stimulated glucose consumption (1.8- to 2.3-fold), reduced gluconeogenesis (60–85%), inhibited mitochondria respiratory chain complex I and activated AMPK indirectly. In a db/db mice model, compounds 4bq and 4bv lowered fasting blood glucose at a dose of 120 mg/kg/day. In addition, compounds 4bq and 4bv were found to possess improved pharmacokinetic profiles (bioavailability 45 and 106%, respectively) compared to berberine. Compounds 4bq and 4bv exhibited no obvious hERG inhibition (IC₅₀ > 10 μM).

Using a rational design strategy for isoform-selective inhibition of PI3Kα, two series of novel 2,3,4,5-tetra-substituted thiophene derivatives containing either diaryl urea or N-Acylarylhydrazone scaffold were designed and synthesized. The most promising compound 12k was demonstrated to bear nanomolar PI3Kα inhibitory potency with 12, 28, 30, 196-fold selectivity against isoforms β, γ, δ and mTOR. Besides, it also showed good developability profiles in cell-based proliferation against a panel of human tumor cells as well as ADME assays. We herein report on their design, synthesis, SAR and potential developability properties.

Using a rational design strategy for isoform-selective inhibition of PI3Kα, two series of novel 2,3,4,5-tetra-substituted thiophene derivatives containing either diaryl urea or N-Acylarylhydrazone scaffold were designed and synthesized. The most promising compound 12k was demonstrated to bear nanomolar PI3Kα inhibitory potency with 12, 28, 30, 196-fold selectivity against isoforms β, γ, δ and mTOR. Besides, it also showed good developability profiles in cell-based proliferation against a panel of human tumor cells as well as ADME assays. We herein report on their design, synthesis, SAR and potential developability properties.

Clear link between DDSA mutations and Developmental Delay
with Sleep Apnea

DDSA mutations are gain-of-function even as ‘heterozygous’
channels

They have a higher open probability caused by a defective Xgating

They are less sensitive to GPCR mediation

DDSA mutations still retain sensitivity to inhibition by clinically
relevant drugs e.g. BAY1000493. Similar drug used in KOALA trial
(NCT04236440

When determining the potencies of drugs in single-cell assays it is a major concern that the employed assay is affected by factors such as the density of the applied cell suspension. It is envisaged that a high density of cells may lead to titration of test compound molecules (by receptor-binding and adsorption) by cells in the vicinity of the targeted cell. This would cause a rightward shift in the concentration-response relationship, and consequently, incorrectly increased IC₅₀ values. We have tested whether IC₅₀ values determined with the QPatch 16 automated patch-clamp system are dependent on the density of the applied cell suspension. We assume that cell density determines the number of cells present in the extracellular flow channel.

It is predicted that increasing the cell suspension density may reduce the time it takes to position a cell on the patch-clamp site. The cell positioning step is accomplished by suction. We tested the effect of the cell suspension density using a wide range of densities (from 0.5 to 16 million cells per ml) on the cell positioning time. For the tests we used two expression systems: (1) CHO cells expressing hERG potassium channels, and (2) HEK cells expressing Na_v1.2a sodium channels. We used test compounds with very different oilwater partition coefficient (log(P)): astemizole and verapamil with log(P) values of 6.43 and 3.45, respectively, (source: www.syrres.com/esc/physdemo) for hERG assays, and tetrodotoxin (TTX) with log(P) of -6.21 for the Na_v1.2a assay.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC₅₀ values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug’s cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [I_Kr], hCav1.2 [L-Type I_Ca], peak hNav1.5, [Peak I_Na], late hNav1.5 [Late I_Na]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC₅₀ variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Background and Purpose Preclinical identification and understanding of drug-induced cardiotoxicity is still a major challenge. The ICH S7B Q&A promote human in silico drug trials for proarrhythmia risk assessment. However, additional evidence is needed to support further regulatory impact and for their integration in the current preclinical assessment pipelines. This study aims to provide a comparative evaluation of drug-induced electrophysiological effects on in silico and in vitro cardiac Purkinje and to assess the accuracy of these models for clinical risk predictions. Experimental Approach The effects of 14 reference compounds were quantified in a population of in silico human cardiac Purkinje models, and compared with results obtained in in vitro rabbit Purkinje preparations. For each drug dose, five electrophysiological biomarkers were quantified at three pacing frequencies, and results compared with clinical proarrhythmia reports. Key Results i) In silico, repolarisation abnormalities in human Purkinje simulations predicted drug-induced arrhythmia for all risky compounds, showing higher predicted accuracy than rabbit experiments; ii) Drug-induced electrophysiological changes observed in human-based simulations showed a high degree of consistency with in vitro rabbit recordings at all pacing frequencies, and depolarisation velocity and action potential duration were the most consistent biomarkers; iii) discrepancies observed for dofetilide, sotalol and terfenadine are mainly caused by species differences between humans and rabbit. Conclusion and Implications In this study, we showed the high degree of consistency and higher accuracy of in silico methods compared to in vitro animal models, demonstrating the high regulatory impact of in silico trials for proarrhythmia prediction.

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.

Background

G protein-coupled receptors (GPCRs) regulate the pathological and physiological functions of the heart. GPCR antagonists are widely used in the treatment of chronic heart failure. Despite therapeutic advances in the treatments for cardiovascular diseases, heart failure is a major clinical health problem, with significant mortality and morbidity. Corticotropin releasing hormone receptor 2 (CRHR2) is highly expressed in cardiomyocytes, and cardiomyocyte-specific deletion of the genes encoding CRHR2 suppresses pressure overload-induced cardiac dysfunction. This suggests that the negative modulation of CRHR2 may prevent the progression of heart failure. However, there are no systemic drugs against CRHR2.

Findings

We developed a novel, oral, small molecule antagonist, of CRHR2, RQ-00490721, to investigate the inhibition of CRHR2 as a potential therapeutic approach for the treatment of heart failure. In vitro, RQ-00490721 decreased CRHR2 agonist-induced 3′, 5′-cyclic adenosine monophosphate (cAMP) production. In vivo, RQ-00490721 showed sufficient oral absorption and better distribution to peripheral organs than to the central nervous system. Oral administration of RQ-00490721 inhibited the CRHR2 agonist-induced phosphorylation of cAMP-response element-binding protein (CREB) in the heart, which regulates a transcription activator involved in heart failure. RQ-00490721 administration was not found to affect basal heart function in mice but protected them from pressure overload-induced cardiac dysfunction.

Interpretation

Our results suggest that RQ-00490721 is a promising agent for use in the treatment of chronic heart failure.

Recent increasing evidence suggests that the currently-used platforms in vitro I_Kr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade.

Tactile information is detected by thermoreceptors and mechanoreceptors in the skin and integrated by the central nervous system to produce the perception of somatosensation. Here we investigate the mechanism by which thermal and mechanical stimuli begin to interact and report that it is achieved by the mechanotransduction apparatus in cutaneous mechanoreceptors. We show that moderate cold potentiates the conversion of mechanical force into excitatory current in all types of mechanoreceptors from mice and tactile-specialist birds. This effect is observed at the level of mechanosensitive Piezo2 channels and can be replicated in heterologous systems using Piezo2 orthologs from different species. The cold sensitivity of Piezo2 is dependent on its blade domains, which render the channel resistant to cold-induced perturbations of the physical properties of the plasma membrane and give rise to a different mechanism of mechanical activation than that of Piezo1. Our data reveal that Piezo2 is an evolutionarily conserved mediator of thermal–tactile integration in cutaneous mechanoreceptors.

The voltage-gated sodium ion channel Na_V1.7 is crucial in pain signalling. We examined how auxiliary β2 and β3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human Na_V1.7α subunit was co-expressed with either β2 or β3 subunits in HEK-293 cells. The β2 subunits and the Na_V1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the β2 subunits did not change the biophysical properties of the channel. In contrast, β3 subunit was clearly associated with Na_V1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated β3 subunit was associated with the Na_V1.7α. Electrophysiological characterisation revealed that the β3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the β3 subunit reduced the susceptibility of Na_V1.7α to several sodium channel blockers. In addition, we assessed the functional effect of Na_V1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of β subunits with Na_V1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with β2. The β3 subunit significantly influences the function of Na_V1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of Na_V1.7 channel in pain signalling.

We have prepared three alkaloids from the Agelas sponges, clathrodin, hymenidin and oroidin, and a series of their synthetic analogues, and evaluated their inhibitory effect against six isoforms of the K_v1 subfamily of voltage-gated potassium channels, K_v1.1-K_v1.6, expressed in Chinese Hamster ovary (CHO) cells using automated patch clamp electrophysiology assay. The most potent inhibitor was the (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (6g) with IC₅₀ values between 1.4 and 6.1 μM against K_v1.3, K_v1.4, K_v1.5 and K_v1.6 channels. All compounds tested displayed selectivity against K_v1.1 and K_v1.2 channels. For confirmation of their activity and selectivity, compounds were additionally evaluated in the second independent system against K_v1.1-K_v1.6 and K_v10.1 channels expressed in Xenopus laevis oocytes under voltage clamp conditions where IC₅₀ values against K_v1.3-K_v1.6 channels for the most active analogues (e.g. 6g) were lower than 1 μM. Because of the observed low sub-micromolar IC₅₀ values and fairly low molecular weights, the prepared compounds represent good starting points for further optimisation towards more potent and selective voltage-gated potassium channel inhibitors.

Cardiac safety side-effects remain the leading cause of new compound attrition during the drug discovery process (Valentin and Redfern, 2017), suggesting that more robust preclinical in vitro, ex vivo and in vivo assays and models are required to predict human clinical risk. Currently, the industry is moving away from an over-reliance on the human Ether-a-go-go related potassium channel (hERG, KV11.1) assay and QT prolongation readouts by developing new initiatives that provide a more balanced assessment of patient risk, focusing in particular on cardiac arrhythmia liability. The FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative and Japanese regulatory authorities (JiCSA, CSAHi) aim to more accurately model and predict arrhythmia risk by including additional human cardiac ion channels in patch clamp electrophysiology screening panels. This broader dataset is then used in sophisticated in silico models of the human ventricular action potential to predict arrhythmias. After 5 years of extensive collaborative effort by working groups in the UK, EU, US and Japan, it has become apparent that incorporating data obtained from the ‘big 6’ panel of cardiac ion channel assays (hERG, Na_V1.5, Ca_V1.2, K_VLQT1, K_V4.3, and K_ir2.1) into standard in silico action potential models allows the accurate prediction of proarrhythmic liability of most, but not all, clinical drugs with well characterised risk profiles.

The voltage-dependent sodium channel, Na_V1.5, was tested on the QPatch in single-hole and multi-hole mode. In this study, we wanted to determine the best test protocol to distinguish between two antiarrhythmic drugs with different modes of action.

The sodium ion channel Na_v1.5 is expressed as in integral membrane protein and contains a tetrodotoxin-resistant voltage-gated sodium channel subunit.The encoded protein is found primarily in cardiac muscle and is responsible for the initial upstroke of the action potential in an electrocardiogram. Mutations in the gene are associated with long QT syndrome type 3, Brugada syndrome, primary cardiac conduction disease and idiopathic ventricular fibrillation.

The slowly activating, delayed rectifier K+ channel is among other activities important for regulating the repolarization phase of cardiac action potentials. The KvLQT1/minK channel consists of two transmembrane proteins. Mutations in the genes KCNQ1/KCNE1 coding for KvLQT1/minK are associated with predisposition to deafness, cardiac arrhythmia syndromes including long QT syndrome, atrial fibrillation and sudden infant death syndrome.

The voltage-gated potassium channel K_V1.5 is a homotetrameric protein present in the heart. It is a delayed rectifier, participating in the early phase of the heart action potential. This report shows data from CHO cells stably expressing K_V1.5 tested on the QPatch platform.

Current clamp recordings provide more physiologically relevant measurements of ion channel activity (in comparison to voltage clamp), allowing the contribution of different ion channel subtypes to resting membrane potential to be evaluated.

Here we demonstrate that:

• Due to their mode of action, some compounds display different
potencies in voltage clamp and current clamp.
• Qube 384 enables drug screening in current clamp mode,
here demonstrated in a cell line stably expressing the KV1.3
ion channel (success rate out of 384 wells of up to 65%).
• Compounds blocking the KV1.3 channel are evaluated for
their ability to depolarize the cell membrane.

Na_V1.7 has moved in the focus of the drug discovery industry in the last years as numerous studies provided strong proof for the efficacy to alleviate both neuropathic and inflammatory pain (Sun, Cohen, & Dehnhardt, 2014). We demonstrate high reproducibility and precision of Na_V1.7 data recorded on Qube

95% of all tested cells had a GΩ seal in the beginning and
61% at the end of a long, 70 min experiment

V_1/2 is stable over time with only a slight shift

Experiments can be performed in physiological ringers,
however fluoride in the internal solution significantly
increases success rate without any altering biophysical
parameters.

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

In this report the applications of the CHO TREx-P2X₃ cell line (hereafter called CHO P2X₃) are described. The purpose was to determine the EC₅₀ value for ɑβ-methyl ATP, and the IC₅₀ value of PPADS on the P2X₃ receptors. All data presented were obtained with QPatch and analyzed with the QPatch assay software.

CHO-TRPM8 and HEK-hTRPM8 cells were tested on QPatch which both provided high success rates, high-quality gigaseals and good recordings of the ion channel current. The EC₅₀ and IC₅₀ estimations for menthol, icilin and capsazepine were in agreement with reported literature values.

The risk of fatal arrhythmias is the major concern for using chloroquine (CQ) or hydroxychloroquine (HCQ) to treat coronavirus disease 2019 (COVID‐19), but the reported number of life‐threatening arrhythmic events or deaths is relatively small. The objective of this study was to assess the arrhythmogenic risk of these two drugs using a multiscale heart simulation, which allows testing even at high concentrations, including those that cause fatal arrhythmias. We measured the inhibitory action of CQ, HCQ, and HCQ with 30 μM azithromycin (AZ) on six ion currents (fast [INa] and late [INa,L] components of the sodium current, L‐type calcium current [ICa,L], rapid [IKr/hERG], and slow [IKs] components of delayed rectifier potassium, and inward rectifier potassium [IK1]) over a wide range of concentrations using the automated patch‐clamp system. Using the concentration–inhibition relationship that was thus obtained, we simulated the drug effects while increasing the concentration until the life‐threatening arrhythmia, Torsade de Pointes (TdP), was observed. The obtained threshold concentrations for TdP were 12.5, 35, and 22.5 μM for CQ, HCQ, and HCQ with AZ, respectively. Adding therapeutic concentrations of mexiletine or verapamil successfully prevented the occurrence of TdP, and verapamil was more effective. CQ, HCQ, and HCQ with AZ thresholds for TdP were larger than both antiviral concentrations that were reported by in vitro experiments and free plasma concentrations that were attained by the clinically used dosage. The current simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ.

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of K_v2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks K_v2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with K_v2.1 clusters in live cells and are released from channels activated by voltage stimuli. K_v2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing K_v2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.