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.

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.

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.

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.

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.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into multiple cell types, including neurons and cardiomyocytes. This gives rise to a novel way of establishing human disease models, which in turn can be used for drug development in vitro. Ion channels represent 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 labour-intensive and low-throughput nature of patch-clamp electrophysiology. Here we use our automated patch clamp systems Qube 384 and QPatch 48 in order to increase throughput and reduce timelines.

Our observations include channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. Na_v and K_v), identification of ligand-gated receptors, and recordings of action potentials using the current clamp feature. Also, we show the electrophysiology of a spinal muscular atrophy (SMA) and an amyotrophic lateral sclerosis (ALS) model. The disease model for SMA was derived by mutations in the SMN1 gene and shows enhanced sodium channel activity but no shift in the normalized current-voltage relationship. ALS was here mimicked by a single point mutation in the superoxide dismutase 1 protein (SOD1), D90A, which had previously been identified in recessive, dominant and seemingly sporadic pedigrees. Cells carrying this point mutation displayed larger sodium currents, which eventually led to neurofilament aggregation, neurite degeneration and other phenotypes. We could confirm that the electrophysiological effect could be reversed by point mutation to D90D.

Our measurements validate the feasibility of measuring hiPSC ion channel currents using the APC platforms Qube and QPatch. Altogether, these results can facilitate evaluating the use of hiPSC for early drug development and in extension personal medicine.

Voltage-gated sodium channels (VGSC) are in the spotlight of drug development as strong evidence is available linking different subtypes to various disease states.

VGSC is responsible for the initiation and propagation of action potentials in excitable cells. During this process, the VGSC transitions from a closed to an open into an inactivated state. Interestingly, inhibitory compounds often exhibit different pharmacological profiles dependent upon the conformational state of the ion channel.

In the present work, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX), amitriptyline and tetracaine on 8 different VGSC subtypes (NaV1.1-8). A first step was to determine the half-inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce the heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

Voltage-gated sodium (Na_V) channels are studied extensively due to their potential as targets for several indications, such as pain, epilepsy, cardiac and muscle paralysis. Some Na_V channel modulators show state-dependence and bind preferentially to the inactivated state of the channel. The potency of state-dependent compounds is known to vary depending on the percentage inactivation of the channels.

To calculate accurate compound activity the precise value for the VHalf of inactivation should be used for each cell. The adaptive protocol block for the Sophion Qube 384-well automated patch clamp platform has made it possible to separately define the voltage applied to individual wells for both the activation and inactivation of the channels. This enables the generation of more precise data for voltage-gated ion channels.

Kv4.3 α-subunits associate with ancillary β-subunits of the KChIP2 family in heart muscular tissue to channel transient outward (Ito) currents. Decreased expression or dysfunction of Kv4.3 channels following myocardial infarction or during heart failure can contribute to abnormal repolarization, which may result in ventricular arrhythmias. Therefore, drug-induced inhibition of Kv4.3/KChIP2-mediated Ito exposes to potential cardio safety liabilities which are important to document early during the drug discovery process. In this study, we have characterized basic electrophysiological properties pertaining to Ito currents in CHO cells expressing recombinant Kv4.3 and KChIP2.2 subunits. The currents obtained were validated pharmacologically by assessing the efficacy of small molecule and peptide toxin inhibitors known from manual patch-clamp studies to block Ito. We then examined the inhibitory activities of 28 drugs with clinically documented high, medium or low pro-arrhythmic risk encompassing the test- and validation-sets of the Comprehensive in vitro Pro-Arrhythmia (CiPA) panel. While none of the CiPA drugs inhibited Kv4.3 at submicromolar concentrations (IC50 < 1 µM), two drugs (loratadine and nitrendipine) displayed intermediate potency (1 µM < IC50 < 10 µM), and four drugs (bepridil, quinidine, chlorpromazine and astemizole) revealed weak, but significant (10 µM < IC50 < 30 µM) inhibitors.