Automated Patch Clamp and Large Molecules

While automated patch clamp (APC) systems have been used for small molecule drug discovery and characterization for the past 20 years, the use of APC systems for large molecule characterization has lagged.

However, large molecules – and hence their characterization by APC – are becoming increasingly important in drug discovery. Currently, more than 90% of approved drugs are small molecules, but large molecules (>1 kDa, also known as biologics) are rapidly rising in prominence and importance in drug discovery and already constitute the lion’s share of the top 10 selling drugs worldwide.

Classes of large molecules

• Venom, toxins and peptides (~3-4 kDa, 30-40 amino acids)
• Wnt peptides (~35-45 kDa, 350-400 amino acids)
• Knotbodies and nanobodies (~50-60 kDa, 400-500 amino acids)
• Antibodies (~150-170 kDa, ~1400 amino acids)

Venom, toxins and peptides

Animal and plant venoms have evolved as potent ion channel modulators to interfere with critical roles in physiological pathways e.g. action potential initiation and propagation. Several toxins are highly potent and specific to individual ion channels. They have been widely used tools in research to elucidate structure and function of ion channels. Increasingly, toxins have been used in the development of novel therapeutics as peptide frameworks for new bioactive molecules or targeting scaffolds for drug conjugates.

If you would like to learn more about using automated patch clamp for venom and peptide characterization take a look at the plethora of publications from the University of Queensland using QPatch. Also, you can watch the Sophion webinar on a venom toxin library screen using Qube 384.

See below list of selected relevant publications or search for more publications here.

Wnt peptides

Wingless-related integration site (Wnt) comprises a diverse family of secreted signaling glycoproteins that are 350–400 amino acids in length and act as close-range signaling molecules. Wnt signal activation initiates a complex downstream signal cascade in eukaryotic cells and is critical in the development of many diseases, including cancer. Wnt peptides activate K⁺ currents by elevating intracellular Ca²⁺ and trigger Ca²⁺ release from intracellular stores. Wnt peptides have significant implications for gene transcription and open novel avenues to modulate this critical pathway.

If you would like to learn more about using automated patch clamp for Wnt characterization, a great place to start is to watch the Sophion webinar on ion channels in oncology together with Kings College London. Also, check out our application report on large molecules and Wnt signal activation, the poster on Wnt peptides control mammalian cancer cell membrane potential or Wnts control membrane potential in mammalian cancer cells.

See below list of selected relevant publications or search for more publications here.

Knotbodies and nanobodies

Knotted peptides present a wealth of structurally diverse, biologically active molecules. The inhibitor cystine knot/knottin class is 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.

If you would like to learn more about using automated patch clamp for knotbodies and nanobodies check out the posters from IONTAS on Generating ion channel blocking antibodies by fusing knottin to peripheral CDR loops and Generating potent and selective inhibitors of Kv1.3 ion channels by fusing venom derived mini proteins into peripheral CDR loops of antibodies.

See below list of selected relevant publications or search for more publications here.

Antibodies

Antibodies display high specificity, selectivity, and affinity for their target antigen, thereby having the potential to target ion channels very precisely. Nonetheless, isolating antibodies to ion channels is challenging. This is due to the difficulties in the expression and purification of ion channels in a format suitable for antibody drug discovery and due to the complexities of screening for function.

If you would like to learn more about using automated patch clamp for antibody characterization, please give us a call. We can share tips and tricks that will ensure you accurate results and good success rates. You can also read the publications on In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom, Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies or Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies.

See below list of selected relevant publications or search for more publications here.

Selected materials on APC and large molecules

Presentations

• Webinar: APC and large molecules – A Venomtech/Charles River Laboratories venom toxin library screen

Application Reports

• High throughput electrophysiological screen of antibody-based antivenom on Qube 384
• Large molecules: Scorpion toxin block of BK channels on Qube 384
• Large molecules on QPatch: IgG antibodies against α-cobratoxin as novel antivenom
• Large Molecules: Wnt signal activation

Posters

• Automated Patch Clamp Evaluation of Snake Neurotoxins and Recombinant Antibody Antivenoms. Boddum, Ledsgaard, Busk, Bell and Laustsen. Poster. Gordon Research Conference, Lucca, Italy. 2022
• High throughput electrophysiological screen of large molecules: evaluating snake neurotoxins and the neutralization potential of recombinant antibodies. Boddum, Ledsgaard, Busk, Bell and Laustsen. Virtual Poster. Society for Neuroscience 2021
• Toxin pharmacology and MoA data against Nav 1.X channels: Multi-parameter analysis using Sophion Qube high-throughput automated patch clamp platform. Duncan, Williams & Kammonen (Charles River Laboratories). Poster. Society for Neuroscience 2021
• Wnt peptides control mammalian cancer cell membrane potential. Ashmore, Olsen, Sørensen, Thrasivoulou, Ahmed (Kings College London, University College London, Sophion Bioscience). Poster. 2019

Selected Publications

– Find abstracts & links in our publication database

Aqwa et al., 2018 Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7.

Ashmore et al., 2019 Wnts control membrane potential in mammalian cancer cells.

Agwa et al., 2020 Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.

Chow et al., 2019 Venom Peptides with Dual Modulatory Activity on the Voltage-Gated Sodium Channel NaV1.1 Provide Novel Leads for Development of Antiepileptic Drugs.

Colley et al., 2018 Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies.

Deuis et al., 2017 Pharmacological characterisation of the highly Na v 1.7 selective spider venom peptide Pn3a.

Gonçalves et al., 2019 From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei.

Gonçalves et al., 2018 Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches.

Inserra et al., 2017 Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1.

Israel et al., 2018 The E15R Point Mutation in Scorpion Toxin Cn2 Uncouples Its Depressant and Excitatory Activities on Human NaV1.6.

Jin et al., 2015 δ-conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails.

Klint et al., 2015 Rational engineering defines a molecular switch that is essential for activity of spider-venom peptides against the analgesics target NaV1.7.

Ledsgaard et al., 2021 In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom.

Petrou et al., 2017 Intracellular calcium mobilization in response to ion channel regulators via a calcium-induced calcium release mechanism.

Prashanth et al., 2017 Pharmacological screening technologies for venom peptide discovery.

Robinson et al., 2018 A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene family.

Schwalen et al., 2021 Scalable Biosynthetic Production of Knotted Peptides Enables ADME and Thermodynamic Folding Studies.

Shcherbatko et al., 2016 Modulation of P2X3 and P2X2/3 receptors by monoclonal antibodies.

Shcherbatko et al., 2016 Engineering highly potent and selective microproteins against Nav1.7 sodium channel for treatment of pain. J Biol Chem. 2016;291(27):13974–86.

Sousa et al. 2017 Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi.

Tran et al., 2020 Enzymatic Ligation of a Pore Blocker Toxin and a Gating Modifier Toxin: Creating Double-Knotted Peptides with Improved Sodium Channel NaV1.7 Inhibition.

Yang et al., 2016 The snake with the scorpion’s sting: Novel three-finger toxin sodium channel activators from the venom of the long-glanded blue coral snake (calliophis bivirgatus).