Review written by the Editor
Ion channels continue to rise in prominence in both academic and commercial areas. Metrion Biosciences, alongside Sophion Bioscience, Nanion Technologies, Evotec and SB Drug Discovery sponsored the “Ion Channels in Drug Discovery XIX” Satellite meeting held on Friday 1st March at the Baltimore Convention Centre, as part of the 63rd annual meeting of the Biophysical Society (BPS).
The meeting was attended by around 100 delegates from a variety of backgrounds, including academia, pharma, small to mid-sized biotech companies and the service industry.
Keynote speaker Professor William Colmers
After a brief welcome and opening remarks from Niels Fertig, CSO of Nanion Technologies who highlighted the merits of Automated Patch Clamp (APC) and the growing number of publications centred around APC data, David Dalrymple of SB Drug Discovery introduced the keynote speaker. This was Professor William Colmers from the University of Alberta, who focused his presentation on the role of NeuroPeptide Y (NPY) and Ih HCN1-mediated currents play in stress modulation across different regions of the brain. NPY may underlie behavioural stress resilience and long-term structural changes in neurons. Work continues into the biological role of NPY with regards to energy balance, obesity, anxiety and cachexia.
Use of APC platforms to support ion channel discovery
Dr Stephen Hess then presented a three-part overview of Evotec’s use of APC platforms to support ion channel drug discovery. Stephen first described a hit profiling study of voltage-dependent Kv1.3 allosteric modulators, followed by a study of the on-off rates of Nav inhibitors using a complex assay protocol they perfected.
Stephen finished by highlighting the emerging power of CryoElectron Microscopy (Cryo EM) as a tool for structure-based ion channel studies and drug discovery. In less than five years, at least one structure from each of the 7 TRP families have been determined, and in total around 117 human ion channel structures have now been elucidated.
The mechanism-of-action and liabilities of local anaesthetics
The next speaker was James Ellis from Nocion Therapeutics, a pre-clinical stage biotech company developing novel small molecules to selectively silence nociceptors involved in cough, itch, pruritis, inflammation and pain responses. Jim spoke about the mechanism-of-action and liabilities of local anaesthetics such as Lidocaine and explained that an ideal Nav channel blocker should maintain efficacy across indications, be administered topically to minimise systemic exposure and CNS redistribution, be cell impermeant and devoid of painful or irritating TRPA1/ TRPV1 agonism.
Nocion are developing a strategy originally described by their Harvard co-founders whereby charged Nav antagonists can selectively enter over-active neurons through the large pores of ligand-gated pain receptors (TRPx, P2X, ASIC) activated by exogenous agonists or endogenous ligands and inflammatory mediators.
Tianbo Li then presented an overview of Genentech’s work on Nav1.7, one of the best validated and characterized pain targets. A key challenge for Genentech is devising new chemical matter with high potency and selectivity, and here he presented a case study on Pro-Toxin II (ProTx-II), a tarantula cysteine knot peptide toxin. Through a combination of Alanine mutant scanning of a Nav1.7-NavAb chimera, charge alterations across the binding face of ProTx II and determination of the cryoEM structure of ProTx-II bound to the channel chimera, it was shown that ProTx-II binds electrostatically to specific residues in the S3-S4 linker of voltage sensor domain II (VSD2). This information was used to create higher affinity analogues of ProTx-II and another spider venom, demonstrating the potential to aid future Nav channel antagonist design.
The rationale for a new type of ion channel screening platform
Hongkang Zhang spoke on behalf of Qwell Therapeutics, a spin-out from Q-State Biosciences who work on non-opioid treatments for pain. Hongkang outlined the rationale for a new type of ion channel screening platform, comparing the costs, temporal resolution, mechanistic detail and throughput available from conventional plate-based readers (e.g. FLIPR) and APC machines.
Qwell Tx are developing the single site Optopatch optogenetic platform from Q-State into a higher throughput 24 well device suitable for target-based screening of ion channels. Using Nav1.7 channels, they have preliminary data to support claims for fast, sensitive, linear read-outs of ion channel activity that combine the advantages of plate-based readers and APC platforms.
In search of a suite of GABA-A receptor cell lines and validated APC screening assays
GABAA receptors are a complex ligand-gated ion channel family with at least 16 subunits. They are important drug targets for anxiety and epilepsy and the next speaker, David Dalrymple, highlighted SB Drug Discovery’s efforts to create a suite of GABAA receptor cell lines and validated APC screening assays. SB generated 19 human GABAA receptor cell lines of various α1-α6, β1-3, γ and δ subunit combinations which they validated pharmacologically on the Syncropatch384 using stacked tip protocols. Several positive allosteric modulators were found after a plate-based screen of SB’s compound libraries, most of which were confirmed by APC electrophysiology.
The study of gating currents
Professor Francesco ‘Pancho’ Bezanilla was the afternoon keynote speaker, providing an overview of his recent work on genetically-encoded voltage indicators and optocapacitance techniques to probe the structure and function of voltage- dependent ion channels and transporters. The study of gating currents requires new types of fast voltage indicators, and Pancho described their work with ASAP-1, an ADP-ribosylation factor GTPase-activating protein from chicken.
Pancho then explained optocapacitance, whereby infrared light can depolarise biological membranes and excite muscle cells, Xenopusoocytes and neurons. This local heating and activation can be focused spatially and in terms of tissue-penetrating wavelengths through application of gold nanoparticles, graphite, and carbon nanotubes to cells, and even directed to sub-cellular sites and specific receptors and channels through conjugation to toxins (e.g. Ts1 toxin for Nav) and antibodies (P2X, TRPx), enabling exquisite optical control of cellular excitability.
Drug discovery collaboration update
Marc Rogers, CSO of Metrion Biosciences, then presented an overview of an 8-year drug discovery collaboration with a global pharma partner, for which Metrion provided in vitro and ex vivo screening services using their manual patch and automated patch clamp expertise. During the collaboration they developed high-quality voltage-gated ion channel assays to reliably identify and profile novel, potent, safe and efficacious state-dependent modulators of a pain-related ion channel target. This yielded a development lead compound and a back-up series with therapeutic potential equal or superior to current clinical treatments.
Knottin-antibody fusion proteins (KnotBodies)
Iontas specialise in mammalian phage display and antibody discovery and affinity maturation. Aneesh Karatt-Vellatt summarised their work on Knottin-antibody fusion proteins (KnotBodies), a novel antibody format which enables the targeting of ‘difficult’ drug discovery proteins such as ion channels and GPCRs by combining antibody light chains with ICK toxins and other small peptides to achieve increased specificity and half-life. Iontas are using Nav1.7, ASIC1a and Kv1.3 channels as case studies, and Aneesh gave an overview of Kv1.3 as a target for auto-immune disease, which affects 2-3% of the worlds’ population and may have a market value around $45.5 Bn by 2022.
As the sea anemone toxin analogue ShK-186 recently showed efficacy in a clinical trial on psoriasis, Iontas incorporated several Kv1.3-targeting toxins into their knotbody template, yielding potent and selective Kv1.3 blockers capable of reducing T-cell cytokine release.
Sam Goodchild then presented an overview of Xenon Pharmaceuticals’ program to develop Nav1.6 modulators to treat epilepsy and rare forms of encephalopathy such as IEEE13. The Nav inhibitors currently used have a low therapeutic index and are poorly selective, often being dosed at concentrations that can cause adverse side effects.
Based upon their extensive work on aryl sulphonamide gating modifiers of Nav1.7 (in collaboration with Genentech), Sam described their precision medicine approach which identified two novel compounds that selectively target Nav1.6 (XPC-224) and Nav1.6/1.2 (XPC-462) through a greater than 1000- fold preference for the inactivated state via binding to specific residues in VSD-IV. The compounds are efficacious in mouse seizure models at 3x in vitro IC50 and exhibit 100-fold TI over acute toxicity effects. Thus, these compounds promise to provide superior efficacy and side-effect profile to current anti-convulsant medications such as phenytoin and carbamazepine.
Developing higher throughput mechanistic and translational assays for CNS drug discovery
The final speaker was Fern Toh from Alkermes, a global pharmaceutical company working primarily on CNS disorders including MS, schizophrenia, depression and addiction. They are interested in developing higher throughput mechanistic and translation assays for CNS drug discovery. Fern outlined ongoing work to correlate molecular profiling and plate-based multi-electrode array data from cultured rodent cortical and hypothalamic neurons with functional recordings of ion channels and receptors on an APC platform. Alkermes hope to build upon these studies to look at GPCR modulators and human stem-cell derived neurons to aid the translation of screening results to CNS drug candidates.