Written by Marc Rogers PhD and Sophie Rose PhD

A global pandemic wasn’t going to stand in our way

Nothing was going to prevent this annual gathering of ion channel enthusiasts and drug discovery professionals from taking place. Planning got underway in late 2020 to facilitate the virtual organisation and smooth running of the event. The event is usually held as a Satellite meeting at the Biophysical Society Annual conference, but could not be hosted virtually by BPS this year. Past sponsors Nanion, Sophion, Fluxion, SB Drug Discovery and Metrion Biosciences stepped in to prepare a schedule of ion channel experts from across the globe to present their recent work.

This year’s meeting brought together both academic and industry speakers from three different continents to talk about GABA(A) receptors and disease, potassium channel openers, neurotoxic venom peptides, with a keynote talk on viroporins that included recent data on drug repurposing against the SARS-CoV2 envelope (E) protein channel. To try and capture as wide an audience as possible, the meeting was held at 8am PST, 11am EST which captured audiences from both East and West Coast USA, the UK and Europe.

Hosting a virtual meeting with improved networking opportunities

Hopin was chosen as the meeting platform due to its enhanced networking capabilities compared to standard webinars we have all watched during lockdown. As well as an online text chat option for questions to be posed by attendees for the Speakers, visible online discussions could take place around each talk and session, and presentations were also interspersed with breakout sessions focusing on various areas of ion channel drug discovery which also promoted some enthusiastic discussions. A highlight for some of the attendees scattered over the globe was a video chat ‘roulette’, which allowed random pairings of attendees. The meeting was opened with a warm welcome from Marc Rogers (Metrion), followed by an introduction to the Hopin platform by Alexandra Stevens (Fluxion) and moderation of the event by Jason Villagomez (Nanion).

Keynote talk – An insight into viroporin virus ion channels as potential drug targets 

The Keynote talk was presented by Dr. Stephen Griffin (SG) from the University of Leeds who highlighted his wide-ranging work on viroporins, a group of small, multi-functional transmembrane proteins in disease-causing viruses which display ion channel like activity. SG introduced viewers to key viroporins including Hepatitis C p7, Zika M and influenza virus M2 proteins, and used both published work and unpublished data to illustrate how structure-function based rational development of inhibitors can be used to develop potent antivirals, including recent work to identify novel, drug-like modulators of the SARS Cov2 E3 envelope ion channel protein.

Development of antivirals has traditionally focused on disrupting DNA-RNA synthesis and replication (e.g. remdesivir), or as we are now all acutely aware, designing vaccines and antibodies to reduce virus infection (e.g. for HPV as well as SARS CoV2 coronavirus). Although viroporins are key to virus cell entry, vesicle trafficking and replication, they are under-exploited antiviral drug targets as little is definitively known about their structure. This is due largely to troublesome expression, and many of these putative ion channel proteins have multiple redundant functions, and reference ligands are promiscuous and non-selective, all of which can confound mutagenic studies and structure-activity relationship (SAR) screening. Several viroporins such as the influenza M2 and hepatitis p7 protein may first act as a pore rather than as channels, preventing the acidification of endosomal compartments that would prevent virus trafficking and release. However, there is mounting electrophysiological evidence that many viroporins exhibit the selectivity (permeation) and biophysical (gating) features of ion channels, and combinations of crystallographic data, NMR structures and molecular dynamic (MD) modelling are now revealing multiple binding sites and mechanisms amenable to further viroporin drug development.

The role of p7 viroporin in Hepatitis C function and drug development

SG started working on Hepatitis C in 2001 with a focus on p7 as a possible novel drug target as this viroporin acts during assembly, envelopment and secretion of viral particles. His group developed crystallographic and NMR structures and applied MD modelling to identify the binding sites of non-selective ligands such as rimantadine and amantadine, which were confirmed in functional liposome dye release assays. This binding site model was used in a virtual HTS to identify a diverse set of small molecules with improved potency and efficacy against viral particle release. SAR around the hit series has led to the development of the lead compound, JK3/32, which has similar potency to Sufosbuvir, a licenced antiviral to treat Hepatitis C. Significantly, this novel ligand pharmacophore is unaffected by mutations reducing rimantadine binding, raising the prospect of a new drug class less prone to developing drug resistance. SG also used their selective and potent lead compound to reveal a role for p7 viroporin in viral cell entry, suggesting that such small molecules could be used as prophylactics to prevent viral infection, which is especially important in patients unable to receive vaccinations.

Developing a synergistic approach to the treatment of Influenza M2

The next case study focused on the Matrix-2 (M2) protein, a viroporin located in the viral envelope of the Influenza A virus which causes seasonal ‘flu infections as well as the Spanish Flu pandemic 100 years ago. M2 was one of the first viroporins discovered and harbours mutations making it resistant to amantadine and rimantadine. Structure-based drug development against M2 was hampered by conflicting x-ray and NMR viroporin structures, but SG’s group sort to exploit this by looking for combination therapies that might mitigate evolution of resistance and reduce antiviral drug dosages. They generated two different families of M2 inhibitors, one targeting the viroporin lumen and the second targeting the peripheral channel binding site in the extended conductance domain, with selective binding modes and synergistic antiviral effects in cell culture against pandemic influenza virus strains. The novel compounds also showed favourable resistance profiles compared to adamantine derivatives.

Does the Zika Virus small membrane (M) protein form a Viroporin?

Zika virus is a recent mosquito-bourne disease vector which can cause neonatal microcephaly. The small envelope protein M forms a non-conducting dimer complex with the E envelope protein. However, this is known to dissociate in acidic endosomes to form a homomultimer which displays viroporin activity in liposomal assays, is pH activated and inhibited by rimantadine, as-is Zika virus cell entry and in vivo infection.  Molecular modelling with the zika M protein viroporin structure has identified known and novel ligands that bind to luminal and peripheral pockets with nM potency and inhibit Zika virus replication in culture.

Drug repurposing targeting the SARS-CoV2 envelope (E) protein viroporin 

Covid-19 is the most pronounced virological event in recent history and has had a significant impact on the entire world. As viroporins are varied with little homology, drug discovery efforts against each virus must rely on functional screening and reliable protein structures for molecular modelling, all of which kicked-off in early 2020 at the start of the coronavirus pandemic. SG explained that the SARS CoV2 virus may possess three different viroporins, but the most important pathogen is the E3 envelope protein. This has some similarities to related SAR and MERS pandemic coronaviruses envelope proteins in terms of function during viral entry, assembly and egress.

Before the release of verified E3 viroporin structures in mid-late 2020, suitable for MD modelling and virtual HTS, SG’s group used their liposome dye release assay to screen a library of FDA approved drugs for potential hits. Although amantadine and rimantadine were identified, their potencies against SARS-CoV2 (200 mM) are even lower than against other viroporins. In contrast, the repurposing screen identified a number of more efficacious inhibitors of viroporin activity at 400 nM, with diverse SAR. A recent membrane lipid structure of the E3 viroporin shows consistent virtual binding of many of the repurposed hits in a luminal pocket at the top of the tetrameric ion channel complex. This is in addition to showing activity in cell-based virological assays, paving the way for additional structure-based drug design and potential clinical development of small molecule antivirals for covid-19.

David Baez-Nieto – High-throughput automated patch clamp analysis of disease-related Ca_v3.3 channelopathies

The important role of Ca_v3.x channels

Dr Baez-Nieto, from the Stanley Center for Psychiatric Research at the Broad Institute in Boston presented a talk detailing the biophysical analysis of rare mutations in the neuronal CACNA1I gene that are associated with schizophrenia. So-called T-type or low voltage activated Ca_v3.x channels play important roles in sub-threshold oscillations and excitability in the peripheral and central nervous system, and through this function and disease-associated mutations they are viewed as validated drug discovery targets for pain and epilepsy.

Work from the Stanley Centre and others has now implicated the Ca_v3.3 subtype in schizophrenia, based on GWAS studies and patient genotyping that have revealed ~60 ultra-rare missense variants. The challenge is to determine what effect each mutation may have on Ca_v3.3 channel function, and how this may relate to disease risk, occurrence and severity. Each rare mutation was expressed heterologously and the Syncropatch384 automated patch clamp (APC) platform was used to determine the subtle biophysical differences between each Ca_v3.3 mutant, such as voltage-dependence and recovery from inactivation. Meaningful differences were found based on a large number of recordings made from many cells in parallel to allow for sophisticated data analysis and statistical comparison.

Significantly, common schizophrenia variants were well tolerated and generally did not affect Ca_v3.3 biophysics or expression. Unexpectedly, several of the ‘unaffected’ patient mutations reduced Ca_v3.3 current expression but had little effect on channel biophysics, suggesting that function was maintained by those channels reaching the plasma membrane of thalamic neurons in vivo. Finally, there were a range of subtle differences in expression as well as activation and inactivation biophysics for many of the rare schizophrenia-associated mutations. These differences were visualised with ‘spider web’ or radar plots, which revealed no significant changes in recovery from inactivation for any mutant group.

Studying schizophrenia mutant Ca_v3.3 biophysics 

In collaboration with Diane Lipscombe at Brown University, schizophrenia mutant Ca_v3.3 biophysics were added to a computational model of thalamic neurons to predict functional effects on firing. As expected, common mutations did not affect thalamic firing, but altered Ca_v3.3 activation seen in rare schizophrenia mutants changed action potential burst firing latency, threshold and spike output. This revealed groups of unaffected and rare disease mutations that were either gain or loss-of-function in terms of thalamic firing; the latter mutations may be protective in relation to schizophrenia symptoms.

Such functional and integrative multi-parameter data is critical for the correct annotation of rare channelopathies, and improved pharmacogenomic treatment of individual patients. This talk reveals the utility of high throughput gigaseal APC platforms as ‘biophysical machines’ for disease-related channelopathy investigations, alongside their proven role as ‘compound screening’ machines for ion channel drug discovery and safety pharmacology profiling.

John Atack – GABAA Receptor Modulators, The Story Continues

GABA-A receptors and modulators

Prof. Atack is Director of the Medicines Discovery Institute at Cardiff University in the UK and has worked in the field of ligand-gated GABA-A receptors for many years. He started his talk with an overview of the structure-function of GABA-A receptors, noting the various key heteromeric isoforms implicated in various disease states and human physiology such as anxiety, epilepsy, anaesthesia, and their modulatory ligands such as barbiturate activators and benzodiazepine (BZP) and neurosteroid positive allosteric modulators (PAM). He highlighted several recent FDA approvals for GABA-A modulators, notably the neurosteroid allopregnanolone PAM Brexanolone for post-partum depression (Sage Therapeutics), and the BZP sedative anaesthetic Remimazolam (Acacia). Although BZPs can be very effective anxiolytics and muscle relaxants, chronic use for long-term disease is problematic owing to their sedatory side-effects and development of tolerance and addiction.

Medium and high throughput screening of small molecules

Development of non-sedating BZPs has been facilitated by recent cryoEM structures of mammalian GABA-A receptors comprising the relevant a (1, 2, 3, 5), b3 and g2 subunits. Thus, new BZPs with reduced sedation should be designed to be less active at a1-containing receptor heteromers that are widely distributed across the CNS, whilst anxiolytic and cognition-enhancing efficacy of BZP ligands can be achieved through selective PAM of a2/a3 and a5 subunit-containing heteromers, respectively. Interestingly, whilst some groups have attempted to create scaffolds with selective binding affinity for certain GABA-A receptor subtypes, more success was seen using a selective efficacy approach such that binding to undesired subunit stoichiometries was ‘silent’. The latter methodology forms the basis of the work in Cardiff, which started screening small molecules in-house on the medium-throughput QPatch-16 automated patch clamp platform, and has recently expanded to use the Syncropatch 384 HTS robot in collaboration with Scottish Biomedical. Both assay platforms were validated using preclinical and clinical candidates (e.g. AZD7325 and PF-06372865) and showed very similar isoform selectivity and potentiation efficacy profiles, which also aligned with literature ‘gold standard’ manual patch data.

Test compounds from Prof Atack’s medchem group were normalised to those of diazepam, revealing a lead compound MDI-595 with ~50% of the efficacy of diazepam at a2/a3 but only 20% activity against a5 and no activity against sedatory a1 GABA-A receptors even though it bound with pM affinity. This compound is brain penetrant and achieves good GABA-A receptor occupancy with an ED₅₀ of ~40ng/ml plasma exposure, and produces significant anxiolytic activity in the elevated maze with an MED < 1 mpk after oral dosing without any side-effects in a rotarod assay up to 30 mpk. This promising profile should support the preclinical IND profiling of a lead series candidate in 2022 followed by clinical testing in 2023.

Julie Klint – Drug discovery of K_v7.2/7.3 openers

Our next speaker was Dr. Julie Klint, who is Head of the Ion Channel section (Molecular Screening) at Lundbeck in Denmark. She and her colleagues had just published a paper describing the successful preclinical development of a selective M channel (K_v7.2/7.3) modulator for neuropsychiatric disease, and here she went into detail on the complex multi-parameter voltage protocols employed at Lundbeck (oocytes) and Metrion Biosciences (CHO cell lines) to profile the potency, selectivity and mechanism-of-action of their lead series compounds, and how they compared to the reference clinical drug, Retigabine. This family of ion channels are widely expressed in cardiac tissue (KCNQ1) and neurons (KCNQ2 – 5), where their activation at resting membrane potentials can strongly modulate single and repetitive action potential firing. Thus, K_v7.1 modulators carry cardiac risk, whilst neuronal K_v7x openers may have utility to reduce firing in pathophysiological conditions and diseases such as pain, epilepsy, ALS and depression.

Screening for K_v7 modulators

Due to the complex biophysics of K_v7 channels and the difficulty in aligning a particular voltage protocol readout or experimental parameter to functional effects on neuronal firing, many groups have used a variety of end points to assay for K_v7 modulators. These include measurements of maximal current or activation, shifts in the voltage-dependence of half-activation, or alterations in current deactivation kinetics. It is unlikely that just one parameter is sufficient, and using a single readout for SAR screening runs the risk of missing useful chemical scaffolds. Thus, more recent efforts have tried to use complex protocols, as exemplified by Lundbeck’s testing in Xenopus oocytes where they included an action potential-like waveform alongside measures of sub-threshold and maximal activation, as well as the voltage-dependence and rate of current activation. Similarly, Metrion developed a complex voltage protocol for screening test compounds on the QPatch platform which is able to distinguish between several different reference compounds with different K_v7 channel binding sites and modulatory mechanisms. Thankfully, the modulatory profile of the Lundbeck compounds aligns well between the client and CRO sites and their respective complex voltage protocols.

The in vitro patch clamp efficacy of the Lundbeck lead compound Lu-AA411178 matches that of the clinical reference Retigabine, and mutagenesis studies suggest that Lu-AA41178 binds to the same or similar site to Retigabine on neuronal K_v7 channels. The lead compound demonstrates in vivo efficacy at low doses (but a narrow therapeutic index) in a panel of relevant CNS disease assays such as MES and PTZ seizure, depression and anti-psychotic models, thanks to brain penetrant PK. Significantly, the lead compound is relatively selective within the KCNQ family (including cardiac K_v7.1), has no other cardiac ion channel liability, and is clean in a wide profiling screen including the lack of any GABA-A activity seen with Retigabine. It is believed that in vivo CNS disease efficacy of the promising Lundbeck compound is likely driven by subtle changes in activation, but a more drug-like molecule will be needed for clinical development.

Julie’s talk elicited some spirited discussions around the ability of a HTS patch clamp platform to deliver such high content data at an early stage of screening (shout-outs to Sarah Lilley at Sussex University, Damian Bell at Sophion and Tim Strassmeier at Nanion), a level of diagnostic detail that is frequently missing from traditional plate-based screening assays.

Irina Vetter – Neurotoxic venom peptides from the giant Australian stinging tree

As if Australia didn’t have enough dangerous animals ready to eat, bite and sting people, Prof. Irina Vetter’s group at the Institute for Molecular Bioscience & School of Pharmacy at the University of Queensland have described another venomous assailant, namely stinging trees. What is particularly fascinating about this story is that it could be another example of convergent toxin evolution, as the plant peptides bear a striking structural resemblance to a class of spider toxins known to modulate Na_v channels.

The gympie gympie tree and a new family of ‘gympietides’

Gympie gympie trees are related to European stinging nettles but grow to a much larger size as bushes or trees, and their leaves and stems are covered in stinging hairs (silica-rich trichomes) that can inject venom which elicit profound and long-lasting pain. Irina’s group used activity-guided HPLC fractionation to identify a single 4 kDa fraction that elicited nocifensive reactions in mice, and subsequently identified a new family of disulphide-rich ‘gympietides’ and venom gene sequences. Although these peptides were dissimilar in sequence to known plant or animal-derived toxins, their triple Cys-loop 2D and 3D structures were analogous to inhibitory cysteine knot (ICK) toxin peptides found in spiders and cone snails. Functional testing of synthetic gympietides confirmed their predicted action to modulate the activity of mammalian TTX-sensitive Na_v channels in rodent DRG neurons via impaired current inactivation.

Gympietides as potential tool compounds

Biophysical experiments using mammalian cell lines on the QPatch revealed opposite shifts in the voltage-dependence of activation and inactivation to produce persistent window currents close to DRG neuron resting potentials, with more potent activity seen for the more painful stinging tree peptide. Gympietides activate Ca²⁺ signalling in isolated DRG neurons, increase peripheral sensory nerve firing in an ex vivo rodent model, and produce profound pain behaviours when injected into the footpad of mice which are reversed by TTX and a selective Na_v1.7 inhibitory toxin Pn3a.  Thus, although gympietides may not be particularly selective for Na_v1.7 (question from Sam Goodchild at Xenon and Tianbo Li from Genentech), their novel sequences and potential as a tool compound could be useful for Na_v-specific target engagement in pain assays and other drug discovery applications.

Andrew Jenkins – GABA(A) receptors and disease

Prof. Jenkins is in the Dept. of Anesthesiology, Pharmacology & Chemical Biology at Emory University School of Medicine in the USA. As we heard in Prof. Atack’s talk, there is a great deal of interest and research around GABA-A PAMs such as benzodiazepines, neurosteroids and anaesthetics, and their potential utility in certain neurological diseases vs their sedatory side-effects which affect the balance between excitatory and inhibitory signalling in brain regions that regulate arousal and sleep.

Prof. Jenkins is interested in the potential role of GABA-A receptors and synaptic signalling in patients with idiopathic hypersomnia, who suffer from excessive daytime sleepiness and poor sleep patterns but lack a definitive clinical, genetic or biological cause of their ailment. His earlier work had identified a small peptide fraction in patient CSF that potentiated GABA-A currents and was sensitive to a benzodiazepine binding site antagonist, whilst a smaller effect was seen in control patient CSF samples, suggesting that there is a normal sleep-modulating peptide in human brain that may be dysregulated in hypersomnia patients.

Screening patient samples

As CSF sampling and testing against human GABA-A receptors may provide a definitive diagnosis for such patients, and have potential for pharmacological profiling and patient stratification, it became clear that a higher throughput and more reproducible electrophysiology screening system was needed to move the project beyond manual patch clamp. Joe Lynch’s group in Australia were obtaining similar results with the medium throughput Patchliner platform that correlated very well with the Emory manual patch data, but throughput was still insufficient to test multiple replicate samples from a growing list of hypersomnia patients. Accordingly, Prof. Jenkins turned to the microfluidic Ionflux APC platform, which offers both higher throughput and reduced sample volumes, helping scale-up patient screening and deliver reproducible data across multiple and sometimes highly variable CSF samples. As well as patient CSF profiling and diagnosis, Prof Jenkins and his collaborators began work to identify the specific sleep-modulating peptide(s) in patient samples using the IonFlux Mercury 384 platform. Of 62 up-regulated proteins identified from patient vs ‘ultra clean’ control CSF proteomes, several have been confirmed to high statistical significance, including a wildtype peptide (which may be up-regulated in patients vs controls) as well as several patient-derived peptide variants. He also used the same Ionflux screening assay to identify potential negative allosteric modulators of sleep-inducing GABA-A receptors to treat hypersomnia patients through a drug repurposing approach, which has identified hydroxychloroquine and clarithromycin (both hits against covid-19 infection as well). Finally, a metabolomics analysis of CSF samples from 44 patients identified over 200 GABA-A potentiators and over 300 negative modulators, which are now being confirmed by electrophysiology. Whilst it had taken 10 years to screen 600 patient samples by manual patch clamp, utilisation of a microfluidic APC platform now allowed a 10-300 fold reduction in sample volume use and screening time (e.g. 600 patient samples in 12 weeks), greatly facilitating patient diagnosis and insights into the underlying disease mechanisms.

Prof. Jenkins finished his talk by describing recent work showing the preferential expression of functional a5 containing GABA-A receptors in cancer cell lines, and evidence that a5-preferring benzodiazepine PAMs can accelerate cancer cell death. Thus, GABA-A PAMs offer the potential to be used alongside conventional chemo- and radio-therapy treatments to increase efficacy and reduce the well-known side-effects of cancer drugs.

Written by Dr Marc Rogers

Introduction

Ion channels are well validated drug discovery targets based on their widespread distribution in the human body and involvement in many critical activities within the brain, heart, smooth muscle and endocrine organs. Additional validation is provided by channelopathy mutations associated with common diseases such as epilepsy and cardiac arrhythmia as well as in many rare disease patient populations.¹ 

In addition, ion channels are expressed in human disease vectors (e.g. malaria parasite) and infective agents such as bacteria and viruses, presenting new areas where ion channel drug discovery can deliver novel therapeutic approaches and agents to improve human health.² 

The creation of novel therapeutics

The SARS-CoV-2 virus is a very topical example of where this second approach can be applied to the creation of novel therapeutics, as the sequencing of the COVID-19 genome in early 2020 revealed the presence of several envelope E protein and putative ion channel genes.³ Numerous academic groups have begun to target COVID-19 viral ion channels (viroporins) during the course of the pandemic to understand their structure–function and utility as anti-viral drug substrates, including a group from Massachusetts Institute of Technology (MIT) that recently published the NMR solution structure of the E ion channel protein that they used for initial ligand screening.⁴

This study from MIT illustrates some of the challenges of modern ion channel drug discovery techniques, which can affect the identification of novel ligands for both human and viral ion channel proteins. Firstly, having a novel gene sequence may allow homology mapping onto the structure of related and better-characterized protein structures, but brings with it issues of evolutionary and functional diversification. For example, although the influenza virus M2 cation channel has been studied for many years,² the MIT group found that the COVID-19 coronavirus E protein has a very different structure once expressed in a biological membrane, and thus inferences from homology modeling may be misleading.

Secondly, it is now possible to obtain high-fidelity protein structures, including that of SARS-CoV-2 viroporins,⁵ and use sophisticated computing software to implement structure-based drug design approaches whereby known drug structures are virtually bound to the target of interest. The MIT study provides an example of the risk in using such an approach for a novel protein, as the prediction that anandamide, a relatively potent inhibitor of the ‘flu M2 cation channel, could also efficaciously inhibit the COVID-19 ion channel was not borne out as only weak micromolar affinity was seen in their NMR binding experiments.

The way forward towards discovering effective inhibitors

This recent study does however point the way towards discovering effective inhibitors of SARS-CoV-2 viral ion channels with therapeutic potential. The MIT group managed to express a viral channel protein in lipid membranes, which will allow for functional screening of modulators that can affect channel biophysics and ionic flux through the pore. It is possible to screen for viroporin modulators using functional bacterial and yeast assays,⁶ but it is unclear at this stage if these hit compounds will deliver antiviral activity in human cells and patients.

Identifying drug repurposing hits that can be used on patients

Nevertheless, identification of selective ligands will enable testing the hypothesis, that modulating SARS-CoV-2 viroporin activity can limit COVID-19 virus infectivity or replication in intact cellular systems, as shown for SARS CoV E protein. This COVID-19 viroporin target and mechanistic validation should also be done genetically, using RNA knockdown or CRISPR editing in human respiratory cell lines and patient lung samples, as it was for the original SARS viroporin. Finally, screening approved clinical drugs against COVID-19 viroporins could rapidly identify drug repurposing hits that can be used on patients, especially those not suited to vaccination owing to compromised immune systems.

Conclusion

The ultimate hope is that academic and industry groups can identify repurposed and novel drugs to inhibit a range of viral ion channels and thus offer an alternative strategy to treat both current endemic viral diseases as well as pandemic infections of the future.

Article originally published on Technology Networks.

Written by Dr Marc Rogers, Chief Scientific Officer, Metrion Biosciences.

References

1. Imbrici P, Liantonio, A, Camerino GM, et al. Therapeutic approaches to genetic ion channelopathies and perspectives in drug discovery. Front. Pharmacol.2016. doi:10.3389/fphar.2016.00121
2. Charlton FW, Pearson HM, Hover S, et al. Ion channels as therapeutic targets for viral infections: Further discoveries and future perspectives. Viruses.2020;12(8):844. doi:10.3390/v12080844
3. McClenaghan C, Hanson A, Lee S-J, Nichols CG. Coronavirus proteins as ion channels: Current and potential research. Front. Immunol.2020. doi:10.3389/fimmu.2020.573339
4. Mandala VS, McKay MJ, Shcherbakov AA, et al. Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers. Nat Struct Mol Biol. 2020;27:1202-1208. doi:10.1038/s41594-020-00536-8
5. Kern DM, Sorum B, Hoel CM, et al. Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs. bioRxiv. 2020. doi:10.1101/2020.06.17.156554(preprint)
6. Tomar and Arkin. SARS-CoV-2 E protein is a potential ion channel that can be inhibited by Gliclazide and Memantine. Biochem Biophys Res Commun.2020;530(1):10-14. doi:10.1016/j.bbrc.2020.05.206

Written by Dr Charlotte Hill

Metrion Scientist Dr Charlotte Hill explains how she first discovered the wonderful world of patch clamp electrophysiology and how she came to write the chapter “An Introduction to Patch Clamp Recording” alongside Dr Gary Stephens. This chapter is featured within the book “An Introduction to Patch Clamp Recording” edited by Dr Damian Bell and Dr Mark Dallas. The book is available via Springer here.

“I graduated from King’s College London with a BSc in Pharmacology in the summer of 2008, which was at the height of the financial crisis. It was not a great time to find a job and my personal tutor recommended that I pursue an MSc in Toxicology at the University of Surrey. This intensive, one-year course not only introduced me to my future husband, but also electrophysiology. However, I didn’t get to do any electrophysiology until I started my PhD at the University of Reading in early 2011. The project was funded by GW Pharmaceuticals and Otsuka Pharmaceutical, with Prof. Ben Whalley and my supervisors Prof. Gary Stephens and Prof. Claire Williams overseeing the research. My PhD project focused on trying to elucidate the anti-convulsant mechanisms of action of non-psychomimetic components of the cannabis plant.

The first task of my PhD was to learn how to do current clamp recordings from acute brain slice preparations, which I now know to be incredibly difficult, even for a seasoned patch clamper and I remember many people telling me that it was “character-building”. In my second year, I learnt how to do extracellular recordings from brain slices using multi-electrode arrays (MEAs). In my third year I was doing both patch clamp and MEA recordings in brain slice epilepsy models (not at the same time, but it would have been awesome).

In 2014, while I was still writing my thesis, I started working in the Ion Channels group at BioFocus, which had recently been acquired by Charles River. It was there that I was first introduced to automated patch clamp with the PatchXpress, and I remember thinking, “Yikes, one of these machines is the equivalent to sixteen of me”. I took several opportunities to work in other departments, such as High-Throughput Screening, Compound Logistics and Informatics, so needless to say this job taught me a lot.

I moved to Dublin in early 2017 because my husband was offered a post-doc at the Royal College of Surgeons in Ireland and I subsequently got a job as a technician at University College Dublin. I was given the opportunity to update and design new practicals for the undergraduate students studying Pharmacology and Neuroscience, and I used my experience in industry to give lectures about Drug Discovery.

In the summer of 2019, I was contacted by my PhD supervisor Prof. Gary Stephens with the offer to co-author a book chapter on patch clamp methods, as he thought parts of my thesis would form a useful base for it. The chapter is an introduction to the patch clamp method, which includes its history, the various configurations and its many applications. There are even a couple of photographs of my old rig, on which sits my electrophysiology totem – a miniature rubber duck called Static.

Working on this chapter made me realise how much I had missed doing electrophysiology., therefore when Metrion advertised for an electrophysiologist, I applied straight away. Getting back into e-phys. (especially automated patch clamp) and drug discovery after a few years away felt like putting on an old, cosy jumper. However, do not assume that I sit back and take it easy, I’m conscious that I still have a lot to learn and working at a CRO is just as busy as I remember it.”

Written by the Editor

Metrion Biosciences have had a longstanding relationship providing drug discovery research services to Grünenthal GmbH, a privately owned pharmaceutical company and global leader in pain management and related diseases. Grünenthal develop medication for patients with severe and debilitating diseases and high unmet medical needs and have a long track record of bringing innovative treatments to market.

Pain and the need for therapeutic intervention 

Pain is a major global health problem, with one in five adults estimated to suffer from pain at any one time and one in ten diagnosed with chronic pain each year. Whilst medication exists, issues with efficacy and dependency associated with some classes of drugs, means there is a substantial opportunity to develop new, safe pain medicines. Grünenthal has identified four key areas with significant unmet medical need for research and development into novel pain medicines. The areas include peripheral neuropathic pain, chronic post-surgical pain, chronic lower back pain and osteoarthritis.

What does the new agreement entail?

Recently, Grünenthal and Metrion, who have been working together since 2015, have signed a new ion channel drug discovery research agreement. Under this new agreement, which lasts a further two years, Metrion continues to provide dedicated ion channel expertise and will create new assays to support the development of novel therapies for the treatment of pain. This will include electrophysiology screening of medicinal chemistry compounds generated by Grünenthal, provision of translational assays and access to Metrion’s extensive knowledge of the pain research field.

Metrion Scientists and project co-ordination teams look forward to continuing the successful ongoing ion channel drug discovery programme with the Team at Grünenthal, to assist with the development of novel pain therapies which will change the lives of those most in need.

Written by Artem Kondratskyi

Introducing Damian Bell: as a new member of the team at Metrion we are using an abridged version of an interview by Artem Kondratskyi for ionchannellibrary.com. A big thank you to Artem for sharing this interview and a quick plug for his website – we highly recommend it as an excellent resource for anyone interested in ion channels.

Artem spoke with Damian about his career, the current state of ion channel industry, automated patch clamp, ion channel antibody development, the new book on methods in patch-clamp electrophysiology, and more.

So, Damian, let’s get to the point right away. Why ion channels? How did you get into ion channels?

Whilst doing my undergraduate degree, I did an industrial placement year at Eli Lilly [Erl Wood Manor, UK]. I was initially pencilled in to work in the animal behaviour unit. However, I’m allergic to animal fur. Lilly had recently hired David Bleakman to set up an ion channel group. So, serendipitously my allergy took me into the world of ion channels.

So, it appears that you’ve got a passion for ion channels directly from the industry, not academia. I would say it’s rather unusual.

Though Lilly and David Bleakman provided the initial spark (pun intended), it was really my PhD and postdoctoral work that fuelled my fire for ion channels. After I finished my undergraduate studies at the University of Nottingham, I went on the usual academic route by doing a PhD in Annette Dolphin’s lab at UCL and then a postdoc at Columbia University in New York under Steven Siegelbaum. Two world-class ion channel labs, two brilliant scientists that understandably were huge influences on my early research and understanding of ion channels.

Well, if you liked your academic route so much, how come that you ended up in industry?

Nearing the end of my postdoc we were witnessing the birth of a revolution in ion channel recording capabilities – various manufacturers were starting to develop automated patch clamp. And that’s what took me from academic ion channel research into industrial drug discovery settings. As my postdoc at Columbia was finishing, I had a great opportunity to become one of the first adopters of automated patch clamp at AstraZeneca in the UK. Divining how this seismic shift would change the landscape of ion channel R and D, I jumped at the chance.

What are your thoughts on the current state of the ion channel industry? Are there opportunities for ion channel electrophysiologists?

I’m optimistic and think the ion channel industry is in a very healthy state. Despite a number of large pharma withdrawing significant resources and capabilities from neuroscience, I still feel that it’s in a healthy state because where the big pharma have pulled out, a lot of smaller pharmas, biotechs (including virtual drug discovery programmes) and CRO’s have sprung up to fill those gaps. And I think that’s good for the field: those smaller, more diverse range of companies are driving ion channel research in more efficient and innovative directions.

Smaller, more diverse companies are driving ion channel research in more efficient and innovative directions.

Damian Bell, Senior Scientist, Metrion Biosciences

Another aspect is we’ve now had nearly two decades of automated patch clamp technology development. And with the vastly improved capabilities and throughput that automated patch clamp has given us, we should soon be reaping the fruits in terms of the greater potential for more fully developed, clinically approved drugs. Consequently, I believe an ion channel blockbuster is imminent. As with any predictions, it’s hard to accurately read the runes, but I’d say within the next two to three years we are going to get one of these big ion channel blockbusters coming to market. And once that hits, the ion channel R&D will suddenly be in the spotlight: I think a lot of big pharma ion channel programs will either be resurrected or they’ll be starting entirely new programs; once you get one ion channel blockbuster to market the ion channel field will really explode on the back of it.

Wow, that’s so optimistic. I really hope your predictions come true. But with such a development of automated patch clamp is there any threat of unemployment for ion channel electrophysiologists? Will automated patch clamp robots replace electrophysiologists in the end?

Of course, this is a typical concern, in any industry, as things become automated. But I still think you need the expertise to fully understand and analyse the data you get from an automated patch clamp platform and, of course, even if you can run a well-defined assay you still need someone who will design, develop and build those assays in the first place. Automation certainly takes you a long way down the road in terms of increasing the number of ion channels and drugs you can test on those ion channels. But you still need expert eyes and minds really looking at the data in detail and designing protocols for automated patch clamp. And, even the best automated patch clamp machines still cannot necessarily do everything you can do on a manual patch rig. A lot of companies, even though they might have several automated patch clamp platforms still often have at least a manual rig or two to do some deeper dive experimentation. Despite automated patch clamp potentially taking over the manual capabilities there is still a need for good electrophysiologists, there is still need for their input and creativity in terms of how you apply certain tests, how you design them, how you run them most efficiently on the automated patch clamp platforms; manual patch still has a very important and useful role in any good ion channel lab.

In your LinkedIn profile you’ve mentioned that you worked with different automated patch clamp platforms. What’s the difference between those platforms? Do you have a preferred one?

I personally have a preference for the QPatch from Sophion. However, that’s based on my own career path: I started off on a QPatch and I’m still working with a QPatch; it’s a very good machine. But over my 16 years of automated patch clamp use I’ve worked with several other platforms from different manufacturers and they all have their uses in different circumstances, in different R and D programmes. Some platforms might be more flexible, some might have higher throughput, but no one platform is perfect. They all have advantages and disadvantages. Personally, I would say that Nanion and Sophion are neck and neck in terms of their capabilities in making and developing automated patch clamp systems; both make very good machines. I also think that competition between those two main players – and other APC specialists like Fluxion – is very healthy, constantly pushing and developing the capabilities and advancing the field.

At IONTAS you worked in an antibody engineering company, could you tell me what the main players in ion channel antibody engineering business are?

Over the years, there have been numerous companies that have looked at ion channel antibodies but highlighting the three companies with the most compelling and innovative programs: IONTAS (take with a pinch of bias), Tetragenetics and Ablynx.

At IONTAS we developed the KnotBody^TM technology where we fuse a venomous species knottin toxin into an antibody background. Essentially, we combine the ion channel modulating capability of a knottin with the therapeutic functionality of an antibody. This KnotBody^TM format has numerous benefits over existing formats and it won’t be long before this will be one of the key technologies for generating ion channel specific antibodies.

Tetragenetics’ R&D scientists are doing some great work: in Tetrahymena they have a very robust expression system giving them the capability to express ion channels at high quantities and quality, allowing them to develop antibodies against this antigenic ion channel material.

Ablynx have been making some nice molecules, taking advantage of the modular building blocks of antibodies: the heavy and light chains, the CDRs (the complementarity determining regions), etc. Using the modular ‘business end’ sequences of an antibody they’ve managed to ‘shrink’ an antibody down to its minimal functional binding components – dubbed a Nanobody. They have some interesting ion channel Nanobodies.

As for big pharma – for instance J&J, Amgen, Genentech, MedImmune/AstraZeneca – they’ve all had some great programmes, but many of them were either shelved or binned. And I haven’t seen much data coming out those labs for quite a while now.

And what about ion channel antibody engineering in academia? You know that various labs in academia try to develop their own ion channel antibodies. What are your thoughts on quality and future of those antibodies?

A number of academic labs over the last decade or two have made very good ion channel antibodies. However, there have also been some well documented problems with academic ion channel antibodies – when industrial labs tried to replicate those antibodies they haven’t worked. I’m not quite sure why that is. For instance, there was a very interesting and intriguing Na_v1.7 antibody that came out of Duke University several years ago, however both Genentech and Amgen couldn’t get it to work the way that the academic group at Duke had [ed. since the interview this publication has been retracted – https://www.cell.com/cell/fulltext/S0092-8674(20)30751-0?dgcid=raven_jbs_etoc]. I think more and more researchers will be turning to antibodies targeting ion channels, but it’s clearly a very challenging target class. Many researchers have been trying, but they haven’t created a particularly good modulating ion channel antibody as yet. Leaving aside the Duke story, there are some good ligand-gated ion channel antibodies but for the voltage-gated ion channels it’s a different story: they’ve been very difficult to make good, modulating antibodies against. The problem is that though you can make a good, bindingantibody to a voltage-gated ion channel, it might not necessarily modulate the ion channel. And that’s where a significant hurdle comes in – how to make good, modulating ion channel antibodies? One problem is a very limited number of externally facing epitopes that voltage gated ion channels have, compounded by the fact that those limited epitopes are often highly fluid and mobile and could be changing rapidly over the gating cycle of an ion channel – one epitope may only be available for a few milliseconds every minute or two.

All of these issues make ion channels a difficult target class for antibody drug discovery. Nonetheless, as we improve our capabilities to express purified, stable, functional ion channel protein in a membrane-like environment, we can potentially develop antibodies to those short-lived, external epitopes that might give rise to better binding and, critically, modulating ion channel antibodies.

If you were to start a company in the ion channel field what would it be?

This question has certainly crossed my mind a few times over the last few years. My focus is routinely in the lab and on my team, my thinking’s not usually on the building-a-business side of things, and yet I do see obvious areas and gaps where I think there could be some great ion channel research and drug development. Much of my background has been in chronic pain. And I still believe ion channels are going to be key in chronic pain therapies. There has been a lot of research going into it and yet we are still to get that true chronic pain ion channel drug. So, if I was to start a new ion channel company it would probably be in the chronic pain field. It would likely involve Na_v1.7, but also other ion channels like Na_v1.8, Na_v1.9, HCN2, TRPA1 and TRPV1. However, it might not necessarily just be the ion channels themselves, it would also be the upstream/downstream proteins and pathways around the ion channels. It’s also becoming clear that like cancer before it, chronic pain is a vast umbrella term covering over a hundred pain states, diseases and pathologies, so future chronic pain medications are going to be increasingly tailored and personalised to highly selected and defined pain patient cohorts. There won’t be a single magic bullet to solve chronic pain but dozens, potentially even mixed and matched, to a specific patient’s ‘painome’ – not sure that’s even a term, think I might’ve just coined a whole new field of medicine. Finally, considering my experience with IONTAS, my new company would most probably involve antibody drug discovery. I would be looking to use these larger, in vivo longer lasting molecules with the multi-specificity and multi-functionality that you can build into an antibody as opposed to the narrow chemical confines of a small molecule.

What’s your opinion on the COVID-19 crisis? Will it influence ion channel research and business? 

As for any lab-based work, the way we do ion channel research is going to be changed substantially. Our work style will become more flexible. It should become more environmentally friendly because you won’t be doing as much commuting and travelling for work or for various meetings, with more virtual meetings and conferences. Overall, I think a lot of people will be re-evaluating the way they do things and changing their lifestyle and their work capabilities accordingly.

Another point is with COVID-19 enveloping us all across the world, the attention of many people, me included, has switched to: “How can I help? What can I do?”. And, since my expertise is in ion channels, I started thinking in terms of where ion channels might have an impact. For instance, there are ion channels highly specific to viruses. And considering that viruses only carry the bare minimum of what they need to replicate, then these ion channels must be critical in their replication cycle. Consequently, viral ion channels will become targets for antiviral drug development. Another key aspect is that ion channels are involved in the immune inflammatory response, and so ion channels will have a key role in the response of an organism to infection by pathogenic viruses. Antivirals are clearly going to be increasingly important now and in the future pandemics: ion channels are likely to be targets for antivirals, which will stimulate ion channel R&D.

From your LinkedIn posts I learned that apparently there is a new book on ion channels to be published very soon. What will be that book about?

Together with Mark Dallas [University of Reading] we have been asked to edit a book, compiling different chapters on methods in patch clamp electrophysiology. The book has a really broad scope and aims to be a guide to methods for novice and expert alike: we obviously cover manual patch clamp through to automated patch clamp, and onto more recent advances in techniques and applications like optogenetics. We’ve brought together experts across the world to each write a chapter on their specific technique, their specific area of expertise in the field of patch clamp methods. So, yes, that book will be published by Springer Nature in August this year – we’re looking forward to that coming out and hope it will be a useful, practical resource for any lab wanting to make ion channel recordings.

In one of your profiles in the internet I read that you are “passionate about ion channels, bordering on an obsession”. Could you comment on this?

Yep: I’m a bit of an ion channel obsessive. Ion channels are so critical, they control so much of our physiology, so much of what makes us ‘us’. And this goes from sensory perception to how those perceptions are collated and processed, how your brain determines and defines what those sensory perceptions are telling you.

We can’t think without ion channels. And so, if you can’t think then how can you have consciousness? Consciousness is the kernel of our humanity. If you really want to get deep you could even argue that ion channels are the molecular source of the soul. Well, maybe that is a little too deep, but think about it: literally everything we do, and see and feel, how we perceive everything – it’s all driven through ion channels. Even my memories – like the memory as a four year old helping my dad in the garden, putting the garden fork clean through my sandal, grazing the skin between big and second toe – all come through ion channels.

Maybe I’m stretching it too much but our entire perception of the world, including our previous perceptions and our histories, they all come through ion channels. For instance, some of the strongest, most visceral memories you can have are tied to smells, like Proust’s proverbial madeleines. You can have a smell twenty years ago, twenty years later when you have that same smell it transports you to that original smell, to that location, to everything that you perceived at that point in time – it’s incredible, fantastical even.

Obviously, there is a lot more going on than simply ion channels. Nonetheless, ion channels are key elements in your perception of the world, in your personal history within the world. And it’s not just about the sensory perception, it’s also about how you perceive yourself, how you perceive others, your place in the world past, present and future. To mangle Descartes’ beautifully pithy dictum: I have ion channels, therefore I think, therefore I am. In other words, ion channels really put the ‘I’ into consciousness … sorry, like Icarus my flight of fancy went too far.

Written by Anna Mikhailova

My name is Anna Mikhailova. I recently started my internship in Sales and Marketing at Metrion Biosciences.

I was born in Moscow, Russia, and moved to study in the UK when I was 15 years old to start my GSCEs. My interest in science was clear to me back then and I selected to study high-level Biology and Chemistry for my International Baccalaureate Diploma. My first piece of coursework in year 12 focused on anti-bacterial properties of Manuka honey which introduced and immersed me into a versatile scientific world. I then started a BSc in Biomedical Sciences at UCL, an emerging field researching technology used in healthcare.

My degree immersed me into the complexity of molecular and structural manifestations of neurodegenerative disorders.

Anna Mikhailova

During my 3 year course, I studied a broad range of subjects from many fields of Biology focusing on Control Systems of the body. My degree was centred around the Structure and Function of the Nervous system, Medical Biophysics, and Investigative Methods of Neuroscience, which included my final laboratory-based project at Great Ormond Street Children’s Hospital in their Research Unit. My dissertation was focused on examining adult mouse hippocampal astrocytes post neonatal injury. Though my degree immersed me into the complexity of molecular and structural manifestations of neurodegenerative disorders, it also introduced me to future public health problems and vectors of potential development.

Captivated by the prospective biomedical tools to treat and diagnose diseases, I have decided to concentrate on Management and Enterprise for my Masters. I was very happy to receive an offer from the University of Cambridge to study an MPhil in Bioscience Enterprise (MBE) which combines technical biomedical development with an innovation and business focused curriculum. Recently graduating from UCL with a First Class Honours Degree, I am excited to start the course at Cambridge, which will allow me to acquire new expertise in commercial and legal dimensions and convey novel research advances into healthcare practice.

Whilst I remain committed to the promise of science, I have come to realise that I am far more invigorated by the questions asked within the areas of sales, marketing, and business development. Metrion Biosciences has offered me a perfect position. While being close to the science I will have a chance to explore and gain new experience in Sales and Marketing practices directly before starting my MBE at the University of Cambridge. During this Sales and Marketing internship, I will acquire knowledge and expertise from the professionals leading the way in ion channel science. I am honoured and very excited to start my placement here and hope to bring a lot to the team and company!

Written by John Ridley PhD

The regulatory guidelines

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines were introduced in 2005 to evaluate the proarrhythmic liability of new drugs. They were implemented in response to the discovery that inhibition of a cardiac potassium channel (encoded by hERG) is associated with prolongation of the QT interval and a potentially deadly arrhythmia, Torsades de Pointes. ICH S7B and ICH E14 utilise hERG inhibition and QT interval prolongation as surrogate markers of proarrhythmic liability, which are highly sensitive and have proven effective at preventing proarrhythmic drugs from reaching the market.

However, these markers have low specificity, with only a modest correlation between hERG inhibition, QT prolongation and proarrhythmic liability⁶. Indeed, hERG inhibition can be mitigated by concurrent inhibition of depolarizing currents³, indicating that screening against a wider panel of cardiac ion channels would provide better proarrhythmic liability profiling.

This notion is supported by the observation that some drugs with low proarrhythmic liability (e.g. verapamil) are potent hERG blockers, but also inhibit depolarising currents such as I_Ca. Unfortunately, an over-reliance on hERG selectivity has potentially led to the removal of many efficacious and safe drugs from development pipelines. Consequently, this casts some doubt on hERG inhibition and/or QT prolongation as suitable markers for predicting proarrhythmic liability.

The launch of a new CiPA initiative

To address these limitations, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was launched by the FDA in July 2013. The CiPA initiative aims to improve the accuracy and reduce the cost of predicting cardiac liability using three ‘pillars’:

• Compounds will be profiled against a panel of human ventricular ion channels
• This in vitro data will be incorporated into an in silico model of a human action potential (AP) to provide a proarrhythmic risk classification
• Compounds will be tested using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) to confirm the risk classification derived from the in silico model

Three Working Groups composed of members from academia, pharmaceutical companies and CROs were established to test and validate each of the CiPA pillars. A common toolbox of 28 compounds, separated into high, intermediate and low proarrhythmic risk groups, was used across all working groups. The toolbox was subdivided into sets of 12 training compounds and 16 validation compounds.

The role of the Ion Channel Working Group

The Ion Channel Working Group (ICWG) was tasked with establishing robust and reproducible assays against a panel of seven ventricular currents. A large component of the work includes generating standardized assays and identifying the most applicable readout parameters to support the In Silico Working Group (ISWG). ICWG is exploring the variability of data obtained across research sites, and comparing data generated from conventional manual patch clamp and automated patch clamp platforms.

An initial study was conducted using the 12 training compounds and standardised experimental methodologies to verify their accuracy, which was followed by blind testing of the 16 validation compounds. The training compound data collected for some ion channels revealed prominent variation in IC₅₀ values across research sites⁴. Some of this was related to inter-site differences in experimental methods. Therefore, the ICWG continues its efforts to improve reproducibility across research sites and to devise best working practices for CiPA, whilst providing additional data to help test and validate the in silico AP model.

The role of the ISWG

The ISWG is developing an in silico model to allow prediction of proarrhythmic risk, focusing its efforts on the O’Hara-Rudy (ORd) model, which is based on human ventricular AP recordings. The model recapitulates early after depolarisations, which are crucial to accurately predict proarrhythmic risk⁷. The FDA optimized the ORd model by incorporating the late component of Na_v1.5 current, as well as a dynamic hERG blocking model that more accurately accounted for the higher proarrhythmic risk profile of compounds that become trapped within the hERG pore during repolarization.

The optimized ORd model was calibrated using IC₅₀ data from the training set supplied by ICWG⁵. The in silico readout includes a net charge metric “qNet” and incorporates an uncertainty quantification method to account for experimental variability⁵. The mean qNet value averaged across 1–4 fold of the C_max drug plasma concentration is provided as the Torsade Metric Score (TMS).

The ISWG has proposed two TMS thresholds that classify drugs into the three proarrhythmic risk categories. The calibrated model was then used to evaluate the validation compound set⁶, showing that the model met all pre-specified measures for ranking and classifying the drugs according to their clinical arrythmia risk classification.

The ICWG studies revealed that inhibition of hERG, Ca_v1.2 and late Na_v1.5 current have the most significant impact on proarrhythmic risk prediction⁶. This opens the possibility that the in vitro ion channel panel (Pillar 1) may be reduced to these key currents.

Testing compounds in iPSC-CM

The third Pillar involves testing compounds in a complex human cellular system (iPSC-CM) that contains all of the ion channels in the CiPA ion channel panel, as well as additional ion channels and pumps that could affect a compound’s proarrhythmic liability profile.

The two favoured approaches for studying the effect of compounds on iPSC-CM include measuring extracellular field potentials (using multielectrode array platforms) and fluorescence signals from voltage-sensing dyes or genetically encoded voltage indicators. The CiPA cardiomyocyte consortium used the test¹ and validation² compounds  across several iPSC-CM cell lines and plate-based readouts, revealing excellent specificity and sensitivity that matched, or exceeded, that of animal models (100% specificity and up to 79% sensitivity). There was greater variation between cell types than between different screening platforms, suggesting that further work is required to optimise the iPSC-CM reagents.

A considerable amount of effort has been expended towards validating the three pillars of CiPA and using this data to revise ICH guidelines⁸. Additional testing and validation is ongoing for all three pillars to further improve the excellent sensitivity and specificity of these assays, with further work required to optimise iPSC-CM reagents to allow the development of more reproducible assays.

References

1. Blinova et al., (2017) Comprehensive Translational Assessment of Human- Induced Pluripotent Stem Cell Derived Cardiomyocytes for Evaluating Drug-Induced Arrhythmias. Tox Sci 155: 234- 247.
2. Blinova et al (2018) International Multisite Study of Human-Induced Pluripotent Stem Cell- Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment. Cell Reports 24: 3582–3592.
3. Kramer et al., (2013) MICE Models: Superior to the HERG Model in Predicting Torsade de Pointes. Nature Sci Rep 3: 2100.
4. Kramer et al., (2020) Cross-site and Cross-Platform Variability of Automated Patch Clamp Assessments of Drug Effects on Human Cardiac Currents in Recombinant Cells. Sci Rep. Mar 27;10(1):5627.
5. Li et al., (2017) Improving the In Silico Assessment of Proarrhythmia Risk by Combining hERG (Human Ether-à go go-Related Gene) Channel–Drug Binding Kinetics and Multichannel Pharmacology. Circ Arrhythm Electrophysiol 10: e004628.
6. Li et al., (2018) Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative. Clin Pharm Therapeutics: 10.1002/cpt.1184.
7. Mirams et al., (2011) Simulation of multiple ion channel block provides improved early prediction of compounds’ clinical torsadogenic risk. Cardio Res 91: 53-61.
8. https://database.ich.org/sites/default/files/E14S7B_IWG_Concept_Paper.pdf

Acknowledgements

Thank you to Dr John Ridley, Metrion Biosciences’ Business Manager for writing this article and to MedNous for publishing this work.

This article was published in the June 2020 edition of MedNous, a publication of Evernow Publishing Ltd.

Written by the Editor

Metrion Biosciences is open to discuss your ion channel drug discovery and safety profiling requirements via info@metrionbiosciences.com.   

In response to the global COVID-19 outbreak, and to help us to safeguard the health and safety of our employees and their families, we temporarily closed our laboratories in April 2020 as advised by the UK Government. After making significant changes to our laboratory space and work patterns we recommenced laboratory operations on 4^th May 2020 and the team is back at Granta Park providing high quality ion channel screening services to our customers.  

To help us maintain responsible levels of social distancing some of our team are operating from home, they can be contacted at info@metrionbiosciences.com and will be happy to help.

We would like to thank our customers for their support at this unprecedented time and our team looks forward to providing data to drive customer drug discovery projects forward during this challenging period for all of us.

Written by Andrew Southan PhD

During the second half of 2019 Metrion’s CEO Andy Southan spent three months on the Goldman Sachs 10,000 Small Businesses programme. In this blog he reflects on the aims and content of this initiative, plus the value he believes it will bring to Metrion Biosciences’ business.

What is the 10,000 Small Businesses programme?

It is a programme launched by Goldman Sachs in 2011 to help accelerate the growth of small businesses by increasing their potential for job creation and enhancement of the UK economy. The intensive three-month course has been designed by business education experts and is run in partnership with leading UK universities and business schools.

The course content helps business leaders to analyse their company, to identify strengths and opportunities and then create an ambitious business plan for growth. The performance statistics for companies run by graduates of the programme are impressive, with UK participant companies consistently doubling revenue and creating 50% more jobs for their companies within two years of completion.

Furthermore, metrics such as revenue growth (16x), job growth (13x) and productivity (28% higher) are all significantly increased after course completion, when compared to similar UK small businesses in general.

As Metrion Biosciences’ CEO why did you apply?

I was initially encouraged to consider applying by Metrion’s Chairman Keith McCullagh. With Metrion Biosciences qualifying under Goldman Sachs criteria and operating in a highly competitive global marketplace, the timing was good to use the potential opportunity and use the time to take a close look at the business and establish the best opportunities to accelerate our growth; whilst continuing our focus on highly specialised, high quality ion channel drug discovery services. After successfully passing the application and interview process I joined the course in September 2019.

What did the course entail?

A mix of online resources, online meetings, three residential sessions, three personal study weeks, 1:1 sessions with a business mentor and regular discussions with other course attendees over a three month period.  The course was very intensive and spread over nine distinct modules covering initial in-depth analysis of the business and customer needs, strategies to identify growth opportunities, branding and marketing, recruitment and employment law, leadership and building a team, funding opportunities, cash flow analysis, financial statements and metrics and internationalisation strategies.

I joined UK Cohort 11 (~70 participants), split into two sections for the residential sessions and also smaller ‘growth groups’ for detailed discussion, with a business mentor leading and advising each group.  In my growth group our businesses specialities were extremely diverse – spanning a housing design and build company, a recruitment agency, a sports equipment supplier, an industrial tool supplier, an audio-visual technology and conferencing company and Metrion, an ion channel specialist drug discovery company. It was interesting that, although our companies had little in common in terms of speciality, we had many areas of shared experience and the support and insight from the group proved to be highly valuable as the course progressed.

Would you recommend the course to other small business leaders?

Absolutely, it was an extremely well run and highly professional course and an excellent opportunity to meet business professionals outside of my specialist field and gain a perspective from their experience. Tuition and residential session accommodation fees are all fully funded by Goldman Sachs and the Goldman Sachs Foundation and participation enables you to interact with world class business coaches and academics. 

The residential teaching sessions, predominantly located at the Saïd Business School in Oxford, were intense, information rich and often challenge your preconceptions and potential for personal bias.  The course does require a significant commitment, with a considerable amount of personal time required to complete the reading, thinking, work package submissions and to attend the webinars, growth group meetings and residential sessions. Participants also need to ensure they approach the course with an open mind and able to take on board constructive criticism. And don’t forget, the day job is still there to be done in parallel.

After completing the course what are Metrion’s plans for 2020?

The course gave me the opportunity to look at the last three years of our business and analyse the evolution of our customer segments and the territories we work in; also to examine the services we consider to be successful, services in low demand and new areas we should focus upon to achieve rapid growth, whilst maintaining our high quality standards and recognition as experts in ion channel drug discovery and safety profiling.

Some of the information assembled in the early stages of the course was already apparent to me and our management team beforehand, but it was good to compile the data set to back it up and to subsequently complete a formal business plan for the next three years, which was a requirement for successful completion of the course. Since formation in September 2015, Metrion Biosciences’ vision has been to become the leading provider of ion channel drug discovery research services and expertise to the worldwide pharmaceutical industry.

Our team has established firm foundations to help us meet this ambitious goal and by providing our clients with reliable, high quality services we will continue to build our company into a leading choice for companies outsourcing ion channel drug discovery and safety profiling. A number of new initiatives from the business plan have already been implemented and we have begun the process for other changes to our business that will be introduced as the year progresses.

Through the work of our team to date, and the new initiatives we have begun, we believe that Metrion is now very well placed to meet our goals and, importantly, to provide new job opportunities for the UK life sciences community and enhanced outsourcing choices for customer preclinical discovery research programmes.

Interview with Dr Marc Rogers

Thank you to the ELRIG Committee and to BioStrata for allowing us to share the below content on our website.

The British Pharmacological Society (BPS) ran a special symposium at the ELRIG Drug Discovery 2019 conference yesterday, focused on the importance of targeting ion channels for drug discovery.  As a unique platform to foster the discovery of new ion channel targets, the symposium featured talks from various experts working at the forefront of this field.

The line-up included Dr Marc Rogers, Chief Scientific Officer at Metrion Biosciences, a UK-based specialist CRO offering ion channel focused drug discovery services. Marc has spent the last three decades researching ion channels within both academia and industry environments. Marc has some intriguing insights into this promising field of drug discovery, which he shares below.

Q: What’s motivated you to focus your entire career on ion channel research?

A: It all started when I was an undergraduate in New Zealand doing my physiology degree. I attended a lecture on neuronal excitability and performed some hands-on animal experiments  (back in the day before the 3Rs came into effect). From that point, the light bulb in my brain went on and has stayed on for the past 35 years!

I’ve worked all over the world (Australia, New Zealand, the US and now the UK) but my motivation has largely remained unchanged. I’m fundamentally driven by science, as well as my fascination with how ion channels work and what effects they have on cell function. As I’ve spent the last 15 years in industry, my work is now more applied than theoretical, but my whole career has focused on studying a range of ion channels and their role in human disease.

Q: Can you explain why ion channels are important to study for drug discovery?

A: Ion channels facilitate the flow of ions such as sodium, potassium and calcium across cell membranes and are a site of action for neurotransmitters like GABA (gamma-aminobutyric acid). This function underpins crucial processes in a vast array of human cells, tissues, and organs, ranging from nerves in the brain and the peripheral nervous system to hormone-secreting cells in endocrine glands. As they play such a key role in so many physiological processes, ion channels have long been validated prime targets for drug discovery.

In fact, ion channel pharmaceuticals are already a successful drug class. One example is Lyrica (or Pregabalin), which was ranked as high as #17 in the top 200 drugs by US retail sales in 2018. However, there’s still an ongoing challenge to design and develop more potent and selective therapies with fewer side-effects and greater clinical efficacy.

Q: What challenges are being overcome in the search for novel ion channel targets?

A: We’re now able to better identify and validate both existing and new ion channel genes in human disease. This is thanks to the huge databases of patient blood, tissue samples and DNA sequences coupled with long-term demographic and epidemiological data.

Excitingly, we’re already seeing scientists advancing research using this data. If we look at rare diseases, for example, the genotyping of patients is linking genetic mutations with abnormal ion channel function, such as Na_v (voltage-gated sodium channels) in erythromelalgia and small fibre neuropathy. Furthermore, in common diseases, SNP (single nucleotide polymorphisms) profiling and GWAS (Genome Wide Association Studies) of large patient databases and epidemiological cohorts are revealing the involvement of a broad range of ion channel genes. One notable example is the use of GWAS to confirm a likely contribution of the TRPM8 ligand-gated ion channel to migraine, which should boost the subsequent development of TRPM8 antagonists to treat chronic pain and migraine.

However, in this pharmacogenomics era, we still face significant challenges in target validation. Although GWAS is a valuable technique, it can only tell you if a genetic variant is associated with a specific trait—it doesn’t reveal the underlying cause. Genetic hits therefore need to be robustly replicated and then functionally validated before committing huge resources to using them in major drug discovery efforts.

Q: What underpins the successful discovery of ion channel ligands and modulators?

A: The design of more useful and effective ion channel ligands and modulators has emerged through structure-based drug design, which combines two recent advances. First, the development of more specific and selective pharmacology (e.g. small molecules, toxins, antibodies) and second, the elucidation of ion channel protein structures through cryogenic electron microscopy (cryo-EM) techniques. With more potent and selective ligands, we can more reliably validate the role of specific ion channels in disease through functional assays and animal models.

Q: Can you describe some recent advances in targeting ion channels to treat disease?

A: Disease modelling is a burgeoning area, driven by developments in 2D and 3D human stem cell-derived cultures and CRISPR gene editing. Validated translational assays, such as iPSC (induced pluripotent stem cell) cardiac or neuronal models of an ion channel disease phenotype, can be used to screen for drugs to more effectively treat both common and rare diseases, even on a personalised patient-by-patient basis.

There’s also a growing push to develop biologics that modulate ion channels, which promises greater selectivity and potency, coupled with lower costs of production. One example is the fine-tuning of animal toxins that can target various domains of ion channels. Some of these have made it into the clinic, such as the cone snail toxin Ziconotide for pain. However, administering peptides cost-effectively and easily to patients remains a challenge.

Q: What’s next for ion channel screening and research?

A: My first choice for the ‘next big thing’ is the advancement in cryo-EM techniques and computer modelling. These techniques facilitate the faster generation of high-resolution images of native human ion channel proteins from every major voltage- and ligand-gated family. In fact, the images obtained are already yielding novel chemical ligands and facilitating structure-based drug design.

My second choice is optogenetics, enabled by the development and optimisation of genetically encoded channels and voltage sensors (such as channelrhodopsin and GCaMP) in academia that is now delivering ion channel screening reagents to the drug discovery world. Essentially, optogenetics allows the sophisticated control and measurement of cell membrane potential, excitability and functional outputs in a scalable fashion in native cells. The cells are in a more physiologically relevant state compared to, say, dissociated heterologous cells on an automated patch clamp platform, allowing us to gain insights that are more translatable to the in vivo environment.

I strongly believe that optogenetics could facilitate phenotypic screening with high throughput and high content, but lower cost than traditional techniques. It should also be amenable to study both native rodent tissue and human stem-cell-derived cultures – I imagine that such use could occur both at the top of a screening cascade (for phenotypic screening and target validation), as well as part of mechanistic studies for lead compounds as they move towards the bottom of the drug discovery process..

Q: Do you have any advice for students and early career researchers?

A: It’s essential to find something you’re passionate about and use that drive, as well as mentors and organisational resources, to figure out a career path that allows you to pursue your interests. Ultimately, the onus is on you to do your due diligence and seek out as much advice as you can—and make sure you have a ‘plan B’ to turn to if all else fails!

I’d also encourage students to take advantage of industrial placements and pharma-sponsored postgraduate degrees available here in the UK. Experiencing an alternative R&D environment outside academia is very smart and useful, as it can widen your training and teach you how to ‘do’ science in a way that books and classes can’t.

Q: Do you think attending events like ELRIG’s Drug Discovery 2019 conference can benefit drug discovery scientists?

A: I feel that focused and multi-disciplinary conferences like ELRIG’s Drug Discovery 2019 conference are crucial to disseminating and promoting the enthusiasm and excellence we have for science. It is vital to have a good mix of academic insight and commercial applications.

Additionally, given the uncertainty over Brexit and possible impacts on scientific and commercial links with Europe, I believe it is important to support local experts, laboratories and companies that are part of the thriving R&D sector in the UK. I think events and networks such as the ELRIG Drug Discovery conference are key to making sure that happens.

Q: What can we expect from your BPS symposium talk at the Drug Discovery 2019 conference?

A: I will be presenting a case study from an eight-year drug discovery collaboration we had with a German pharma company to discover and profile selective small molecule inhibitors of the Cav2.2 channel, which is a well-validated non-opioid pain target. My talk represents the effort of a large team of chemists and biologists over many years and several companies, and a celebration of a successful international collaboration in drug discovery.