Written by Dr Sophie Rose and Professor David Sheppard
Transformative therapies targeting the root cause of disease are now available for around 90% of individuals living with Cystic Fibrosis (CF) following the recent FDA and EMA approval of the triple drug combination of Elexacaftor, Tezacaftor and Ivacaftor. Professor David Sheppard (DS) from the University of Bristol presented work undertaken in collaboration with both the University of Manchester and Pfizer investigating the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel in CF and its rescue with small molecules. The presentation focused initially on targeted therapies for the most common cause of CF, the F508del variant in the CFTR gene. This was followed by a summary of work undertaken with Pfizer on a novel CFTR potentiator, which enhances CFTR activity and its use in conjunction with Ivacaftor to restore greater function to faulty channels in CF.
Identification of the defective gene responsible for CF
DS began by highlighting the long road to developing therapies for CF that target the root cause of disease. Work by an army of researchers led by Collins, Riordan and Tsui led to the identification and cloning of the defective gene responsible for CF in 1989. With the CFTR gene identified, researchers raced to identify the function of the CFTR protein and understand how disease-causing CFTR variants lead to a loss of function of this protein.
The structure of CFTR
When the CFTR gene was identified, it was recognised that its protein product was a membrane protein composed of five domains: two transmembrane domains, each with six a-helices which span the lipid bilayer; two nucleotide-binding domains, containing amino acid sequences known to interact with ATP and a fifth regulatory domain, a unique feature of CFTR distinguished by multiple consensus phosphorylation sites. This structure of CFTR placed it in a large family of transport proteins called ATP-binding cassette transporters, found in bacteria, plants and animals including humans. Advances in structural biology, led to the publication in 2016 of the first atomic resolution structure of CFTR, showing a dephosphorylated, ATP-free configuration of CFTR. In this configuration, the transmembrane domains form an inverted V-shape closed towards the outside of the channel, and the nucleotide-binding domains are separated by the regulatory domain. The structure of CFTR in a phosphorylated, ATP-bound configuration reveals that upon phosphorylation, the regulatory domain moves out of the way allowing the nucleotide-binding domains to dimerise and the transmembrane domains to align vertically.
Understanding how the protein functions
In contrast to most ATP-binding cassette transporters, CFTR is an anion channel with complex regulation. DS reviewed the ATP-driven nucleotide-binding domain model of CFTR channel gating developed by Vergani and Gadsby to explain how ATP controls CFTR activity before atomic resolution structures of CFTR were solved. Two ATP-binding sites are formed at the interface of the nucleotide-binding domain dimer. These ATP-binding sites have distinct properties. The first is a site of stable ATP binding, whereas the second is a site of rapid ATP hydrolysis. Once the regulatory domain has been phosphorylated, cycles of ATP binding and hydrolysis at the nucleotide-binding domains control channel gating. ATP binding at both ATP-binding sites is required for channel opening, whereas ATP hydrolysis at the second ATP-binding site leads to dimer separation and prompt channel closure. DS highlighted how transitions between the closed and open channel observed in single-channel recordings represent conformational changes in the CFTR protein driven by cycles of ATP binding and hydrolysis at the nucleotide-binding domains.
How does F508del cause loss of CFTR function?
The CFTR variant F508del (in frame deletion of the phenylalanine residue at position 508 of the CFTR amino acid sequence) primarily causes CFTR dysfunction because it is missing from its correct cellular location, the apical membrane of epithelia lining ducts and tubes in the body. However, if F508del-CFTR reaches the plasma membrane two further defects are observed: defective channel gating and reduced plasma membrane stability.
DS discussed electrophysiology data his group had captured with colleagues at the University of Manchester to demonstrate the impact of the F508del-CFTR variant on channel gating and plasma membrane stability. They used the excised inside-out configuration of the patch-clamp technique and baby hamster kidney (BHK) cells stably expressing wild-type and F508del-CFTR. To deliver F508del-CFTR to the plasma membrane, they incubated BHK cells expressing the variant at 27 °C for 24 hours prior to study. To magnify the small size of CFTR channel openings, a large chloride concentration gradient was used and voltage was clamped at -50 mV. ATP and protein kinase A (PKA) were continuously present in the intracellular solution to activate and sustain CFTR channel activity. By studying CFTR channels at 37 °C, the impact of the F508del variant on channel rundown, a measure of the plasma membrane stability of CFTR was assessed.
F508del slows CFTR channel opening
Low temperature-rescued F508del-CFTR has a severe gating defect which greatly slows channel opening. As a result, the open probability (a measure of channel activity) of F508del-CFTR is greatly reduced compared to that of wild-type CFTR. Using prolonged channel recordings, DS demonstrated that at 37 °C F508del-CFTR is unstable and runs down within 10 minutes even in the continuous presence of PKA and ATP in the intracellular solution. He explained that the rundown of F508del-CFTR reflects not only changes in channel gating, but also current flow through the channel evident by openings to a sub-conductance state during rundown. DS emphasized that channel rundown makes studying the function of F508del-CFTR and its rescue by small molecules particularly challenging.
DS summarised that F508del-CFTR has multiple mechanisms of CFTR dysfunction including defective delivery of protein to the plasma membrane, perturbed channel gating and reduced protein stability at the plasma membrane. He emphasized that most CFTR variants that had been studied to date disrupt CFTR function in multiple ways. Very few variants, including the CFTR gating variant G551D, cause CFTR dysfunction by only a single defect.
Combinations of correctors and potentiators repair F508del-CFTR
To rescue F508del and other disease-causing CFTR variants, requires two types of CFTR-targeted therapies, correctors and potentiators. Correctors, such as Tezacaftor and Elexacaftor, allow misfolded CFTR variants to escape from the endoplasmic reticulum and traffic to the Golgi apparatus for maturation before delivery to the plasma membrane. By contrast, potentiators, such as Ivacaftor, enhance CFTR channel gating once the protein is phosphorylated by PKA. The combination therapy Elexacaftor-Tezacaftor-Ivacaftor is a CFTR-targeted therapy for most people with CF.
A novel CFTR potentiator – CP-628006
DS then spoke about the characterisation of a new efficacious CFTR potentiator developed by Pfizer, CP-628006 (referred to as CP). CP was identified by Pfizer following a high-throughput screen of a 150k compound library. It has a distinct chemical structure to known CFTR potentiators and efficaciously potentiated F508del- and G551D-CFTR in Fischer Rat Thyroid (FRT) epithelia heterologously expressing CFTR and human bronchial epithelia from individuals with CF and the genotypes F508del/F508del and F508del/G551D.
Using single-channel recording, CP was shown to have reduced potency, but similar efficacy to Ivacaftor, enhancing channel activity by increasing the frequency and duration of channel openings. Interestingly, CP restored wild-type CFTR levels of channel activity to F508del-CFTR, but not G551D-CFTR.
To learn about how CP enhances CFTR activity, the group at Pfizer/ University of Bristol examined the ATP-dependence of channel gating for WT-CFTR, F508del-CFTR and G551D-CFTR in the absence and presence of either CP or Ivacaftor. For F508del-CFTR, channel activity was weakly ATP-dependent. Both potentiators restored some ATP-dependent channel gating to F508del-CFTR. In the case of G551D-CFTR, channel gating was ATP-independent. Ivacaftor potentiated G551D-CFTR activity similarly at all ATP concentrations tested, demonstrating that it enhances ATP-independent channel gating of G551D-CFTR. By contrast, potentiation of G551D-CFTR by CP was ATP-dependent. This result indicates that CP restores some ATP-dependence to G551D-CFTR.
The distinct effects of CP compared to Ivacaftor suggest a different mechanism of action. This encouraged the group to test combinations of the two potentiators. They found that CP and Ivacaftor together enhanced the channel activity of G551D-CFTR but not that of F508del-CFTR. DS explained that studies by other investigators have also demonstrated that some CFTR variants are receptive to combinations of two potentiators, whereas others are not. DS speculated that greater clinical benefit might be achieved by combinations of CFTR potentiators.
CFTR-targeted therapies have transformed the treatment of CF. Around 90% of people with CF will likely benefit from Elexacaftor-Tezacaftor-Ivacaftor combination therapy. However, DS emphasized that there is still much research to be done. An urgent priority is to develop drug therapies for the last 10% of individuals with CF who have CFTR variants unresponsive to current CFTR-targeted therapies. Ultimately, the aim is to cure CF.
I completed my undergraduate degree with a BSc in Natural Sciences at the University of Bath in Summer 2021. I chose to major in pharmacology and minor in biology which was an excellent way to explore a breadth of different subjects, whilst also providing a depth of scientific understanding.
I particularly enjoyed studying various neuroscience units and learning about the intricate mechanisms underlying the physiological processes in the brain. I also developed an interest in the ways in which the brain can malfunction and how this can lead to a range of neurological diseases.
A placement at the Barrow Neurological Institute
During the third year of my degree, I was fortunate enough to be able to complete a placement year at the Barrow Neurological Institute in Phoenix, Arizona, where I joined the Whitaker-Lukas lab. I investigated the effects of the modulatory prototoxin, lynx1, on a subtype of nicotinic acetylcholine receptors as part of a project studying nicotine addiction. Through the year, I developed the technique of two-electrode voltage-clamp electrophysiology and thoroughly enjoyed this process! Not only did this experience expand my molecular biology skillset at the bench, but it also helped me to develop an appreciation for the elaborate nature of ion channel function within the brain. It was also an incredible opportunity to live in and explore another part of the world, and I am still very grateful for the experience!
My work so far at Metrion Biosciences
After completing my placement, I was particularly intrigued by the clinical potential of ion channels, particularly in neurological disorders. After graduating, I knew I wanted to continue to develop my knowledge of ion channels – as well as expand my abilities at the bench. I recently joined Metrion Biosciences exactly for these reasons and I am grateful for the opportunity to develop skills within manual and automated patch clamp electrophysiology. There are many fascinating ongoing drug development projects, and I am enjoying the challenge of making recordings from various ion channels. I look forward to continuing to develop within this role and contribute to various projects across the company.
The second instalment in the ‘Ion Channels in Drug Discovery’ webinar series was presented by Professor Stephen Tucker from the Department of Physics, University of Oxford. Stephen delivered a presentation titled:
“Defective X-gating caused by de novo mutations in the TASK-1 K2P channel (KCNK3) underlies a developmental disorder with sleep apnoea”.
The work presented by Stephen is available as a pre-print in MedRxiv. The work was supported by Bayer who provided the TASK-1 blockers which were used in the study and are currently being advanced through clinical trials.
The origin of leak conductance
Stephen has spent much of his career working on the two-pore domain (K2P) and inward rectifier (Kir) families of potassium channels. K2P channels produce background leak currents that contribute to regulating the resting membrane potential. The first reported mammalian K2P channel, TWIK-1 (Tandem of pore domains in Weak Inward rectifying K+ channel 1, often referred to as KCNK1 or K2P1) was identified over 25 years ago and is now known to be one of 15 members in the human genome. These channels can be highly regulatable and finely tuneable; many exhibit polymodal regulation by diverse stimuli and cell signalling pathways, including GPCRs.
When K2P channels go wrong
K2P channels are expressed in a wide range of different tissue types where they have important roles in controlling physiological processes. Therefore, it is not surprising that variants that affect the function of K2P channels can result in a wide variety of disorders, which range from migraine to pulmonary arterial hypertension. The membrane topology of K2P channels differs significantly from ‘classical’ potassium channels, as each K2P channel subunit contains two pore forming domains and functional channels are composed of two subunits.
TASK channels are a subset of the K2P channel family, which are expressed in many excitable and non-excitable tissues, including the pancreas, brain, lung, heart and kidney. Loss-of-function mutations in TASK-3 (KCNK9) can lead to a condition known as Birk Barel syndrome, an inherited condition characterised by intellectual disability, hyperactivity and unusual facial features. Most cases result from a mutation in the M4 helix, which produces almost complete loss-of-function. The condition demonstrates dominant inheritance with paternal imprinting. Furthermore, loss-of-function mutations in TASK-1 (KCNK3), (either homo- or heterozygous) have been linked to the pathophysiology of pulmonary arterial hypertension; a rare, progressive disorder.
What is the link with sleep apnoea?
TASK-1 (KCNK3) is also implicated in sleep apnoea, which is a disorder where breathing repeatedly stops during sleep. Sleep apnoea affects up to a billion people worldwide. and there are two main types:
Central which is caused by defective circuits that control beathing;
Obstructive which is caused by a physical obstruction of the airways.
TASK-1 channels are expressed in the chemosensitive regions of the brain that are involved in the control of breathing. Research showing the involvement of TASK-1 in sleep apnoea led Bayer to develop highly potent, highly selective inhibitors of TASK-1 channels that are currently in clinical development (phase 2).
A new channelopathy associated with TASK-1 (KCNK3)
The link between sleep apnoea and TASK-1 came from a recent publication based on the results of a large study of 31,000 parent-offspring trios. During the study, 28 novel genes were identified, including KCNK3 (TASK-1), that were associated with neurodevelopmental disorders such as developmental delay. A total of 9 probands, each heterozygous for one of six separate de novo missense mutations in KCNK3 were identified. Patients shared a common phenotype consisting of developmental delay with various limb abnormalities. Additionally, they also suffered from sleep apnoea that necessitated the use of nocturnal oxygen. The authors of the study termed the disorder, Developmental Delay with Sleep Apnoea (DDSA). Due to his expertise with TASK-1 channels, Stephen was contacted by the study leader to help characterise the mutant TASK-1 channels.
Obtaining structural information about the TASK1 channel
Stephen explained how his team obtained crystal structures of the TASK-1 channel (published in Nature in 2020). Interestingly, they identified that TASK-1 contains a lower gate, which they designated as an ‘X-gate’, which is created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This region is known as the halothane response element (HRE) and is important for TASK channel gating. The structures were co-crystalised with the Bayer inhibitors and the drug binding site was found to sit just below the selectivity filter. The six most prevalent mutations identified in DDSA are clustered around the X-gate.
Probing the functional characteristics of the channel using electrophysiology
Using the voltage clamp technique, it was observed that the mutant TASK-1 channels displayed a prominent gain-of-function phenotype, where dramatic increases in current amplitude were identified. The gain in function occurred irrespective of whether one or both of the channel subunits contained a mutation.
Single channel recordings performed from channels expressed in Xenopus oocytes, demonstrated a higher open probability for the TASK-1 mutants. N133S (TM2 mutation) and L239P (M4 mutation), were associated with a ten-fold increase in single channel open probability, but with no change in conductance. These mutations appear to be involved in interfering with interactions that stabilise the X-gate in its closed state. For example, N133 generates a backbone hydrogen bond with the M4 helix. When this residue was mutated, the H bond was disrupted, which led to a gain-in-channel function.. The group confirmed this result using a molecular dynamics simulation of the TASK-1 structure; wild type versus N133S and L239P. The two mutations were found to promote opening of the X-gate.
What regulates the X-gate?
Previous studies which were performed almost 20 years ago, showed that mutating the X-gate renders TASK-1 resistant to GPCR mediated inhibition. Interestingly, all of the TASK-1 mutants associated with DDSA were resistant to GPCR inhibition, which may underlie what is happening with the L239P mutation. It was also found that the mutant channel, N133S, is not inhibited by either diacylglycerol or anandamide.
Can the mutant channels still be pharmacologically modulated?
A critical question regarding the mutant TASK-1 channels associated with DDSA, is whether they can be modulated by inhibitors of wildtype TASK-1. Bayer’s TASK-1 blocker, BAY 2253651, which is being developed for the treatment of obstructive sleep apnoea, inhibits TASK-1 with a sub nanomolar IC50. Fortunately, electrophysiological assessments identified that the mutations associated with DDSA are still sensitive to BAY 2253651; the compound is capable of significantly inhibiting the mutant channels.
Possible genotype/phenotype correlation
Mutations located in the M4 region produce the smallest increase in whole cell current amplitudes; patients with mutations in the M4 region presented with less severe phenotypes in general, exhibiting fewer limb abnormalities and less severe developmental delay. It will become clearer, with further research, whether this observation is consistent with other mutations that are identified in the M4 region. However, further work is required to further characterise the mutant channels, as these experiments were performed using Xenopus oocytes, rather than mammalian cells, and do not take into account factors such as hetero-multimerization with TASK-3 or coupling to different GPCRs.
Wild type TASK-1 channels or heteromeric TASK-1/ TASK-3 channels cause hyperpolarization of the cell and play an important contribution in controlling the resting membrane potential.
GPCR stimulation results in inhibition of these channels, which may lead to depolarization of the resting membrane potential.
Gain-of-function TASK-1 mutations have been identified in patients with DDSA.
The gain-of-function phenotype is caused by a defective X-gate, which also renders the channels resistant to GPCR induced inhibition.
Fortunately, the mutant channels can be modulated by inhibitors of wild type TASK-1 channels. Therefore, there is a potential opportunity to treat younger patients.
Importantly, the results generated in Stephen’s lab strengthen the link between overactive or dysregulated TASK-1 channels and sleep apnoea.
It will be very interesting to see the final outcome of the Bayer clinical trials.
Metrion Scientist Dr Alexander Howarth summarises below his undergraduate and postgraduate studies and how he came to join Metrion Biosciences.
“In 2015, I graduated from the University of Sheffield with a BSc in Biomedical Sciences. The degree was fascinating and provided an in-depth insight into human biology, from endocrinology to neuroscience, immunology to cancer biology, and even human dissection! I was fairly certain on wanting to explore research from early on in my degree. I completed a research placement in the Summer of 2014 within the department investigating the role of purinergic signalling in mast cell chemotaxis, which reassured my aspirations to proceed with a career in research.
My next dilemma was deciding which field or area of research to pursue. I was most interested in neuroscience and cancer biology, as well as learning as many techniques as I could manage. Eventually, I successfully applied for a BBSRC-funded PhD in the Brackenbury lab at the University of York, researching the role of voltage-gated sodium channel function and regulation in breast cancer cells. I employed a range of techniques to investigate the sodium channel β1 subunit, specifically, including fluorescence imaging, biochemistry and above all, patch clamp electrophysiology.
I employed a range of techniques to investigate the sodium channel β1 subunit, specifically, including fluorescence imaging, biochemistry and above all, patch clamp electrophysiology.
DR ALEX HOWARTH
I graduated in 2020 after presenting at multiple conferences and publishing a first author article and was faced with the scary prospect of finding my first full time job, compounded by the pandemonium of the COVID pandemic. I wanted to utilise the skills that I had learnt during my PhD to find a stable job in research. I came across the advert for an electrophysiologist position at Metrion Biosciences, which promised a meaningful, industrial application for my electrophysiology skills alongside continual personal development, and instantly applied.
I started in November 2020 and have been enjoying the job ever since!”
Metrion Scientist Dr Peter Matthews summarises below his path into electrophysiology and recounts his experiences during his undergraduate and postgraduate studies and beyond.
“I first became interested in the physiology and pharmacology of ion channels during my undergraduate MSci Pharmacology degree at the University of Bristol. This course introduced me to the diverse world of voltage-gated and ligand-gated ion channels, later focussing on the mechanisms by which these membrane proteins function, and how small molecule drugs can correct aberrant activity present in various neurological disorders.
Having developed a passion for studying ion channel function, I sought to gain hands-on laboratory experience with recording their activity. This led me to undertake a 12-month research placement in neurophysiology at the University of Exeter, under the supervision of Prof Andrew Randall and Dr Jonathan Brown. This placement year equipped me with experience in in vitro electrophysiology, performing extracellular field-potential recordings with multi-electrode probes in brain slice preparations studying the propagation of epileptiform activity. This placement introduced me to the fundamentals of not only electrophysiology, but also surfing, having enjoyed summer trips to north Devon. I then returned to Bristol for the 4th year of my studies, intent on completing an ion channel-based neuroscience PhD.
In the final year of my MSci degree, I developed a keen interest in ionotropic glutamate receptors (iGluRs) (AMPA, NMDA, kainate) and their contribution to excitatory synaptic transmission, responsible for information transfer and storage in the brain. Lectures from Prof David Jane sparked my interest in glutamate receptor function and drug design, and a series of lectures from Prof Graham Collingridge on synaptic plasticity highlighted their role in the process of learning and memory. These lectures provided me with an appreciation of iGluR function from a molecular level to physiological function at excitatory synapses – ultimately shaping the choice of my PhD project.
“This role has given me the opportunity to further improve my skillset, having established single-channel recording from outside-out patches, and become familiar with a diverse selection of voltage-gated and ligand-gated ion channels.”
Dr Peter matthewS
I then secured a place on the PhD programme at the MRC Laboratory of Molecular Biology (LMB), University of Cambridge. Here, I studied the molecular mechanisms of AMPA receptor function at excitatory synapses under the supervision of Dr Ingo Greger and Prof Ole Paulsen. Ultimately, my PhD aimed to understand the molecular processes of fast point-to-point neuronal communication in the mammalian brain. More specifically, this project aimed to identify and characterise the physiology of protein interactions with the AMPA receptor (recent review: https://doi.org/10.1016/j.neuropharm.2021.108709). This invigorating project introduced me to a wealth of novel molecular biology and biochemistry techniques and further expanded my electrophysiology toolset. In between punting on (and falling into) the river Cam, my time at the LMB was spent performing dual whole-cell patch clamp recording in hippocampal brain slices and applying glutamate onto excised outside-out patches of AMPAR expressing cells. These electrophysiological techniques were used to elucidate the function of AMPA receptors at hippocampal synapses, crucial for understanding signal transduction in the brain. Nearing the completion of my PhD, I sought to next apply my experience with electrophysiology to a fast-paced drug development setting to immerse myself in a new, energising scientific environment.
Recently, I started as an electrophysiologist at Metrion Biosciences, where over the past 3-months I have been involved in several different drug development projects. This role has given me the opportunity to further improve my skillset, having established single-channel recording from outside-out patches, and become familiar with a diverse selection of voltage-gated and ligand-gated ion channels. I am thoroughly enjoying my time at Metrion, working with experienced, friendly scientists, whilst constantly challenging myself to learn new skills. I look forward to developing as a scientist in this role and being a part of the company’s continued growth!”
Metrion’s first “Ion channel drug discovery” webinar
Metrion Biosciences’ inaugural ion channel webinar took place at the end of June and featured a presentation by Dr Jon Lippiat, from the School of Biomedical Sciences at the University of Leeds. Jon presented an inspiring and thought-provoking talk focusing on his recent work which involved using structure-based virtual screening techniques to identify novel inhibitors of the hKNa1.1 channel. Prior knowledge of the structure of the channel, including insight into the potential binding domains had been gained using cryo-electron microscopy. Attendees gathered from across the globe and there was a brief introduction prior to Jon’s presentation and a spirited question and answer session afterwards, both presented by Metrion’s CSO Dr Marc Rogers.
The importance of the KNa1.1 channel and the journey to find novel inhibitors
Jon focused his presentation on the KNa1.1 ion channel, which is encoded by the KCNT1 gene and is one of the 4 SLO-related, RCK-domain containing human potassium channels. The channel is tetrameric and comprised of six transmembrane segments. Jon’s group have been studying the epileptic disorders associated with mutations of the KCNT1 gene, which unfortunately are not well controlled by anti-epileptic drugs. Sadly, many children suffering from these symptoms are severely affected and their life expectancy is reduced so there is a high unmet clinical need. Generally, all mutations are heterozygous and dominant with mutations resulting in gain-of-function. Prior to the year 2020, the only known inhibitors of this channel were Quinidine, Bepridil and Clofilium, all of which are rather non-specific, lack potency, and are associated with significant side-effects. The only inhibitor trialled in clinical use is quinidine, in an attempt to suppress over-active channels in these patients. However, due to its effects on ion channels in the heart, quinidine dosing is very limited. To understand how inhibitors interact with the human KNa1.1 channel and identify new inhibitors, the published structures of the chicken KNa1.1 homologue have been used for virtual docking and mutagenesis studies.
Uncovering the mutation required for channel inhibition
KNa1.1 can be studied using whole-cell recordings from HEK293 cells transiently transfected with wild-type or mutant human KNa1.1 channel proteins. Channel activation is weakly voltage-dependent and slowly time dependent compared with other potassium channels. It was clear from Jon’s work that phenylalanine residue 346 (F346) is required for KNa1.1 inhibition by both Quinidine and Bepridil. A disease-causing mutation (Phe to Leu switch) has since been published. Mutations of F346 cause an increase in the IC50 to at least ten-fold higher concentrations with both Quinidine and Bepridil, in line with the docking studies.
Screening of a large virtual compound library
To further probe the structure of the active pore domain, Jon then discussed how a virtual library of 100,000 compounds was screened to give predictions of free energy changes and suggest which drug compounds may induce channel inhibition. They supplemented this with a ligand-based approach whereby compounds similar in structure to Bepridil were selected and purchased for further functional validation. They completed functional analysis of 17 compounds at 10mM against the wild-type channel, using manual patch clamp electrophysiology whole cell recordings.
The active compounds were counter-screened against the channel carrying the core F346 mutation and evaluated in concentration inhibition experiments. IC50 values versus the wild-type channel and the Y796H disease mutation were plotted and compared to the data observed for both Quinidine and Bepridil. 6 of the 17 compounds showed inhibition of the human KNa1.1 channel at 10mM, and so additional compounds with similar structures were purchased and tested. Small changes to the basic structure resulted in a loss of inhibition of the channel. This demonstrates the specificity of the docking approach and the significant involvement of the inner side of the channel pore. Terminal trifluoromethyl groups are a common feature of the compounds in their virtual screens and this motif is known to position itself within the pore of the channel.
Preliminary safety/ toxicology findings
This structure-based approach delivered compounds with potential to inhibit other potassium channels, such as hERG. This potential cardiac safety risk was probed in more detail and it was found that only three of these compounds exhibited strong hERG inhibition, demonstrating good potential for developing potent, safe and selective KNa1.1 modulators. Jon’s group also investigated cytotoxicity using HEK cells incubated overnight in a cell viability assay, using positive controls 10% DMSO and Blasticidin. The selected compounds showed very little cytotoxic effect across the concentration range, increasing their potential as starting points for further drug discovery efforts.
KNa1.1 channel inhibitors
Jon then discussed further KNa1.1 channel inhibitors which have been published. He highlighted studies published by a group at Vanderbilt University (this involved a thallium flux fluorescent HTS assay) and by Praxis Precision Medicine (Cambridge, MA). Praxis employed high throughput rubidium flux and atomic absorption spectroscopy to identify hits and develop their lead compound. These compounds appear to have efficacy in reducing rodent CNS epileptic activity in vitro and in vivo.
The structures of the various KNa1.1 channel inhibitors described in the presentation were given, which have been identified and published over the past year and a half. Compounds denoted “BC5”, “BC6” and “BC7” came through the ligand-based approach and are structurally similar to Bepridil, but strongly inhibit hERG channels. The frequent appearance of the trifluoromethyl group in docked compounds remains important and two of these compounds displayed this motif. One of the compounds was particularly potent at inhibiting wild-type channels, but less so for those containing disease causing GoF mutations.
Progressing structure-based approaches
Having demonstrated that structure-based virtual screening can deliver novel inhibitors of the hKNa1.1 channel, this work has been expanded to another virtual screen using a 9 million compound ZINC library on the High-Performance Computing ARC3 system located at the University of Leeds. This adds an extra layer to the workflow and enables the screening of much larger virtual compound libraries and the further characterisation of additional and novel hit compounds. The group have also developed a concatemeric construct KNa1.1 WT-T2A-Y796H and a stable cell line which will be used in medium throughput fluorescence and automated patch clamp experiments. Jon’s colleagues have also been generating their own ion channels structures at the University of Leeds using cryo-electron microscopy to determine how drugs interact with a number of different ion channels implicated in human disease.
Jon concluded that high resolution cryo-EM structures of KNa1.1 can be used both to model inhibitor binding and in virtual HTS. There is a high degree of homology between species and strongly conserved domains. This enables the use of non-human protein structures. The potential success of this approach is that a small-scale virtual screen can be evaluated initially using libraries of reasonably priced and readily available screening compounds to ascertain the hit rate before embarking upon a larger functional evaluation. The limitation here however is that knowledge is required of a viable binding site. To escape potential cross reactivity issues, it would be favourable to target other parts of the protein structure, such as sodium binding sites. The study has resulted in a selection of tool compounds for further characterisation and to probe channel function, and Jon’s group hope to be able to address the high unmet clinical need associated with KCNT1 associated epileptic disorders using this combined approach. The project was supported by the BBSRC, UKRI, Autifony Therapeutics and Action Medical Research for Children.
The full presentation including the Q&A session is available to all registrants. If you did not register for the event but are interested in accessing the presentation please email: email@example.com.
Our guest blog, written by longstanding Friend and Collaborator Steve Trim, CSO and Founder of Venomtech is presented below.
An early career at Pfizer, Sandwich
“Why spider man is joining forces with ion man” is a newspaper headline I’ll never forget, and one of the few puns about my work that I really liked. This heralded the announcement of Venomtech partnering with Metrion Biosciences in 2017, but my interest in ion channels goes back much further.
After my degree, I moved to East Kent as a molecular biologist at Pfizer Global Research and Development in 1999. Prior to this I only knew of Sandwich as my favored portable lunch, but now I know it as the nearest town to this cutting-edge research institute. It was an exciting introduction to the drug discovery process and included preparing delicate scientific instruments for the oncoming millennium bug. In the mid 2000’s I got the opportunity to join the pain therapeutics team and this started the journey leading to me writing this blog nearly 20 years later.
The involvement of ion channels in pain
Pain affects most of us at some point or another throughout our lives and if you are lucky, this is only acute pain from the mild trauma of misadventure such as stubbing your toe or a hangover. But for millions of people worldwide, pain is a debilitating disease that is difficult to control. Pain processing is a complex conglomeration of pathways ending with even more complex interpretation and emotional overlay in the brain. Ion channels are critical components all along the process from detection to final interpretation of painful sensations. When this process goes wrong chronic pain can become a debilitating disease in itself and this is the front line of therapeutic development.
The role of venoms in ion channel drug discovery
Ion channels are very complex transmembrane proteins and often have conserved structures around ion pores and drug binding domains, making modulation a significant challenge but also an intriguing one. Drugs like Lidocaine deliver good target engagement and local anesthesia without involving motor neuron ion channels, however Lidocaine also does a good job in blocking cardiac ion channels and hence is not systemically useful. This lack of functional and tissue selectivity highlights the challenge of ion channel drug development and is where venoms come in. Evolution has produced endless forms most beautiful and most wonderful at both the species and protein levels. Natural selection is relatively quick to build on any opportunity and modulating ion channels has turned out to be a very common method of subduing prey and warning off predators.
‘Poisonous’ versus ‘Venomous’
Tetrodotoxin is a famous toxin from the Fugu sp pufferfish which blocks sodium ion channels and thus is a formidable defense, and intriguingly it is also found in other aquatic animals such as blue ringed octopus. However, even though the toxin is most likely produced by similar microbes, these animals differ in their delivery of this toxin. Puffer fish are poisonous (the predator does the biting) whereas the octopus is venomous, actively delivering the toxin through a beak-like mouth. Correcting people on the difference between poisonous and venomous has become a constant amusement having called the company Venomtech (and not poisontech 😊). Evolution has had millions of years to hone venoms to block ion channels to stop prey escaping or open pain signaling ion channels to give predators a strong message. So, it’s not a surprise that having only had about a hundred years of concentrated drug discovery, humans are lagging behind nature in selectively and efficaciously modulating ion channels.
The importance of venom-based screening libraries and the creation of Venomtech
It was the abundance of scientific papers on venom peptides, particularly those from Theraposidae (aka tarantula) spiders, that seeded the idea that venom-based screening libraries were needed to unlock the full potential of venom peptides for drug discovery. So, when I got made redundant from Pfizer, I found myself with an idea and some key skills. This included an understanding of the need for better drugs, an understanding of venoms and venomous animals, combined with pharmaceutical health and safety training. I formed Venomtech in March 2010 with the brave ambition to help research around the world to find new tools to treat the most challenging diseases, especially pain. In order to do this, I had to solve the problem of supply and thus had to go back to the source.
Creating a diverse collection of venomous animals
This involved building a lab with the capacity to safely house a varied collection of venomous animals in order to have the diversity needed in the compound library I was assembling. Amazingly enough, acquiring the venomous animals was actually the easy bit, and innovating the safe systems of work was relatively straightforward with my safety training, but was not in common use. We published several papers and even a patent on these novel safe systems which put us in good stead as I grew the team and our customer base.
The next stage was in separating out this phylogenetically and geographically diverse collection of venoms to get the compound library I needed to hit diverse ion channels and nearly all other targets. People often ask us how do you collect the venom and how safe are the animals to work with? Well, my top answer to the first question is always carefully! And the second answer is venoms are a lot safer in the plate than when they are packaged with a brain and injection system! So, it was a steep learning curve to build a lab and then populate it with a large collection of venomous animals, to then safely and recoverably collect their venom. But we did and eleven years later, I’m still fascinated with ion channels, have developed a cosmetic ion channel blocker and spend a lot of time working to develop venom peptides from hits in plates into drug-like molecules.
Adoration of the natural world
Managing any company through the pandemic is tough, but our venomous chemists still got the same care and attention as always, and the global spotlight on science and drug discovery has also been useful. I still relish those opportunities to work with the animals as it keeps my adoration of the natural world fed at a macro level in between the daily fascination of the molecules of biology. I don’t think I’ll ever loose the fascination of ion channels at the interface between biology and electricity. Even though I work with many targets and disease areas, neuroscience has a special place in my own bundle of neurons producing these words.
Metrion and Venomtech have collaborated on a poster, entitled: “Identification of novel scorpion venom peptide inhibitors of the Kv1.3 ion channel and their potential as drug discovery leads for human T-cell mediated disease.” This can be found here (2nd poster, 2018).
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 Cav3.3 channelopathies
The important role of Cav3.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 Cav3.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 Cav3.3 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 Cav3.3 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 Cav3.3 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 Cav3.3 biophysics or expression. Unexpectedly, several of the ‘unaffected’ patient mutations reduced Cav3.3 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 Cav3.3 biophysics
In collaboration with Diane Lipscombe at Brown University, schizophrenia mutant Cav3.3 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 Cav3.3 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 Kv7.2/7.3 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 (Kv7.2/7.3) 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, Kv7.1 modulators carry cardiac risk, whilst neuronal Kv7x openers may have utility to reduce firing in pathophysiological conditions and diseases such as pain, epilepsy, ALS and depression.
Screening for Kv7 modulators
Due to the complex biophysics of Kv7 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 Kv7 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 Kv7 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 Kv7 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 Kv7.1), 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 Nav 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 Nav 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 Nav1.7 inhibitory toxin Pn3a. Thus, although gympietides may not be particularly selective for Nav1.7 (question from Sam Goodchild at Xenon and Tianbo Li from Genentech), their novel sequences and potential as a tool compound could be useful for Nav-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.
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.
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.
Written by Dr Marc Rogers, Chief Scientific Officer, Metrion Biosciences.
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
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
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
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
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
Marc Rogers PhD
Marc trained as a physiologist and neuroscientist in New Zealand and Australia before graduating with a PhD from the John Curtin School of Medical Research at the Australian National University in Canberra, Australia. He then embarked on an extensive postdoctoral career in the US which included fellowships at Baylor College in Houston, Texas, the University of Hawaii in Honolulu, and UCSF in San Francisco. Marc then left academia to start a new career in ion channel drug discovery, beginning at Exelixis in the Bay Area before moving to the UK in 2005 to work at Xention on voltage- and ligand-gated ion channels involved in atrial fibrillation, immunology and pain. Marc then led the management buy-out of the biology group to create Metrion Biosciences in 2015.
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.”