My interest in Neuroscience started when I was sixteen and I became a volunteer for my local Parkinson’s UK branch. My volunteering with the charity gave me exposure to the world of neuroscience research and motivated me to pursue my scientific studies to degree level. I graduated with a BSc in Biochemistry from the University of Bath in 2017. I was attracted to the degree programme for the breadth of knowledge covered and the ability to select modules from the Schools of Biological Sciences, Pharmacy and Pharmacology, and Chemistry. I chose to focus on modules related to neuroscience, allowing me to study the basics of neuronal function and how this can relate to neurological disorders. As part of my degree, I undertook a placement year at Oxford Immunotec, working in their diagnostics laboratory carrying out routine blood tests for TB. This taught me valuable lab skills and how to work within a heavily regulated industry.
Katie Puddefoot – Scientist, Metrion Biosciences
Industry experience before returning to academia
Following my time at the University of Bath, I moved back to Oxford, where I worked as a Laboratory Assistant at ProImmune Ltd., in their Cellular Analysis Services department. Here, I further expanded my bench skills, learning a variety of molecular biology and cell culture techniques. My experience within industry gave me an insight into the world of biotechnology and made me want to return to academia to gain a PhD, so that I could further climb the industrial ladder.
Trying my hand at manual patch clamp
In 2018, I started my PhD in Neuroscience at the University of Leicester and was finally introduced to the world of electrophysiology. I was fortunate enough to be funded by the Midlands Integrative Bioscience Doctoral Training Partnership, giving me the opportunity to carry out a three-month rotation in the lab of Dr Mark Wall at the University of Warwick. Here, I learnt manual patch clamp techniques in rat brain slices, studying the effects of adenosine on thalamocortical neurons. Taking these newly learnt skills, I returned to the University of Leicester to the lab of Dr Jonathan McDearmid to complete my PhD project. My PhD focused on how pharmacological inhibitors of the proteasome affect the synaptic and intrinsic properties of larval zebrafish motoneurons and neuromuscular junction function and formation. My PhD showed me that of all the techniques I had learnt to date, electrophysiology is the one I felt I had the most affinity for.
My new role at Metrion
I knew that following my PhD I wanted to pursue a career within industry where I could continue to expand on my electrophysiology skills. Hence, I attended the Cambridge Ion Channel Forum in Spring 2022, hosted by Metrion Biosciences and AstraZeneca. I was impressed with the work presented at the forum, so I applied for a Scientist role at Metrion Biosciences. I am excited for the opportunity to expand my electrophysiology skills to the world of ion channel drug discovery and to be working back in industry once again.
In 2014, I graduated from the University of Manchester with a degree in Cognitive Neuroscience and Psychology with Industrial/Professional Experience. In the first weeks of my degree, my classmates and I were asked by our personal tutor (an electrophysiologist) to give a group presentation on Erwin Neher and Bert Sakmann, and their 1991 Nobel Prize for the development of the patch clamp technique. Although my undergraduate studies were very niche and covered a wide range of both neuroscience and psychology topics, ion channels and patch clamp continued to fascinate me whilst others seemed to shy away. Thus, I was very excited to gain an industrial placement position at Novartis, Horsham, where I used the automated QPatch system to screen compounds against TMEM16A and to conduct a mutational study investigating both channel function and compound binding. There, I also learnt a lot about the drug discovery process and the undeniable value of multidisciplinary research.
Catherine Hodgson – Metrion Biosciences
A Different Path
Circumstances led me on a slightly different path post-degree. I first worked in the third sector in research developing a mental health programme for drug and alcohol treatment services, and then on a neuroimaging project at the University of Manchester studying the effects of maternal mental illness on the development of language recognition in infants. Although I value those years for the skills and maturity I gained, I missed life in the lab and ion channels.
Back to the World of Ion Channels!
In 2018, a position opened up at the University of Leeds for a Research Assistant in Ion Channel Pharmacology. The interview for this position introduced me to a couple of academics at Leeds who encouraged me to keep pursuing a PhD rather than one year of postgraduate study. I took their advice and that year I commenced my PhD in Leeds with Drs Jon Lippiat and Ste Muench examining the molecular and structural bases of the Kv4.2 complex. Throughout my PhD, I expanded my lab skillset to include some structural techniques and manual patch clamp. Although I quickly appreciated that you cannot learn patch clamp from a textbook or watching someone else and it had to be a case of failing a thousand times over at first, I soon found my enthusiasm for ion channels again.
Beyond my PhDand starting at Metrion
Considering my career post-PhD, I felt I wanted variety and to venture back into ion channel drug discovery. My PhD supervisor, Jon, gave the first Metrion webinar on KNa1.1 inhibitors and through him, the webinar series and my own research, I was impressed to learn about the range of services Metrion offers, what I could be taught, and the values the company upholds. Thus far, everyone has been very welcoming and using the QPatch again has been like riding a bike!
Prior to joining Metrion in August 2022, I studied at the University of Lincoln for four years. My undergraduate degree was in Criminology, in which I learned many different things ranging from the nature/nurture debate as to whether a criminal is born or made. This encompassed conversations surrounding Freudian theory and how criminality comes about. I also enjoyed studying modules about the prison system and whether it is outdated with the way criminals are treated and dealt with by the criminal justice system. I spent my third year writing my dissertation on the criminal justice system and prisons and their effect on prisoner’s mental health. My findings revealed that the system should try to increase the use of rehabilitation as an alternative to punishment as criminals are more likely to respond better and are less likely to reoffend in the future.
Yasmin Henry – Metrion Biosciences
Business Management
After my undergraduate degree, I decided to do a Master’s degree in business management in which I decided to stay at the University of Lincoln. Within this degree, I studied many different modules such as entrepreneurial capability in which I was able to create my own new product. This was a fitness QR code that could be sold to gyms and individuals and used to see different fitness regimes and track their progress. The QR code would continually update itself to keep the regimes fun and new, as this is where a lot of people lose motivation in fitness. I also studied modules such as international marketing, finance and organisational psychology which I found very interesting. I have come to learn that I use the skills I acquired within this degree as part of my everyday job at Metrion. This includes aspects of the marketing modules, which I am putting into practice regularly, especially the digital side including social media.
My role at Metrion
Since I started working for Metrion I have found that my role is extremely varied and I have the chance to work with many different people, where I can learn a lot of new skills and develop professionally. I have loved every minute of this so far and I can’t wait to see what my future within Metrion brings!
Written by Dr Sophie Rose and Professor David Sheppard
Introduction
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.
Professor David Sheppard
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.
Recent Developments
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.
Adam Young, Metrion Biosciences
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.
Figure 1 – Mutations cluster 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.
Summary
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.
Figure 2 – A summary of the druggability of DDSA mutants by BAY1000493, Bayer’s TASK-2 blocker
Metrion Scientist Dr Alexander Howarth summarises below his undergraduate and postgraduate studies and how he came to join Metrion Biosciences.
Dr Alex Howarth, 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
Metrion Biosciences
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.
Dr Peter Matthews, Metrion Biosciences
“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
Metrion Biosciences
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.
In conclusion
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: webinars@metrionbiosciences.com.
Our guest blog, written by longstanding Friend and Collaborator Steve Trim, CSO and Founder of Venomtech is presented below.
Steve Trim, CSO and Founder of Venomtech
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).
