‘Let’s Cure CLCN4’ rare disease channelopathy drug discovery symposium report

By Dr Marc Rogers and Dr Sophie Rose

Guest blog within the series ‘Ion channel drug discovery insights from the Channelogist’ (@Albion Drug Discovery Services) for Metrion Biosciences


Cure CLCN4 is a charity founded by a patient family to provide support, raise awareness and fund medical research for effective treatments for CLCN4, a rare genetic condition which causes intellectual disability, behavioural and movement disorders and microcephaly. Each year, this important charity holds a conference to highlight the condition, bring together thought leaders in the field (including international academics and clinicians), provide input from patients and their families, and share research insights with the aim of discovering a cure for this rare disease and generating a strong patient-orientated research community and support network.

A link to this years’ conference can be found below:

This years’ event, held earlier this Summer, was organised by Paul McGuiness and Rebeca Ridings Figuero, alongside Dr. Peter Trill, a UK bio-entrepreneur and his wife Dr. Gina Tan, a clinician who founded Cure CLCN4 as their youngest daughter Daphne has a mutation in the CLCN4 gene and suffers from neuro-developmental symptoms. There were around 40 in-person attendees in addition to a varied online delegation of ~30 attendees, plus a closed session focused solely on patient families.

Selected talks are available in video here:

Scientific background

CLCN4-related neurodevelopmental disorder (CLCN4-NDD) is a rare X-linked genetic condition associated with intellectual disability, psychiatric disorders, gastrointestinal issues and epilepsy. The extent and severity of the condition is extremely variable and dependent upon the type of gene change and the gender of the individual, amongst other factors.

CLCN4-NDD is caused by mutations of the CLCN4 gene encoding the CLC-4 channel which is expressed across various tissue types, but most prominently in the brain and skeletal muscle. Significantly, CLC4 is expressed on endosomes and the ER, and likely interacts with related CLC-3 channels which facilitate sorting of CLC-4 proteins into endosomal compartments.

Key points:

  • There are 7 CLCN proteins, split between classical transporters and dual transporter-channels
  • CLCN4 is part of a group of organellar (endosomes, lysosomal) transport proteins
  • CLCN4 expression and function is most notable in neurons, hence CNS deficits in patients
  • CLCN4 appears to heteromerise and require closely related CLCN3 protein for function
  • CLCN4 Cl/H+ anion/proton transporter regulates organellar pH (trafficking, autophagy, etc)
  • Disease symptoms and severity linked to mutation (location, gain/loss function) and sex

The importance of diverse intracellular organellar ion channels in a wide variety of diseases is now gaining greater attention (e.g. TMEM175 in Parkinson’s Disease), making them a new class of ion channel drug discovery target. Cure CLCN4 is also a great example of the growing trend for patient families to get involved in finding treatments for such rare disease channelopathies.     

A summary of the talks presented across the two days is provided below:

Vera KalscheuerMolecular Genetics of CLCN4

Vera is one of the original and leading academics studying CLCN4 genetics (through her focus on X-linked genetic disorders) and is a major part of the CLCN4 foundation. Her group has identified many examples of patients and families with CLCN4 mutations, expanding the picture to include de novo and inherited missense mutations in females and males, respectively. She co-authored a comprehensive review in 2023 (Palmer et al., 2023) that brought together many international researchers and highlighted the emerging genetic diversity in CLCN4 patients. Her previous publications have attracted the attention of other clinicians trying to diagnose and treat rare CLCN4 patients scattered across the globe; Elizabeth Emma Palmer is an Australian clinical researcher who is now also a big part of the CLCN4 collaborative network and another major force in the CLCN4 foundation.

Vera and collaborators now have a growing database of CLCN4 gene mutations from the ClinVar database and publications, which currently shows 90 variants from 49 families which comprise 41 unique (typically de novo) and 18 recurrent (typically missense) mutations which have been predicted to be benign (1), pathogenic (36), or a variant of unknown significance (VUS, 53) which are the most perplexing and require further work to determine if/how they affect CLCN4 protein function, channel activity and patient symptoms. Michael Pusch is working through many of these mutations using Xenopus oocyte TEVC electrophysiology (next talk), and Vera highlighted that many of the in silico predictions for new mutations do not match with functional studies: for example, 30 of the 90 known variants have predicted ‘low’ or ‘benign’ effect, but 13 of these were shown to be either loss- or gain-of-function on CLCN4 currents.

The new data is also significant for showing g-o-f mutations, as CLCN4 was previously thought to be l-o-f (missense and truncating mutations, especially in females);
In males they see truncating l-o-f mutations (de novo, inherited), as well as new g-o-f variants (e.g. Val317x residue);
In females, de novo & inherited mutations are ALL missense, and there are new g-o-f variants (Ala555 residue)

Interestingly, although CLCN4 is an-X-linked genetic disease expected to be limited to females and inherited from the maternal line, males can both pass on inherited mutations and be affected by de novo mutations as X chromosome inactivation can be incomplete, and genome imprinting can occur from the fertilised (i.e. male XY) egg. However, de novo mutations are much less common in males (27%) than females (78%). 90% of variants are missense, and all affected females carry such mutations as truncating or intragenic chromosomal deletions appear to be compensated for by the un-affected X chromosome allele.

Michael PuschFunctional studies of CLCN4 channel mutants
His interest initially stemmed from the fact he trained in Prof. Thomas Jensch’s lab, who cloned the first member of the CLC family. His specialist contribution is Xenopus oocyte electrophysiology of CLCN4 mutants.

There is a major distinction between members of the CLCN family; CLC-1, CLC-2 and the two kidney and inner ear CLC-K variants are ion channels expressed on the plasma membrane where they regulate anion transport, membrane potential and counter-ion movements. The other members (CLC-3, 4, 5, 6 and 7) are expressed in intracellular organelles (endosomes and lysosomes) where they act predominantly as co-transporters (exchangers) of Cl and H+ ions to regulate pH, membrane potential and counter-ion permeability. There are rare human channelopathy diseases associated with 7/8 members of the CLCN family. 

  • CLC-5 is associated with Dent’s Disease
  • CLC-7 is also known as OSTM1 and associated with bone disease (osteopetrosis)

Each of the organellar CLC proteins are specialised/localised to different endocytotic, sorting and recycling endosomes and degradative lysosomes, where they all help to control pH and function.

Interestingly, CLC proteins seem to form channel dimers, with each subunit contributing an independent pathway for the exchanger transport of Cl and protons (similar to dual ‘pore’ Fluc fluoride anion channels).

CLC4 may be unique in the CLCN family as its function, but certainly its trafficking and localisation to endosomes from the ER, requires heteromerization with CLC3 proteins (other CLCs homodimerise). Previous electrophysiology work showed all/most CLCN4 mutations were loss of function, but newer data shows that some (pathological) mutations are like wildtype, while other predicted benign variants are actually loss-of-function, and some rare new mutations are in fact gain-of-function.  

New gain-of-function variants, revealed by studying electrophysiology with altered (acidic, i.e. physiological) pH would be lethal in males, so are only seen in females. Further biophysical work shows that some loss-of-function mutations actually shift voltage-dependence, but could have a dominant negative effect in heteromers.

Emma PalmerCLCN4 clinical genetics and rare patient families

Emma is a key player in the CLCN4 community, and has lead the development of (new) clinical guidelines for treating patients with CLCN4 mutations with Vera and others. She remains a major initial point of contact for many GPs and clinicians who come across patients with CLCN4 mutations after genetic screening for undiagnosed diseases. Therefore, gaining knowledge about new variants, and testing their actual vs predicted significance for function and diagnosis and patient stratification, is key.

In this talk Emma outlined the typical rare disease family experience and clinical journey, patient and symptom progression, and the challenges remaining.

CLCN4 is a complex syndrome, with some sex-specific differences. Common symptoms involve:

  • Language (communication, comprehension)
  • Speech
  • Behaviour and mental health (ADHD, anger, anxiety/OCD, depression)
  • GI (eating, digestion)
  • Neurological (epilepsy in 60% of males but only 25% of female; muscle tone and movement)

Yann HeraultAnimal models of CLCN4
Yann (Inserm) is a key part of the CLCN4 foundation, successfully creating several mouse and rat models of CLCN4 mutations for translational disease modelling and pharmacological screening; this works occurs alongside development of iPSC cell-based models of CLCN4 condition (e.g. by South Korean researchers featured on day 2).

The interesting issue is that the chromosome location of CLCN4 and neighbouring genes is more homologous in rat than mouse, so he decided to make global (exon 4 STOP) and Cre/Lox conditional CLCN4-/- knockout rats for more specific disease modelling, as well as a g-o-f A55V (A449V in rat) knock-in rat (using CRISPR). They have stablished breeding colonies for all 3 rat models and these are now available for collaborators, for free. Also significant is the fact that previous CLCN3 and CLCN4 knockout mice models show no gross symptoms, although other talks at this conference do report some subtle CNS deficits in CLCN4 knockout mice (e.g. Raul Guzman’s work on pyramidal dendrite morphology and function, South Korean autism researcher Yeni Kim).

Anselm ZdebikZebrafish CLCN4 model
Anselm works at UCL, but was previously in Thomas Jentsch’s lab working on CLCN channels. He is currently trying to develop zebrafish loss-of-function (V536M) and gain-of-function (A555V) CLCN4 genetic models, as many symptoms can be measured in this species (psycholocomotor, seizure, cerebral atrophy, anxiety, depression, autism). He is using a CRISPresso technique for zebrafish genetic editing; work is still in progress. He previously made a Kv (KCNJ10) channel knockout for electrophysiological and behavioural studies in zebrafish.

Joseph Mindell (NINDS) – CLC7 gain-of-function mutations in lysosomal storage disease
Joseph is another leading academic researcher in the CLC community and well known to senior participants. He became interested in this channel family as a postdoc in Chris Miller’s lab that studied CLC channels. His speciality is lysosomal disease, and includes study of CLC7 mutants and patient families; CLC7/Ostim1 is implicated in bone disease, but he didn’t talk about this today. Jo favours a pH-sensitive fluorescent dye assay (Oregon Green dextran) to study endosome function, as well as whole-cell and single channel patch clamp recordings from CLC channels expressed in heterologous cells. His group has discovered several new patient families with a g-o-f mutation in CLC7 (Y715C) which affect lysosome acidification, resulting in hypopigmentation (dysfunction in melanocytes) and systemic lysosome storage disease. Three new patients have similar g-o-f mutations but milder disease phenotypes. His recent work suggests that these g-o-f mutations affect the interaction of lysosome-specific phosphoinositide PI(3,5)P2 with CLC7 proteins, which normally inhibits transport activity.

Day 2:

CNS electrophysiology and pharmacology

Jinju Han (KAIST, Korea) – Excitatory neuron survival
The key data in this talk is the development and study of human ESC CLCN4 mutant and knockout stem cell lines, neural progenitors, and (immature) neurons to create translational disease-in-a-dish reagents and assays. Stem cell models using immature human CNS neurons may be more relevant for CLCN4 syndrome than many other CNS diseases  (e.g. schizophrenia, depression, neurodegeneration) as CLCN4 is a neuro-developmental syndrome that affects infants and young children, rather than adults. However, the use of ES rather than iPS cells may affect drug discovery efforts outside of Korea due to ethical issues and regulatory requirements in the US, EU and UK.

Introducing the A555V g-o-f function into stem cell neurons actually decreased CLCN4 mRNA expression by 50%, and lead to rapid cell death (TUJ1+ neurons), indicating severe effects on neurogenesis, especially for excitatory neurons (identified by scRNA transcriptome profiling). Similar effects were seen in 2D hES and 3D organoid mini-brains, with altered dendrite morphology. It may be possible that this loss of excitatory neurons could impact neuronal development and cognition and behaviours, but it remains unclear how a similar mechanism could underlie the seizures that are prominent in CLCN4 patients.

Yeni Kim (Dongguk University, Korea) – Synaptic dendrite function and developmental deficit
Yeni works with Jinju Kim. The key finding here is that the anti-psychotic Risperidone, a mixed DA and 5-HT GPCR modulator, can reverse or ameliorate the CNS neuron morphological and behavioural effects of CLCN4 knockout mice. Previous studies found no gross neurological symptoms in knockout mice, but her group has identified autism-related effects on social interaction, anxiety and repetitive behaviour in 7-8 week old knockout mice. As CLCN4 patients have neuro-developmental deficits and some key features of autism, she postulated that an agent effective in her models of autism may also work for the CLCN4 condition. Knockout of CLCN4 differentially affected cortical expression of genes associated with neuronal plasticity and synapses (PSD95, CDK5), and dendritic morphology (Golgi staining). Risperidone could ‘correct’ these genetic deficits, as well as rescue (excitatory) neuron differentiation, dendritic and synaptic morphology, and behavioural symptoms.

The relevance of a complete and global CLCN4 knockout in mice to patients with gene-modifying mutations that may reduce or increase channel function is not clear, as-is the mechanism of Risperidone action or the signalling cascades affected, so further work is clearly required and is underway with human ES neuron translational models. Also, there was no link shown between these CNS and behavioural effects and the known function of CLCN4 to regulate organellar patho-physiology, although in their talk at the 2020 CLCN4 Symposium they said they saw increased LysoTracker dye labelling, suggesting increased organellar cycling, perhaps due to process and degrade dying neurons?

CLC channel biophysics and function:

Thomas Jentsch (FMP Berlin) – Physical and functional interactions of CLC4 and CLC3
Thomas is famous in the field for cloning the first CLC channel gene in 1990 (CLC1 in Torpedo electric organ), and several leading researchers in the current field are linked to his laboratories in Germany. He has looked at a range of CLC and VRAC anion channels, and their channelopathies and functions in various and varied human diseases.

The fact that human patients with single CLC gene deletions and mutations manifest significant (neurological) symptoms suggest that the CLC gene family members cannot fully compensate for each other, likely as each of them are expressed and function in different organellar compartments and pH states of the endo-lysosomal pathway (see Figure below). CLC3, 4 and 5 seem able to form heterodimers, so knockout of CLC3 probably causes more serious neurological symptoms than CLCN4 knockouts as the former also leads to reductions in CLCN4 protein, but the reverse is not the case as CLC3 is a dominant negative partner, and CLCN4 stability, trafficking and lysosomal function depends on CLC3 but not the other way round.  Significantly, uncoupling or deletion of CLC3 H+ transport in knockout mice only leads to neurological dysfunction in the absence of CLCN4, suggesting the latter can contribute a functional transport function if it forms a dimer with non-functional CLC3 proteins, which can still help with protein complex stability and trafficking.

Taken from Jentsch & Pusch (2018): 10.1152/physrev.00047.2017

Thomas reiterated the theme that in many mouse CLC gene knockouts there are few discernible neurological deficits, and he (but not all others) employs an extensive battery of tests and assays (e.g. elevated maze, Barnes maze and open field for anxiety, home cage activity and sleep: wake cycle) to detect behavioural deficits. The only difference between wild type and CLC4 mutant mice was in a social interaction assay (in a test tube), which may correlate with the autism symptoms detected by South Korean researchers.

Merritt Maduke (Stanford) – CLC proton exchange transporter structure-function
Merritt runs a molecular and cellular physiology lab, where she uses a prokaryotic CLC gene and protein model. Her lab has obtained several new and detailed cryo-EM views, as well as carried extensive mutational analysis, to refine molecular dynamic models to try and determine each of the 4 steps of the Cl/H+ counter-transport cycle, disproving several previous models and proposed protein conformations.  These 4 states or configurations require the Glu residues in the permeation pathway to be in the ‘up’, ‘middle’ or ‘down’ orientation, as well as a new ‘out’ position.

During questions, it was asked whether CLCN researchers had used Google’s AlphaFold software to predict and model CLCN4 protein structure-function and the potential impact and effect of patient mutations. Several speakers replied that they had tried this, but it turns out that the crucial dimer stoichiometry of functional CLC proteins is not well modelled by this software (which was surprising and interesting to hear). Thus, it would appear as though there is still a real bonus to having empirical x-ray and cryo-EM structures and to use these in detailed computational MD studies of ion channel structure-function, rather than rely on purely in silico techniques.

Cecilia George (Nanion) – Investigating CLCs using APC methods
Cecilia showed the attendees how they could augment their current electrophysiology and imaging methods to include automated patch clamp (APC) and Solid State Membrane (SSM) technology and platforms for their CLC and lysosomal disease research, and how these could help them span the gap between basic research and drug discovery. Her talk revealed recent high quality APC data from CLC-1 channels (on the SP384 platform) and CLC-7 channel/transporter recordings (on the Surfe2R SSM platform), and included some data from native, purified lysosome membranes rather than plasma membranes from (mutated) over-expression systems. This is an important feature, as there are marked differences in the membrane lipid composition and protein-interacting partners between organellar and plasma membranes which can affect biophysical behaviour and pharmacology, with important implications for disease modelling and drug discovery efforts.

Rare disease patient organisations:
It is becoming clear in the field of channelopathies and rare disease that there is an increasing role for patient recognition, and family and charity lobbying, fundraising and involvement in the drug discovery process. Academics and clinicians already tap into these resources through diagnosis and treatment, especially given the rapid rise and reduced cost of genome sequencing, and parents in turn are more active in searching for specialists and treatments from the literature and online after their child is diagnosed (sometimes after several frustrating years without much useful information). It is also crucial to acknowledge the potential for drug discovery companies to also get involved with rare disease individuals, families and organisations, as many in this space are now doing. Several talks on Day 1 and Day 2 addressed some of these issues, but more still needs to be done.

Also worth mentioning is that fact that Gina Tan (mother of Daphne and wife and co-founder with Peter Trill of the CLCN4 Foundation) started on her rare disease journey after Daphne’s diagnosis with a single Facebook post that initially connected her to 5 other CLCN4 families. This group was found by the clinician Emma Palmer in Sydney who was looking for new patients and mutations, and has now grown to a support group with over 120 families and an extensive network of academics and clinicians, and an established rare disease charity based in the UK but with connections to groups around the world.

Paul WangSimon’s Searchlight Foundation  
Paul provided an overview of Simon’s Searchlight Foundation, an organisation based in the US which deals mostly with rare disease patient groups. Paul is a neurodevelopmental disease clinician who worked in industry at Pfizer, before getting involved in clinical trial advocacy and rare disease patient groups. Simon’s Searchlight is a charity set up to help bring together the various stakeholders in rare disease, but especially to help patient families and foundations put together the necessary toolbox of experts, databases and reagents to facilitate the search and clinical testing of new treatments for rare disease patients. Their services include patient and family contact lists and co-ordinators, family and medical history surveys, blood and cell sampling to allow creation of iPSC disease models, development of cell line and animal genetic models and reagents (e.g. in academic labs, or by commercial CRO partners paid for by the charities and founders), through to clinical trial design and execution.  A current focus with CLCN4 patients is to acquire clinical EEG recordings, as epilepsy is a common symptom but this data can also help assess other neurological changes, and may provide a biomarker for drug trials. Currently the Foundation is working on over 170 rare disease genes and thousands of patients and families involved with neurodevelopment disease.

Jessica Duis (Colorado Children’s Hospital) – Clinical trial readiness for rare disease
Jessica is involved with Angelman, Prader-Willi, and Pitt Hopkins syndrome patients and families, all of which are rare neurological conditions. She is a clinician who has led and advised investigator-driven clinical trials and now also works with industry to design better rare disease clinical trials and regulatory filings. The main issues are the small patient populations and difficulty in profiling, classifying and stratifying patients to enable a suitably robust and statistically powered trial (more likely a small Ph II than a larger Ph III), and the need for new outcome measures and biomarkers to be developed, validated, and approved by the FDA.

For Angelman patients she helped develop a ‘disease concept’ model using data from care givers, patients and their families, and clinicians to identify the most disruptive symptoms, so that these can be specifically addressed in treatment regimes and drug discovery research and clinical testing. These highlighted speech and motor problems, as well as seizures. Detailed case histories also identified changes in gait, which could then be correlated with genetic changes and aid in genotyped patient classification.

Dr Pangkong FoxCACNA1 Foundation
Dr Fox’s son was diagnosed with a mutation in the Cav2.1 ion channel gene in 2021, and after looking for information online she found the CACNA1 Foundation and by 2022 she was their Scientific Engagement Director. She has a PhD in Cell and Molecular Biology and uses her scientific background to create education support and resources. Cav2.1 mutations are linked to various forms of ataxia, migraine and epilepsies, as well as neurodevelopmental conditions such as intellectual disability and autism. There is a complex geno-phenotype with over 300 pathogenic mutations that lead to loss- or gain-of-function. The CACNA1A Foundation is doing many of the same things as the CLCN4 Foundation and Simon’s Searchlight (via their Research network) to develop a ‘preclinical toolbox’ of patient histories, biobanking, iPS and heterologous cell line generation, animal models, etc. These are and will be used for drug re-purposing screens and the search for new treatments (small molecules, ASOs, gene therapy). They are utilising collaborations with many other charities and disease and research institutions to achieve this cost-effectively and quickly (e.g. NORD, RareX, Citizen Invitae platform), as well as working with industry partners (e.g. Metabolom in Ireland for biomarkers), and receiving funding from US agencies and organisations (e.g. Chan Zuckerberg Initiative).

Many other ion channel channelopathy parents have followed this pathway, but without an established rare disease organisation they created their own charity, advocacy group or foundation – examples include KCNT1, KCNH10, CLCN4, SCN2A, etc.  These groups help to identify and bring together patients, academics, clinicians and industry to facilitate collaboration, raise awareness, increase understanding of the disease and patient and family needs, discover new treatments, and raise funds.

A Family Story
Whilst the main patient and family engagement session on Day 2 was limited to the key clinicians, attendees were able to gain a personal insight into what it was like to have a child diagnosed with CLCN4 syndrome in a short online session with a mum in the Bay Area. Her child was first taken to the GP at 6 months with feeding problems and at 12 months with developmental delays, but was not diagnosed until after genotyping and neurological clinical profiling at 2 years of age. It was a very touching presentation – the child was asleep I think, but we were shown family videos and also some slides that the Mum had created about disease progression, symptoms, and family challenges and highlights. This was a very moving, personal and emotionally affecting presentation.

There was a similar video montage and slideshow of patient family pictures and information on Day 1 (also shown at the 2022 Symposium) illustrating patients and families who were diagnosed at varied ages from months-years-decades old, spread across the globe. This type of patient family insight is a very powerful way to bring R&D into personal focus.

Drug Discovery case studies:

Michael Schwake (University of Bielefeld) – Parkinson’s Disease affects lysosomal function
Michael is another alumni from the Jentsch lab, where he studied CLC5 in Dent kidney disease. He is now working on Parkinson’s Disease, which can be classified as an Inherited Disease of membrane Trafficking (IDT) as there is genetic linkage to TMEM175 and TRPML1 lysosomal channels, as well as to GCase enzyme involved in autophagy and endo-lysosomal cycling and function. Several academic and industry groups are working on GCase treatments for PD, but he noted that while GCase activity is reduced in the brains of PD patients and animal models, there are also PD patient mutations which show no effect on enzyme activity. I wasn’t aware of this but Ambroxol (a non-selective Nav channel inhibitor, cough medicine) is being tested clinically for PD as it has been shown to act as a chaperone for GCase, to boost protein trafficking and function.

Michael is working on human patient-derived iPS neuron models of PD, differentiating TH+ dopa neurons; he stressed the importance of using isogenic controls to confirm disease and gene differences, which he creates using CRISPR gene editing (baculovirus vectors being more effective than other expression methods). His models replicate the loss of GCase expression and activity and formation of a-synuclein fibrils, and also found that another lysosomal protein LIMP2 is reduced in PD. Significantly, he found that LIMP2 activates GCase and it’s associated protein GBA1, so loss of LIMP2 may lead to reductions in GCase in PD. His group is developing cell-penetrating peptides of the LIMP2 helix to promote GCase and GBA1 activity and function as a novel therapeutic approach for PD.  The obvious corollary for potential CLCN4 disease treatments is to use small molecules or genetic treatments to modulate upstream regulators as well as the CLCN4 gene and protein itself, such as CLC3 and other proteins which may augment CLCN4 trafficking and activity.

Laksh AithaniCharm Therapeutics
Charm Therapeutics is a London start-up with US connections; Laksh previously worked at leading AI drug discovery company Exscientia (UK), and the other co-founder is a US academic and entrepreneur focused on in silico deep learning protein folding applications. Other team members have come from Google DeepMind and big pharma.

Their main argument is that genetic sequences do not reliably predict 2D or 3D protein structure (i.e. the AlphaFold approach), so high resolution protein crystal structures should be used; additionally, ligand binding can alter protein structures (i.e. apo state may not be useful for drug discovery).

They have developed a fast deep learning algorithm called DragonFold that is trained on the 25% of PDB crystal structures with ligands bound (GPCRs, ion channels) and which can be used in a virtual screening workflow to create virtual crystals of the target protein and small molecule libraries, using supercomputer hardware (100 NVIDIA GPUs) and optimised AI/ML software.

Their therapeutic interest is mainly oncology and Laksh presented 2 case studies of small virtual screens against a kinase and a nuclear polymerase. Only 100-500 compounds could be virtually bound owing to computing and cost limitations, with each screen identifying a handful of hits with improved selectivity or mM potency respectively, which were validated in functional binding and biological assays.

Marlen Lauffer (Dutch Centre for RNA Therapeutics) – ASO genetic treatments for rare disease
I really enjoyed Marlen’s talk and her enthusiasm to find effective treatments for patients suffering from ultra-rare diseases, for which academic drug discovery is too slow and commercial interest is extremely limited, and any drug to reach market would be extremely expensive.

Her group is a non-profit consortium supported by the Dutch government and EU funding to rapidly find treatments for rare neurodegenerative disease, as effective drugs need to be delivered to young children before life-limiting symptoms take hold. To this end, they are using patient-derived fibroblasts to directly-induce human stem cell iNeurons, as traditional iPS engineering techniques are much slower and more expensive. Their therapeutic focus is on RNA antisense oligonucleotides (ASOs) to modulate faulty gene expression through exon skipping, RNAi or allele-specific mechanisms. She acknowledged that these approaches work best for g-o-f mutants, which have now been identified in a small subset of CLCN4 patients, but others have shown it can work to up-regulate gene and protein expression as well which may be suitable for treating l-o-f CLCN4 mutations as well. Rather than trying to group related patients with similar mutant genotypes for traditional small clinical trials, she suggested that defining each patient as their own population enabled N=1 Ph 0 clinical trials with fewer regulatory requirements, essentially breaking down each rare disease into multiple ‘nano’ rare diseases (also called 1 Mutation 1 Medicine, 1M1M, or N=1 diseases). ASOs can be delivered locally by i.c.v, i.t or injection into the eye, but it is unclear if a single application (injection or transgenic expression) can ‘cure’ a disease (one-and-done), or whether repeated applications may be needed during a child’s development.

This is a high risk and revolutionary approach; there have been recent patient deaths with such ultra-personalised genetic approaches (e.g. a DMD patient family-funded effort in the US), but Marlen is using the example of Milasen which was an ASO developed and tested to treat a single young female patient with Batten’s disease (CLC lysosomal storage disorder). She presented a case study on a patient with the retinal disease Stargardt Syndrome, with ASOs designed to correct intron/exon splicing that are being tested in iPS retinal organoids. I was concerned by the fact that limited safety testing is carried out with these ASOs (as many assume they are incredibly specific and selective, and thereby safe). For example, limited toxicology testing is done in non-human species, which presumably have different gene sequences to the human protein, so the action and efficacy of human-specific ASOs on the target protein and it’s ‘ome partners and signalling cascades may be reduced and under-estimated. For example, several pharma groups and CRO companies (e.g. Amgen, Metrion Biosciences) have shown that siRNA and ASOs and other genetic modulators can have unanticipated safety pharmacology effects, such as long-term (rather than acute) effects on hERG channel activity. The FDA and EMEA are aware of thee issues and may be increasing the threshold for safety testing of such RNA treatments, but as with oncology the seriousness of the disease and small patient numbers (in genetically stratified patients) may allow a more relaxed approval process.


In conclusion, this key conference promoted collaboration and rare disease patient and family support and participation, clearly showing that a varied and muti-disciplinary approach to channelopathy drug discovery can lead to tangible results. Above all there was a feeling of satisfaction that contributions, however small, are not going un-noticed and there is an overwhelming number of people and companies who are dedicating their drug discovery endeavours to accelerate treatments for rare conditions such as CLCN4.

Molly – My Placement At Metrion So Far

Written by Molly Rowlett

I’m Molly, and I am an undergraduate student in Neuroscience at the University of Bristol, currently on my placement year at Metrion Biosciences. I have completed the first 2 years of my studies at Bristol studying the foundations of Pharmacology, Physiology and Neuroscience, in addition to useful and interesting courses in statistics and psychology. My favourite academic experience at Bristol have been the practical sessions where I could get hands-on laboratory experience.

During my first year, I became convinced that my interests lie in the Pharmaceutical/Drug Discovery industry, particularly Neuropharmacology. Therefore, at the end of the year, I decided that gaining relevant work experience would be effective to enhance my exposure to this field. I was delighted to seize the opportunity to apply for the MSci year in industry, where I would spend a year working in a company setting. Due to the fantastic overlap between my interests and the scientific services offered by Metrion in drug discovery, I contacted the company to enquire about the possibility of a placement opportunity. To my pleasure, I received an offer to begin my placement earlier this summer.

So far, I have been exploring the company’s role as a contract research organisation, focusing on ion channel research and drug discovery services. As part of my training, I have been getting trained on a broad range of laboratory techniques such as cell culture, manual patch-clamp, automated patch-clamp, and FLIPR plate-based assays. I’m excited to be able to start  working on my own project next month, where I will get the chance to apply my new skills and experience to drive the project forward. 

I am thoroughly enjoying my time here and I’m grateful for the insight into the industry. I’ve already been able to grow both professionally and personally being part of a welcoming and motivated team of scientists. I’m developing valuable scientific and transferable skills that I’ll be able to bring with me into my final year at university and even beyond my undergraduate studies. I have even started forming an idea of my career progression after my studies, and my work experience at Metrion has been and will continue to be absolutely vital in this process.

Mol Rowlett in lab 1
Molly Rowlett – University of Bristol, during her placement at Metrion

The CICF 2023 – My Perspectives

Written by Faith Zarume

On 10th May , Metrion Biosciences and AstraZeneca co-hosted the annual Cambridge Ion Channel Forum, this year held at the Cambridge Building, Babraham Research Campus. Here, ion channel focused research was shared via a series of presentations, interspersed with networking opportunities. Before hearing from the six speakers, there was time to view the displayed posters, ask the authors questions, as well as catch up with some familiar faces.

First, we heard from Professor Alistair Mathie from the University of Kent on the future of potassium channels as therapeutic targets; predominantly focusing on human gene cloning, identification of channelopathies, and improving structural information on these channels. Professor Mathie began with a description of the role of potassium channels in humans and opportunities for therapeutic intervention. In order to characterise the pharmacology of potassium channels, he and his team studied KCNK9 imprinting syndrome, which is a monogenic disorder caused by an alteration in the maternal copy of the KCNK9 gene. It was found that mefenamic acid, a nonsteroidal anti-inflammatory drug allowed channel activity in a range of mutations where there was a gain or loss of TASK3 channel activity. Within 13,500 families involved in their work, 42% were given the satisfaction of a diagnosis. This research is aided using artificial intelligence (AI), such as the Alphafold protein structure database, which can provide predictions for protein folding.

Professor Mathie’s presentation was an excellent prelude to the talk given by Mr Sam Bourne (LifeArc) on genetic and functional validation of two-pore domain potassium (K2P) channels and their use in the treatment of pain related symptoms and disease, where there is great need for novel pain therapies. Through use of data, encompassing <120,000 K2P gene variants associated with distinct types of pain in both UK and Finnish populations, they were able to investigate the genetic impact of K2Ps in disease. There was a focus on TREK2 channel missense mutations and how this lead to an increased frequency of patients experiencing pain due to significantly reduced function – which was probed using a thallium flux assay.

Next, we heard from Dr. Oliver Acton, Senior Scientist at AstraZeneca. Oliver took us through how Cryogenic Electron Microscopy (CryoEM) has played a role in structure-based drug design with a specific focus on TRP channels. CryoEM allows for assessment of small molecule binding and collects data in just hours to then be processed in only a matter of days. With the help of CryoEM, Oliver’s group now have 320 TRP channel structures, captured  in high resolution. Oliver presented an example where, in TRPA1, two antagonist binding sites can be seen, one closely located to the agonist binding site. This type of structural information  can prove important in structural based drug targeting.

After a coffee break, with more time for networking and to study the posters on display, Professor Iain Greenwood from St George’s, University of London started off the afternoon session. He outlined ERG channels’ relevance in bodily locations, outside of the heart, such as in smooth muscle, allowing spontaneous contractile activity. Iain explained how ERG channels have been a key therapeutic target in studying hereditary arrythmias. Iain presented an example, where lack of effect of ERG channel blockers / activators was observed in the uterus of pregnant mice. This being determined by modification of KCNE2 proteins and their expression in the uterus, presenting altered physiological function. Other ERG channel responses have been explored in the bladder, portal vein, stomach and oesophagus.

Dr. Samantha Salvage from the University of Cambridge then detailed the implications for cardiac conduction from novel insights into Nav1.5α-β3 interaction, specifically the important role for the β3 extracellular immunoglobulin domain. Nav1.5 is heavily involved in cardiac action potential conduction and accordingly, mutations, including some β3 mutations, are known to pre-dispose individuals to arrhythmias such as Brugada and LongQT syndrome. Voltage sensor movement was observed using  fluorescent tags and results indicated a loss of the extracellular domain that led to more negative potentials. Unique structural features of Nav1.5 account for differences in the α-β compared to other α subunits. For example, the presence of an N-linked glycosylation site (N319) on the Nav1.5 extracellular loop 3 influences β1 and β3 binding in a way that forces the Ig domains away from the core Nav1.5 structure and allow β1-mediated trans cell adhesion interactions.

The event was drawn to a close by Metrion Biosciences’ Dr. Alexandra Pinggera who presented on the success of endolysosomal patch clamp in investigating lysosomal storage disorders, such as Alzheimer’s and Parkinson’s disease, particularly when brought about by mutations in TMEM175 and TRPML channels in naïve HEK cells.  It is usually necessary to introduce a mutation that traffics channels to the plasma membrane to utilise conventional patch clamp techniques. Additionally, acidic pH is required for operation of these channels and this cannot be easily regulated on the plasma membrane. But application of a refined technique aided in characterising endolysosomal ion channels in their naïve environments, allowing investigation into potential therapeutic agents. The presentation included a short video showing the lysopatch technique and the expertise required to gain a strong seal and make recordings.

Faith Zarume
Faith Zarume – SGUL, during her placement at Metrion

Jack – My Academic and Professional Career So Far!

Written by Jack O’Donnell

My time studying at Bristol

Before joining Metrion in November 2022, I studied at the University of Bristol for four years. I started my undergraduate degree in 2018, studying Biomedical Sciences which covered a wide range of biologically processes. I particularly enjoyed the pharmacology modules, finding the practical aspects the most exciting. My third-year dissertation involved the molecular modelling of a novel delta opioid agonist SNC80. The project involved investigating the factors that influence how a ligand is docked to a receptor, navigating Chimera software to make 3D models of receptor-ligand complexes to visualise the delta-opioid receptor complex and observe the intermolecular interactions.

I continued studying at Bristol, undertaking a master’s degree by research. My project led to me studying receptor signalling and pathways that have been activated by opioids. I performed binding assays using Bioluminescence Resonance Energy Transfer and Radioligand binding techniques to quantify the affinities and efficacies of eight different µ-opioid receptor agonists. Some of the compounds I studied are well known, such as morphine and fentanyl. I also studied some lesser-known compounds such as isotonitazene and etonitazene which have been appearing more frequently in recreational overdose deaths in USA.

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Jack O’Donnell

My Role at Metrion

During my masters, I enjoyed culturing and being responsible for the preservation of my own cell line. I applied for the Cell Culture Associate Scientist role at Metrion to continue working in cell culture, with the opportunity to work with various different cell lines. While working at Metrion, I have enhanced my cell culture technique, learning new methods and protocols for growing cell lines. I look forward to developing my cell culture skills throughout my time at Metrion.

Zeki – From Ants to Ion Channels

Written by Dr. Zeki Ilkan

From ants to ion channels

My first encounter with science was using a toy microscope that my parents gave me as a present. I remember the immense excitement I got visually analysing onion skins and ants in greater detail. During my high school years in Cyprus, my enthusiastic and committed teachers (and the experience of upgrading to a real microscope) solidified my passion for the sciences, especially chemistry and biology. I graduated from Imperial College London with a BSc in Biochemistry in 2011, and an MRes in Biomedical Research in 2013 with distinction. As part of my MRes degree, I completed two six-month research projects in the field of cardiovascular medicine and have since been fascinated by the physiology of the heart and the vasculature. Motivated by the high morbidity/mortality rates from cardiovascular disease both in my community and worldwide, I wanted to understand the molecular pathophysiology of the cardiovascular system through research. I was awarded a Medical Research Council (MRC) Doctoral Training Studentship to pursue a PhD in cell physiology and pharmacology at the University of Leicester. During my doctoral studies, I discovered that human platelets express the mechanosensitive Piezo1 cation channels which can sense various levels of shear stress in the circulation and respond by elevating intracellular levels of calcium. In pathological conditions, this can significantly increase the possibility of spontaneous platelet activation. This important discovery indicated that these ion channels could serve as possible therapeutic targets in the treatment of thrombosis and stroke.

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Dr Zeki Ilkan – Metrion Biosciences

From academia to industry

My interest in the pathological role of ion channels motivated me to join Mount Sinai Health System in New York to investigate how mitochondrial ion channels and associated membrane proteins can promote cardiac rhythm disorders. I investigated the therapeutic potential of classical pharmacological tools and novel gene silencing techniques in the inhibition of ion channel activity involved in the dissemination of toxic oxidising agents within the diabetic heart tissue which cause cardiac rhythm disorders. Here, I used a range of electrophysiological and molecular techniques, including the high-resolution optical action potential mapping in Langendorff-perfused hearts.         

Having been fascinated with the workings of ion channels, I could not resist the desire to finally learn the ‘gold-standard’ patch-clamp technique. In 2019, I joined the Department of Pharmacology at the University of Oxford as a British Heart Foundation (BHF) post-doctoral scholar. There, I investigated the role of chloride channels in pericytes, contractile cells that surround capillaries, in the regulation of blood flow in the brain and their potential as therapeutic targets in the treatment of strokes. I discovered the role of TMEM16A chloride channels in pericyte contractility in the brain cortex which regulate blood flow through capillaries. This important study identified this channel as a novel therapeutic target for the treatment of ‘no-reflow’ phenomenon which follows cerebral ischaemia. At Oxford, getting first-hand experience in drug discovery projects through collaborations with industry partners sparked an interest in transforming research knowledge into drug development.        

From Oxford to Cambridge (via San Francisco)

I first met Metrion Biosciences through a Metrion-branded stress ball waiting for me at my new office desk in Oxford. It must have been picked up by a colleague or my supervisor and inadvertently placed there. I then received a phone call by a recruiter informing me about Scientist roles at the company during a time I was very busy publishing a paper. About a year later, at a Biophysical Society Annual Meeting in San Francisco where I was a speaker, I had the perfect opportunity to hear more about Metrion and discuss my growing interest in applying for a position. A month after this meeting, I was invited to Metrion for an interview. I am now a Scientist at Metrion and am delighted to be part of this wonderful team and for the invaluable opportunity to expand my research knowledge and electrophysiology skills into the industry world.

Thomas’s Career So Far

Written by Dr. Thomas Hill

My introduction to the world of drug discovery

My scientific career started at around the time I learnt the words “why” and “how”, with my first school report stating that “Tom likes to learn new things and tell people about them”. Fast forward to 2008, in the middle of a financial crisis, I have just finished a BSc in Forensic Science and I am standing talking to my supervisor at graduation asking their advice on how to get a job in Forensics. I was recommended to read for an intensive 1-year Masters in Toxicology at the University of Surrey, which I started 5 weeks later. This was arguably the most important year of my life. I was firstly introduced to the world of drug discovery and to my future wife – Charlotte (Charlotte’s Patch Clamp Journey So Far… • Metrion Biosciences).

Thomas Hill photo
Thomas Hill – Metrion Biosciences

Putting my skills into practice

After the Masters, I worked as an intern in the DMPK department at UCB, where I was able to put into practice many of the skills that I had acquired and really cemented my love of drug discovery. Following this internship, I went on to do a PhD where I helped to develop cannabis-based treatments for epilepsy, sponsored by GW Pharmaceuticals and Otsuka pharmaceutical working alongside Charlotte. The group covered two major disciplines, electrophysiology, and behavioural neuroscience.I worked within the behavioural side of the group, looking over with interest at my wife who was learning both manual patch clamp and multi-electrode array.

After my PhD, I was invited to interview for a position as a Pharmacology Project Manager at GW pharmaceuticals, where I managed the preclinical development of treatments for neurodegenerative disorders, oncology, and epilepsy (as well as dabbling in PK when needed). I loved the science we were doing and developing drugs for people with rare diseases, but I missed the lab.

Getting back in the lab

So, I took up a Post-Doctoral position in the Royal College of Surgeons in Ireland working with Prof. David Henshall, where I worked on microRNA-based treatments and diagnostics for epilepsy, picking up both tethered and telemetry based in vivo electrophysiology. Having had some time to recover from her Ph.D. and rediscovering her enjoyment of electrophysiology while writing a book chapter, my wife wanted to get back to the world of Drug Discovery and so applied for a job at Metrion, which brought me back to the UK just as the COVID-19 pandemic got started.

Helping during the pandemic then starting work in the CRO industry

Upon my return to the UK, I wanted to help with the pandemic in the only way I knew how working in science. I started a job at the Cambridge Covid Test Centre (employed by Charles River), where I prepared samples for PCR testing. I was then promoted to Team Lead of the Technology Development department, where I helped to evaluate new testing methods, optimise the testing process and introduce a novel approach to making samples safe to handle in the lab. After the Government closed the testing labs in Cambridge, I used my knowledge to help optimise a start-up Covid testing lab in Cambridge. After spending more than a year working in Covid labs, I got a job in the Ion Channels Group at Charles River, where I learned how to use both the FLIPR and Qube platforms. I am excited to be working at Metrion and further my career in Ion Channel Drug Discovery.

Katie – Expanding my skills to the world of ion channels

Written by Dr. Katie Puddefoot

Volunteering sparked my interest in neuroscience

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.

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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.

Catherine’s Career So Far

Written by Dr. Catherine Hodgson

Ion Channels and Patch-Clamp

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 PhD and 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!

Yasmin – About Me

Written by Yasmin Henry

My Criminology Studies

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
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!

Professor David Sheppard presents on “Cystic Fibrosis: Rescuing Faulty Channels with Targeted Therapies”

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.

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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.

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