Improving Cardiac Safety Testing Protocols

Written by John Ridley PhD

The regulatory guidelines

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

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

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

Metrion Biosciences Cardiac Safety Screening Services
The launch of a new CiPA initiative

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

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

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

The role of the Ion Channel Working Group

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

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

The role of the ISWG

The ISWG is developing an in silico model to allow prediction of proarrhythmic risk, focusing its efforts on the O’Hara-Rudy (ORd) model, which is based on human ventricular AP recordings. The model recapitulates early after depolarisations, which are crucial to accurately predict proarrhythmic risk7. The FDA optimized the ORd model by incorporating the late component of Nav1.5 current, as well as a dynamic hERG blocking model that more accurately accounted for the higher proarrhythmic risk profile of compounds that become trapped within the hERG pore during repolarization.

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

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

The ICWG studies revealed that inhibition of hERG, Cav1.2 and late Nav1.5 current have the most significant impact on proarrhythmic risk prediction6. This opens the possibility that the in vitro ion channel panel (Pillar 1) may be reduced to these key currents.

Testing compounds in iPSC-CM

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

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

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

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

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

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

John Ridley PhD
John Ridley PhD
Business and Strategic Partnerships Executive

John Ridley PhD

John has more than 10 years’ experience working in ion channel drug discovery. This experience was obtained firstly as a Senior Scientist at Xention, where he was involved in the discovery of ion channel blockers for atrial fibrillation, neuropathic pain and autoimmune diseases, and then as the Business Development Manager at Metrion Biosciences. 

Final PDF MedNous Cardiac article JR June 2020 Cropped
MedNous Cardiac article
John Ridley PhD

Metrion is open for business

Written by the Editor

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

Ion Channel Screening traffic light screening

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

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

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

Metrion’s CEO reflects on participation in Goldman Sachs’ 10,000 Small Businesses UK

Written by Andrew Southan PhD

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

10k SB Cohort 11
The 10,000 small businesses 2019 cohort
What is the 10,000 Small Businesses programme?

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

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

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

As Metrion Biosciences’ CEO why did you apply?

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

What did the course entail?

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


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

Would you recommend the course to other small business leaders?

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

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

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

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

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

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

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

Andrew Southan PhD 1
Andrew Southan PhD
Chief Executive Officer

Andrew Southan PhD

Andy has over 25 years’ experience in life science research, including 14 years in the CRO sector. In 1991 he received his PhD in Pharmacology from the University of London (UK), investigating the effects of anaesthetics and high pressure on CNS potassium channels.

Why Metrion’s CSO spent 35 years unlocking the potential of ion channels for drug discovery

Interview with Dr Marc Rogers

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Marc Rogers PhD Chief Scientific Officer
Dr Marc Rogers PhD
Founding Director and Chief Scientific Officer

Dr Marc Rogers

Marc is a trained neuroscientist with 25 years’ experience in the patch clamp technique from a number of prestigious international universities and 12 years of drug discovery on both sides of the Atlantic. He was an early adopter of automated patch clamp platforms for ion channel screening and has an excellent knowledge of this market and connections with many of the commercial vendors. 

Nav1.5(Late) Cardiac Safety Assay on QPatch

Written by the Editor

The Metrion Biosciences team have recently produced a second application note in collaboration with Sophion Bioscience, describing the use of a ∆KPQ LQT3 mutant to create and validate a Nav1.5 (Late) cardiac safety assay on QPatch 48. Herein, we aim to provide a more reliable and accurate Nav1.5(Late) assay, which removes the requirement for costly activators such as ATX-II and delivers improved screening reliability.

Snip of app note for blog e1567429990651
Why do we need to better predict proarrhythmic risk?

Due to the ongoing need for more robust preclinical in vitroex vivo and in vivo models and assays to predict clinical cardiac safety risks in humans, new initiatives providing a more balanced assessment of patient risk are being developed. The FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm, which aims to more accurately model and predict proarrhythmic risk, is driven by a panel of in vitro ion channel assays (hERG, Cav1.2, Nav1.5 (Peak and Late), Kir2.1, KvLQT1 and Kv4.3), coupled with in silico modelling of human cardiac action potentials (AP).

Why the Nav1.5(late) current?

Due to the small amplitude of native Nav1.5(Late) currents toxin enhancers are often used to increase the signal to noise ratio in electrophysiology assays. However, poor selectivity, cost and batch to batch variability observed when using toxins such as ATX-II means this approach is suboptimal for routine screening.

What did assay validation include?

Metrion therefore tested various voltage protocols typically used to evoke Nav1.5(Late) currents on a newly created Nav1.5(Late) stable cell line, utilising the Nav1.5 LQT3 syndrome KPQ deletion mutant. Assay validation included voltage protocols such as the CiPA protocol, an action potential like waveform and step ramp protocols.

Each was compared on the HEK Nav1.5 ∆KPQ cell line on QPatch 48, with the step ramp protocol selected for optimisation to create a stable Nav1.5(Late) current assay. This assay was then validated by testing two known Nav1.5(Late) blockers, mexiletine and ranolazine, both of which showed a preference for inhibiting the late current compared with peak inward current.

Final remarks

In summary, the need for a reliable Nav1.5(Late) current assay is required on APC platforms to provide accurate cardiac safety data to support in silicomodels of proarrhythmic risk. Metrion created and validated a novel assay using a ∆KPQ LQT3 mutant which should remove the requirement for pharmacological enhancers of Nav1.5(Late) and thereby deliver improved cardiac safety screening reliability and cost per data point.

Learn more

The full application note can be found here. You can contact our cardiac safety experts or request further information via info@metrionbioscienes.com.

Metrion would like to thank Thomas Binzer and the Sophion Biosciences team for their support and involvement in making this application note possible.

Application Report Nav1.5 Late Cardiac
Application Report
Metrion Bioscience and Sophion Bioscience
DOWNLOAD

Opportunities for Ion Channel Targeting Antibodies: Current Landscape and Pipeline

External Speaker Series presentation featuring Dr Catherine Hutchings

Metrion Biosciences’ tenth External Speaker Series presentation featured Dr Catherine Hutchings, a highly experienced biologics drug discovery researcher and independent consultant. Dr Hutchings’ presentation included an overview of membrane proteins as drug targets and the evolution of antibody therapeutics technology, spanning isolation of the first mouse monoclonal antibody (mAb) hybridoma to exploitation of the favourable properties of antibodies for therapeutic purposes.

Dr Catherine Hutchings
Dr Catherine Hutchings
The current landscape of biologics

Dr Hutchings initially reviewed the biologics landscape for membrane bound proteins, focusing on G-protein-coupled receptors (GPCRs) and ion channels (covering 4% and 1.5% of the human genome, respectively). Biologics-based therapies for GPCR proteins are expected to begin a phase of rapid growth, with two GPCR directed antibodies currently approved for therapeutic use and techniques to overcome the technical issue of insolubility in aqueous solution and stabilisation of structure becoming more widely available.  

For ion channels, global therapeutic approved drug sales exceed £12 Bn per annum, targeting just 8% of the available family of ion channel proteins. Approved therapies are all either small molecules or peptides, reflecting both the historical challenges for ion channel drug discovery and untapped potential for biologics approaches. Complex conformational and functional dynamics and, crucially, relative lack of structural information compared to other important drug target classes such as kinases and GPCRs, means that ion channels are still a difficult class of proteins for researchers.

The struggle with biologics

Furthermore, Dr Hutchings outlined multiple additional barriers to overcome for biologics research; spanning the difficulties associated with expression and purification of ion channels, to obtaining suitable amounts for immunisation and technically demanding follow on screening assays.

The generally short and conserved ion channel extracellular domains (ECDs) also limits availability of potential epitopes. Despite these technical complications opportunities do exist, with significant biologics R&D interest focused on channels such as the ASIC and P2X families,  where a large ECD and a proportionally enhanced epitope were seen as an opportunity for researchers. The latter being targeted by Biosceptre (Cambridge, UK), currently progressing a Phase II study for treatment of basal cell carcinoma using their polyclonal antibody BIL010t recognising a non-functional form of P2X7.

Advantages of antibody-based therapeutics

Dr Hutchings also shared the wealth of potential advantages for antibody-based therapeutics over small molecules. This included an improved selectivity profile, limited CNS penetration (advantageous for peripheral targets), lower variability in patient pharmacokinetics and improved half-life.

Recent advances in ion channel structure elucidation via CryoEM, combined with novel platforms for immunisation, recombinent protein expression and post immunisation antibody characterisation should enhance the progression of novel antibody therapeutics targeting membrane proteins. DNA immunization has recently rivalled the more traditional use of peptide immunogens and native protein folding can be maintained by synthetic model membrane system (such as nanodiscs) and SMA lipid particles (SMALPs) for antibody characterisation.

New techniques for enhancing recombinant protein production levels are also emerging, for example TetraGenetics protisit-based system yields high levels of correctly folder recombinant human ion channels. Dr Hutchings also stressed that traditional drug discovery functional assays may need to be adapted when working with antibodies for ion channel targets, sometimes necessitating high quality, long duration electrophysiology recordings to accurately reflect the slower binding kinetics of biologics.

Biologics targeting ion channels

Biologics-based ion channel modulation was also discussed, with a particular focus on the sodium channel Nav1.7 – a target still of great interest to analgesics researchers, despite the multiple Phase II failures for small molecule inhibitors. Nav1.7 blocking peptides, such as GpTx-1 and JzTx-V isolated from tarantula venom, are proving to be useful tools for both experimental research and for antibody conjugation.

Amgen reported JzTx-V as being able to block histamine-induced pruritis in mice following subcutaneous administration and more recently it has been used as the basis of an antibody-peptide conjugate. It will be interesting to see if the early research publicised by Shionogi (mAb) or Amgen (antibody-peptide conjugate) can be more successful than the Nav1.7 blocking small molecules evaluated to date. 

It is also hoped that new, functional biologics targeting ion channels will be discovered in the not too distant future; with increasing availability of structural information for ion channel proteins, coupled to recent advances in purification and assay technology and emerging new computational techniques all contributing towards an enhanced chance of success for this target class.

Looking to the future

At the conclusion of the presentation Dr Hutchings suggested that the ligand-gated P2X family and also voltage-gated Kv1.3 family of ion channels are likely future success stories for mAb targeted therapy, with two-pore domain (K2P) and transient receptor potential cation channels (most notably TRPV1) also showing potential.

References
  1. Hutchings, C.J.; Colussi, P. & Clark, T.G. (2019) Ion channels as therapeutic antibody targets. mAbs, 11:2, 265-296, DOI: 10.1080/19420862.2018.1548232.
  2. Haustrate, A.; Hantute-Ghesquier, A.; Prevarskaya, N. and Lehen’kyi, V. (2019) Monoclonal Antibodies Targeting Ion Channels and Their Therapeutic Potential. Front. Pharmacol. 10:606. doi: 10.3389/fphar.2019.00606

An Interview with Professor Nikita Gamper

Interview by the Editor

Professor Nikita Gamper, the ninth contributor to Metrion Biosciences External Speaker Series, gives a perspective below regarding his ion channel research focus and collaboration activities with scientists from the Hebei Medical University, China.  

Gamper 1 3
Professor Nikita Gamper
Q1. You currently split your time between Leeds University and your role as Adjunct Professor of Pharmacology position at Hebei Medical University in China. What influenced you to collaborate with this team of scientists?  

There is quite a list of reasons actually. But most important are the following: i) robust, dynamic and enthusiastic research group at HebMU; ii) strong cohort of PhD and Masters students; iii) collegiate and friendly environment; iii) generous intramural and extramural funding; iv) booming biotech industry giving access to affordable cutting edge technologies.

Q2. Your publication history involves research into ion channels including GABAA, KCNQ (M-type), Ca2+-activated Cl, sensory TRP channels and voltage-gated Ca2+ channels; including their modulation by protein coupled receptors (GPCRs). If you had to pick one of the above to focus your research upon what would it be and why?  

Even though we do have active projects on all above mentioned ion channel families, our largest endeavour currently is focused on the role of GABAAchannels in peripheral pain pathways. We think that there is a GABA-ergic gate within the peripheral somatosensory ganglia, which controls the nociceptive input to the CNS. We are now working on deciphering the cellular and molecular mechanisms of this phenomenon. I am really excited about this new line of enquiry as it may change the way we think of how somatosensory information is processed by the nervous system. Additionally, it may uncover whole new ways of pain control.

Q3. Are there any recent advances in either scientific technology or understanding that particularly interest you?  

Definitely there is a lot going on in terms of technology. I am particularly fascinated with opto/chemogenetics, novel transgenic and gene delivery approaches and a whole swarm of new ideas in microscopy (super-resolution, expansion, clarity, light-sheet imaging etc.). Of course there is also a Cryo-EM revolution which is changing structural biology at a speed.

Q4. Your research involves a range of ion channels that may regulate nociception. Asking a similar question to the one we asked Alistair Mathie in November 2018, do you believe that specific modulation of ion channels could be a route to new non-addictive analgesics? If yes, where do you see the most promise for a clinical candidate?

I believe that one key approach could be in finding a way to restrict ion channel modulators away from the CNS, such that only peripheral nerves are affected. Most pains are triggered by peripheral input; even in the cases where central sensitization plays a major part, usually peripheral input is necessary to trigger a sensation. Thus, effective analgesia could be achieved by precipitating such input using treatments that do not affect CNS. One area to look at is the fact that spinal ganglia (e.g. dorsal root ganglia, DRG) are not well protected by the blood-brain or blood-nerve barriers. Hence, one can try to target DRGs by drugs with poor BBB permeability. For instance, there is a good evidence that M-type channel activators are efficacious when applied to DRG directly in rodents. Peripherally-restricted GABA-mimetics or other drugs that dampen excitability could also be tested I suppose.

Q5. What are your future research plans?

I mentioned the GABA project already, this is one of the main activities for the immediate future, thanks to some generous funding from the Wellcome Trust and China. I am also very interested in localised intracellular signalling and how it is used by neurons to enforce signal fidelity. Another topic of interest is epigenetic mechanisms of hyperexcitability disorders, such as epilepsy and pain. Generally, ion channels and excitability is still a major theme of our research and I think it will remain important for us within the foreseeable future.

Q6. What made you choose to stay in academia rather than going into industry? 

Blue sky research is what fascinates me the most and I think academia is just a more appropriate environment for this.

Q7. Do you feel that pharmaceutical companies do enough to engage with academics and embrace their findings?

Throughout my career I enjoyed some very good relationships and collaborations with industry, yet there is some divide which cannot be ignored. Interestingly, there is a will on both sides to come together for a stronger partnership, yet, due to some inherent differences in goals and processes, this good will often remains unfulfilled.

Q8. How do you feel that the landscape of academia has changed in recent years?

Yes, the landscape is changing, some of it for the better, some of it – not so much so. Among the good trends I would mention emergence of new technologies, drive to have better and larger scale collaboration and cooperation between academic labs, industry, clinics etc. Also increasing internationalization of research is definitely good. What I’m not so happy about is growing bureaucratization, invasion of metrics and short-termism.

Q9. Why do think that ion channels have been a difficult drug target class for the pharmaceutical industry? 

This is a big question which would be hard to answer in just a few lines, but there are several reasons to it. One being that since ion channels are membrane proteins, it was difficult to get good crystal structures. Hence, the progress in structural biology of ion channels was relatively slow and this hampered modelling of drug interactions. This is now changing rapidly with the emergence of the Cryo-EM though, so perhaps progress here will enable a new level of SAR-based drug discovery.

Another issue is broad expression profiles and high degree of similarity of some important ion channels, which makes it difficult to target them with high selectivity and specificity. A notorious example here is the voltage-gated sodium channels: even though it is pretty clear which subunit to target for pain relief, suitable drugs are still not available.

You can find out more about Nikita’s research activities at his University web page here.

ELRIG: Advances in Cell Based Screening – Gothenburg, Sweden

Review written by the Editor

ELRIG is a not-for-profit organisation aiming to provide access to high level scientific content, promote innovation in drug discovery and provide networking platforms for the life science community. To implement this, ELRIG hosts conferences throughout the year across various sites, bringing together life science focused researchers from across Europe.

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‘Advances in Cell Based Screening’, hosted at AstraZeneca’s impressive Mölndal based facility is one of five 2019 ELRIG hosted conferences. This event was focused heavily on assay precision, a topic which spans screening, target validation and drug development. Multiplexed and phenotypic screening were particularly highlighted within several of the presentations, as were novel techniques such as Cell Painting.

Whilst many of the presentations were not within Metrion’s key focus area of ion channel drug discovery, it was an excellent opportunity to hear about emerging new techniques and the breadth of opportunities to create assays with highly precise readouts for a range of druggable targets.  I will summarise below a selection of the talks including three of the keynote presentations.

Target Engagement and Coherence with Functional Cellular Responses

The keynote on Day one within the session entitled ‘Target Engagement and Coherence with Functional Cellular Responses’ was given by Professor Herbert Waldmann of the Max Planck Institute of Molecular Physiology. He spoke on the topic of ‘Chemotype, Phenotype, Target’. The lab lead by Professor Waldmann are focused in part on the development of natural product (NP) inspired compound collections using Biology Oriented Synthesis (BiOS).

He discussed the utility of assays focused around the conversion of phenotypes and their relevance in locating unbiased novel targets. He confirmed the relevance of natural products in drug discovery, explaining that in their 2014 publication (Nat. Rev. Drug. Disc. 2014, 13, 577), Eder, Sedrani and Wiesmann from Novartis had analysed the origins of drugs approved by the FDA between 1999 and 2013 and found that 30% are biologics, 13% are natural products and 15% are natural substance derived molecules.

Hedgehog pathway inhibitors

Professor Waldmann discussed his work on hedgehog pathway inhibitors. The group created an osteoblast differentiation assay using purmorphamine (the first small-molecule agonist developed for the protein Smoothened (Smo), a key part of the hedgehog signalling pathway) as pathway agonist. Using C3H cells they generated a hedgehog pathway assay and observed Smo binding using Vismodegib (used to treat basal cell carcinoma) stained with BODIPY dye. To find the target they used quantitative proteomics (label free) to look for differences between the inactive and active probe.

The Waldmann lab have also been using cell painting assays (used to monitor changes in cellular features) to stain cellular compartments, generating five different fluorescent channels. Images are then analysed using a cell profiler. They found that many compounds are able to enter the lysosome, as they are protonated and trapped. In general, cell painting is a useful tool due to its ability to identify the phenotypic impact of chemical/genetic perturbations, collecting compounds/genes into functional pathways and identifying disease signatures.

Imaging single cell target engagement

Later within this track, Dr Matt Dubach, from Harvard University spoke about imaging single cell target engagement. He discussed a recent project focused on an in vivo approach to the study of pharmacokinetics and target engagement. The group are specialists at cellular level drug imaging and presented an example in which intravital microscopy can be used to image the PARP inhibitor Olaparib tagged with BODIPY to gain information on drug distribution.

The group use fluorescent microscopy and plate-based assays to further elucidate drug mechanisms and Dr Dubach displayed images of fluorescent drug being added to cells and how nuclear staining can be observed. By calculating the anisotropy, the group were able to measure drug binding. They progressed their study to the use of clinical Olaparib and using a Schild analysis, they could determine KDs accurate with in vitro protein based measurements. This helps to elucidate drug binding mechanisms within cells and helps to deduce the optimal drug type for tumour treatment.

The final step of the project was to image in vivo and they performed controlled delivery to administer clinical and fluorescent drug to tumours. Results indicated that fluorescent drug was essentially competed off by the clinical drug. Dr Dubach discussed their in vivo versus in vitro studies. They saw an increased heterogeneity in vivo and concluded that the experiment is affected by expression levels and drug distribution which is not uniform. In this type of study, to generate the most reliable data, the clinical drug should be injected at different doses and fractional occupancy measured.

Bulk measurements can be misleading, as they can indicate saturation of the target.  However, at the single cell level, experiments showed that cells encounter some protein inhibition, but still possess enough non-inhibited protein to permit function (for example tumour cells can still grow), known as fractional occupancy. With their approach one can look at single cell heterogeneity, this works with non-covalent drugs in vivo, which enables other quantitative measurements.

Precision Medicine in Miniaturized Format

During the afternoon track ‘Precision Medicine in Miniaturized Format’, the keynote presentation was given by Professor Olli Kallioniemi from SciLifeLab and the Karolinska Institute who spoke on the topic of ‘Functional Precision Medicine for Cancer and Beyond’. He explained that disease characteristics are changing as a result of molecular information and hence we can sub-classify diseases to make better treatment decisions.

Prof. Kallioniemi elaborated on a pilot study based around the concept of 14 dimensions of health which looks at 14 correlated molecular features which may contribute to the overall health of individuals. Factors such as diet, the gut microbiome, alcohol consumption and blood pressure are considered with the aim of combining this information, drawn from various diverse sources and considering complex inter-connections of genome, proteome, microbiome, metabolome, diet and lifestyle. People who appeared to be in poor health were offered advice to improve certain characteristics where possible.

Professor Kallioniemi next described the challenges of predicting cancer drug responses in patients. 10-15% of all cancers could be helped by having a druggable clinical mutation, but of course many of the common mutations such as P53 are not ‘directly druggable’. He elaborated on functional precision systems medicine in leukaemia and emphasised the need to better understand the biology of the disease, drug sensitivity and resistance, how to rapidly induce therapies and then to report data back to the clinician.

He described a study in which they took 500 known cancer drugs, performed dose response testing in patient derived cells (plate reader based), looking at viability, toxicity and other characteristics and then compared drug efficacy on normal cells using bone marrow. This is known as ‘ex vivo drug testing’. It allows the user to gain results on the comparative efficacy, patient profile and biomarkers for each drug and to formulate drug efficacy correlations. One can also learn about mechanism of action, resistance and the evolution of the drug response over time. This can be run alongside clinical trials and would also allow the user to identify new effective drugs if resistance has occurred.

Accelerating Drug Discovery through the power of microscopy images

Day Two’s keynote talk was presented by Dr Shantanu Singh of The Broad Institute and was entitled ‘Accelerating Drug Discovery through the power of microscopy images’. Dr Singh described the various imaging techniques used at the Broad to measure and score known phenotypes, profile and characterise samples and analyse the data generated. They can then identify compounds to use in treatments. He described a cell painting assay they developed using six stains, imaging five channels and revealing eight constituents/ organelles.

By using an automated microscope, they can also confirm signatures of genes, compounds and diseases. Dr Singh elaborated on how image-based profiling can predict small molecule activity. He described a 1000 compound screen carried out using cell painting techniques, with the resulting images then being matched to morphological profiles. This project is still underway at the Broad.

Dr Singh explained that the Broad are also focused on rare diseases. Of 7,000 known rare diseases, 4,550 are known to be monogenic and only 6% have FDA approved treatments. He discussed whether it would be possible to screen a vast number of compounds using cell painting techniques, create a database and determine which give the disease signature. This is known as image-based profiling for virtual screens.

Bioimaging is poised for dramatic improvements driven by deep learning. This includes deep learning for the segmentation of nuclei. In collaboration with Peter Horvath’s lab, The Singh lab created a new deep learning tool known as Cyto AI. The lab have also been progressing another project focused on the extraction of features by training networks on auxiliary tasks. This allows compound grouping based on their mechanisms. They are also able to capture the heterogeneity accurately by modelling data statistics and using a wet lab method known as pooled cell painting, can scale up profiling of genes via in situ sequencing.

Advances in Phenotypic screening

Later in the session, Professor Neil Carragher from the University of Edinburgh described ‘Advances in Phenotypic screening: Accelerating the Discovery of New Chemical Entities and Drug Combinations towards in vivo proof of concept’. Professor Carragher explained that the Proteomics Drug Discovery group is predominantly involved in cell and tissue-based screening assays to validate novel targets.

The lab used the image Xpress (PAA robotic), with cell profiler analysis to analyse tens of thousands of small molecules, in order to create phenotypic fingerprints for every compound and then complete machine learning to predict the mechanism of action. He described how they trained the machine learning models in breast cancer cell lines and applied the model to unseen cell lines and discussed a method for comparing the similarity of compounds to differential cellular phenotypic responses across breast cancer cell lines.

Professor Carragher then described a case study collaboration with CRUK (Rebecca FitzgeraldTed HuppRob O’Neill) for a drug discovery project on oesophageal cancer. He explained that they undertook a high throughput screen of 20,000 chemical libraries, using a cell line panel that represents the heterogeneity of disease. Using a machine learning model combined with the feature extraction method, they saw novel phenotypic space and mode of action. Using morphometric profiles, they were able to detect compounds with selectivity for oesophageal cells.

The conference was interspersed with networking sessions and delegates also enjoyed a drinks reception on the first evening. During these more informal breaks, scientists were able to discuss their research and their thoughts on the talks, meet with vendors and make valuable connections in addition to catching up with existing contacts. A tour of AstraZeneca’s facility was offered on the evening of Day two and Day three followed the format of a satellite meeting which was focused around Chemical Biology, namely PROTACs as a novel therapeutic modality and proteomic target identification strategies.

Metrion Biosciences now looks forward to exhibiting and presenting at the largest ELRIG event of the year, ‘Drug Discovery: Looking back to the Future’ to be held at the ACC in Liverpool on 5th and 6th November.

Ion Channels in Drug Discovery XIX – Satellite event at BPS 2019

Review written by the Editor

Ion channels continue to rise in prominence in both academic and commercial areas. Metrion Biosciences, alongside Sophion Bioscience, Nanion Technologies, Evotec and SB Drug Discovery sponsored the “Ion Channels in Drug Discovery XIX” Satellite meeting held on Friday 1st March at the Baltimore Convention Centre, as part of the 63rd annual meeting of the Biophysical Society (BPS).

Biophysics 2019 Marc presenting at Sophion meeitng e1566992758504

The meeting was attended by around 100 delegates  from a variety of backgrounds, including academia, pharma, small to mid-sized biotech companies and the service industry.

Keynote speaker Professor William Colmers

After a brief welcome and opening remarks from Niels Fertig, CSO of Nanion Technologies who highlighted the merits of Automated Patch Clamp (APC) and the growing number of publications centred around APC data, David Dalrymple of SB Drug Discovery introduced the keynote speaker. This was Professor William Colmers from the University of Alberta, who focused his presentation on the role of NeuroPeptide Y (NPY) and Ih HCN1-mediated currents play in stress modulation across different regions of the brain. NPY may underlie behavioural stress resilience and long-term structural changes in neurons. Work continues into the biological role of NPY with regards to energy balance, obesity, anxiety and cachexia.

Use of APC platforms to support ion channel discovery

Dr Stephen Hess then presented a three-part overview of Evotec’s use of APC platforms to support ion channel drug discovery. Stephen first described a hit profiling study of voltage-dependent Kv1.3 allosteric modulators, followed by a study of the on-off rates of Nav inhibitors using a complex assay protocol they perfected.

Stephen finished by highlighting the emerging power of CryoElectron Microscopy (Cryo EM) as a tool for structure-based ion channel studies and drug discovery. In less than five years, at least one structure from each of the 7 TRP families have been determined, and in total around 117 human ion channel structures have now been elucidated.

The mechanism-of-action and liabilities of local anaesthetics

The next speaker was James Ellis from Nocion Therapeutics, a pre-clinical stage biotech company developing novel small molecules to selectively silence nociceptors involved in cough, itch, pruritis, inflammation and pain responses. Jim spoke about the mechanism-of-action and liabilities of local anaesthetics such as Lidocaine and explained that an ideal Nav channel blocker should maintain efficacy across indications, be administered topically to minimise systemic exposure and CNS redistribution, be cell impermeant and devoid of painful or irritating TRPA1/ TRPV1 agonism.

Nocion are developing a strategy originally described by their Harvard co-founders whereby charged Nav antagonists can selectively enter over-active neurons through the large pores of ligand-gated pain receptors (TRPx, P2X, ASIC) activated by exogenous agonists or endogenous ligands and inflammatory mediators.

Genentech’s work on Nav1.7

Tianbo Li then presented an overview of Genentech’s work on Nav1.7, one of the best validated and characterized pain targets. A key challenge for Genentech is devising new chemical matter with high potency and selectivity, and here he presented a case study on Pro-Toxin II (ProTx-II), a tarantula cysteine knot peptide toxin. Through a combination of Alanine mutant scanning of a Nav1.7-NavAb chimera, charge alterations across the binding face of ProTx II and determination of the cryoEM structure of ProTx-II bound to the channel chimera, it was shown that ProTx-II binds electrostatically to specific residues in the S3-S4 linker of voltage sensor domain II (VSD2). This information was used to create higher affinity analogues of ProTx-II and another spider venom, demonstrating the potential to aid future Nav channel antagonist design.

The rationale for a new type of ion channel screening platform

Hongkang Zhang spoke on behalf of Qwell Therapeutics, a spin-out from Q-State Biosciences who work on non-opioid treatments for pain. Hongkang outlined the rationale for a new type of ion channel screening platform, comparing the costs, temporal resolution, mechanistic detail and throughput available from conventional plate-based readers (e.g. FLIPR) and APC machines.

Qwell Tx are developing the single site Optopatch optogenetic platform from Q-State into a higher throughput 24 well device suitable for target-based screening of ion channels. Using Nav1.7 channels, they have preliminary data to support claims for fast, sensitive, linear read-outs of ion channel activity that combine the advantages of plate-based readers and APC platforms.

In search of a suite of GABA-A receptor cell lines and validated APC screening assays

GABAreceptors are a complex ligand-gated ion channel family with at least 16 subunits. They are important drug targets for anxiety and epilepsy and the next speaker, David Dalrymple, highlighted SB Drug Discovery’s efforts to create a suite of GABAA receptor cell lines and validated APC screening assays. SB generated 19 human GABAA  receptor cell lines of various α1-α6, β1-3, γ and δ subunit combinations which they validated pharmacologically on the Syncropatch384 using stacked tip protocols. Several positive allosteric modulators were found after a plate-based screen of SB’s compound libraries, most of which were confirmed by APC electrophysiology.

The study of gating currents

Professor Francesco ‘Pancho’ Bezanilla was the afternoon keynote speaker, providing an overview of his recent work on genetically-encoded voltage indicators and optocapacitance techniques to probe the structure and function of voltage- dependent ion channels and transporters. The study of gating currents requires new types of fast voltage indicators, and Pancho described their work with ASAP-1, an ADP-ribosylation factor GTPase-activating protein from chicken.

Pancho then explained optocapacitance, whereby infrared light can depolarise biological membranes and excite muscle cells, Xenopusoocytes and neurons. This local heating and activation can be focused spatially and in terms of tissue-penetrating wavelengths through application of gold nanoparticles, graphite, and carbon nanotubes to cells, and even directed to sub-cellular sites and specific receptors and channels through conjugation to toxins (e.g. Ts1 toxin for Nav) and antibodies (P2X, TRPx), enabling exquisite optical control of cellular excitability.

Drug discovery collaboration update

Marc Rogers, CSO of Metrion Biosciences, then presented an overview of an 8-year drug discovery collaboration with a global pharma partner, for which Metrion provided in vitro and ex vivo screening services using their manual patch and automated patch clamp expertise. During the collaboration they developed high-quality voltage-gated ion channel assays to reliably identify and profile novel, potent, safe and efficacious state-dependent modulators of a pain-related ion channel target. This yielded a development lead compound and a back-up series with therapeutic potential equal or superior to current clinical treatments.

Knottin-antibody fusion proteins (KnotBodies)

Iontas specialise in mammalian phage display and antibody discovery and affinity maturation. Aneesh Karatt-Vellatt summarised their work on Knottin-antibody fusion proteins (KnotBodies), a novel antibody format which enables the targeting of ‘difficult’ drug discovery proteins such as ion channels and GPCRs by combining antibody light chains with ICK toxins and other small peptides to achieve increased specificity and half-life. Iontas are using Nav1.7, ASIC1a and Kv1.3 channels as case studies, and Aneesh gave an overview of Kv1.3 as a target for auto-immune disease, which affects 2-3% of the worlds’ population and may have a market value around $45.5 Bn by 2022.

As the sea anemone toxin analogue ShK-186 recently showed efficacy in a clinical trial on psoriasis, Iontas incorporated several Kv1.3-targeting toxins into their knotbody template, yielding potent and selective Kv1.3 blockers capable of reducing T-cell cytokine release.

Xenon Pharmaceuticals’ program to develop Nav1.6 modulators

Sam Goodchild then presented an overview of Xenon Pharmaceuticals’ program to develop Nav1.6 modulators to treat epilepsy and rare forms of encephalopathy such as IEEE13. The Nav inhibitors currently used have a low therapeutic index and are poorly selective, often being dosed at concentrations that can cause adverse side effects.

Based upon their extensive work on aryl sulphonamide gating modifiers of Nav1.7 (in collaboration with Genentech), Sam described their precision medicine approach which identified two novel compounds that selectively target Nav1.6 (XPC-224) and Nav1.6/1.2 (XPC-462) through a greater than 1000- fold preference for the inactivated state via binding to specific residues in VSD-IV. The compounds are efficacious in mouse seizure models at 3x in vitro IC50 and exhibit 100-fold TI over acute toxicity effects. Thus, these compounds promise to provide superior efficacy and side-effect profile to current anti-convulsant medications such as phenytoin and carbamazepine.

Developing higher throughput mechanistic and translational assays for CNS drug discovery

The final speaker was Fern Toh from Alkermes, a global pharmaceutical company working primarily on CNS disorders including MS, schizophrenia, depression and addiction. They are interested in developing higher throughput mechanistic and translation assays for CNS drug discovery. Fern outlined ongoing work to correlate molecular profiling and plate-based multi-electrode array data from cultured rodent cortical and hypothalamic neurons with functional recordings of ion channels and receptors on an APC platform. Alkermes hope to build upon these studies to look at GPCR modulators and human stem-cell derived neurons to aid the translation of screening results to CNS drug candidates.

A summary of the Sixth Cambridge Ion Channel Forum

Review written by the Editor

AstraZeneca and Metrion Biosciences again joined forces on April 9 to co-host the 6th Cambridge Ion Channel Forum, held at the Milstein building at Granta Park. The event has been a key feature of the ion channel enthusiasts’ calendar since 2011 and attended by around 65 delegates, who also enjoyed a networking lunch and poster session prior to the keynote talk presented by Professor Sarah Lummis from the University of Cambridge.

CICF image for blog
A study of the molecular function of neurotransmitter-gated ion channels

Sarah’s group study the molecular function of neurotransmitter-gated ion channels, with an emphasis on cys-loop receptors. Her research is focused on both vertebrate and bacterial cys-loop receptors, which includes nicotinic acetylcholine, gamma-aminobutyric acid (GABA), glycine, 5HT3 and glutamate-activated Cl- receptors. Sarah’s talk focused on the prokaryotic ELIC channel from Erwinia Chrysanthemi which is activated by GABA and cysteamine and triggers a stress signal or defence mechanism in plants. CryoEM data for ELIC can be used to probe the structure and function of GABAA receptors. 

Sarah concluded with animal and human data on mutations in glycine receptors that cause startle disease or hyperkeplexia. These disturb inhibitory glycine-mediated neurotransmission but remain poorly understood.

Overview of an 8-year drug discovery collaboration

Marc Rogers, CSO of Metrion Biosciences, then presented an overview of an 8-year drug discovery collaboration with a global pharma partner, for which Metrion provided in vitro and ex vivo screening services using their manual patch and automated patch clamp expertise.

During the collaboration, they developed high-quality voltage-gated ion channel assays to reliably identify and profile novel, potent, safe and efficacious state-dependent modulators of a pain-related ion channel target. This yielded a development lead compound and a back-up series with therapeutic potential equal or superior to current clinical treatments.

“It was exciting and highly rewarding to hear some of the latest developments in the field and to see people from academia and industry come together and share their knowledge”.

Marc Rogers
CSO of Metrion Biosciences
The development of novel therapies for respiratory diseases

The next speaker was Professor Martin Gosling, the CSO of Enterprise Therapeutics and Professor of Molecular Pharmacology at the University of Sussex. Enterprise focus on the development of novel therapies for respiratory diseases by targeting the underlying mechanisms of mucus congestion, with a focus on Transmembrane Member 16A (TMEM16A) calcium-activated chloride channel and epithelial sodium channels (ENaCs).

Martin presented a case study of TMEM16A, which has recently been proven to behave as a key orchestrator of anion secretion in human airway epithelium. The team started a parallel screening campaign to identify low molecular weight potentiators of TMEM16A using plate-based readers and APC platforms, identifying EXT001 as being particularly efficacious.

This TMEM16A potentiator significantly enhanced the secretory current in cystic fibrosis (CF) patient-derived bronchial epithelial (HBE) cells, with EXT001 promoting mucosal fluid secretion to clinically relevant levels. In collaboration with the Cystic Fibrosis Trust and Cystic Fibrosis Foundation, Enterprise are taking several TMEM16A potentiators into clinical development.

The modulation of GABA-A receptors

Paul Miller from the University of Cambridge then provided insights into the modulation of GABA-A receptors, a ligand-gated chloride channel that mediates fast inhibitory synaptic transmission in brain. As well as being a validated target for anxiety, sleep and addiction, Sage Therapeutics received approval for their GABA-A modulator brexanolone in post-partum depression. GABA receptors possess well-known binding sites in the pore and near the ligand-binding pocket where allosteric modulators like diazepam can bind.

There remains a challenge, however, to develop more selective ligands with diverse chemistry and mechanisms-of-action, and Paul described how his group, in collaboration with Professor Jan Steyaert, have recently raised llama nanobodies against the α1β3 GABA-A receptor. Binding of nanobodies to purified GABA-A proteins is detected using biacore/SPR and patch clamp electrophysiology, with one promising nanobody (Nb38) found to favour the GABA-bound activated state, acting as both an agonist and a potentiator.

Binding of Nb38 helped elucidate the GABA-A receptor structure to 5 angstrom resolution, which was improved to 2.5 angstrom using CryoEM, revealing the two binding sites of Nb38. Paul explained that this work is being extended through generation of a 108 nanobody library.

New cardiac ion channel assays

The session was brought to a close by Verity Talbot with an overview of AstraZeneca’s efforts to develop new cardiac ion channel assays to meet the requirements of the FDA’s CiPA initiative (Comprehensive in Vitro Proarrhythmia Assay). CiPA aims to promote a novel paradigm for assessment of clinical cardiac risk, especially Torsades de Pointes, by combining in vitro ion channel assays and in silico modelling techniques together with translational human tissue assays to facilitate early drug development and compound selection.

Verity described how the FDA selected a training set of 28 compounds of known TdP risk which were screened by pharma companies, CROs (including Metrion), platform providers, academic collaborators and regulatory agencies as part of the CiPA consortium. AZ selected 12 compounds from the toolbox and used them to validate an APC ‘dynamic hERG’ assay utilising the Milnes voltage protocol. They were able to generate kinetic parameters for use in in silico models of action potential duration and to determine qNet scores, finding that combinations of very different hERG binding parameters could estimate similar qNet scores of cardiac arrhythmia risk.

Verity explained that these results are driving ongoing work to assess whether APC kinetic hERG data is predictive of proarrythmic risk and how AstraZeneca will use the CiPA paradigm in their drug discovery process.

It was exciting and highly rewarding to hear some of the latest developments in the field and to see people from academia and industry come together and share their knowledge.  We very much look forward to hosting the Cambridge Ion Channel Forum again in 2020, which by then will be in its seventh year, please keep an eye on the Metrion website for further details.