External Speaker Series presentation featuring Professor Annette Dolphin

Professor Annette Dolphin, Metrion Biosciences Scientific Advisory Board member and seventh presenter in our external speaker series, gives her perspective on her research into neuronal voltage-dependent calcium channels.    

How and when did you first become interested in neuropharmacology?

In the second year of my Biochemistry degree in Oxford, during my Diploma in Chemical Pharmacology. As far as I remember, it was mostly about the differences between the effects of hexamethonium and decamethonium, but it seemed fascinating to me at the time.

What attracted you to your current research focus of neuronal voltage-dependent calcium channels?

I did postdoctoral research on G-protein coupled receptors and their downstream effects. Many Gi/o-coupled receptors act at synaptic terminals to cause presynaptic inhibition, and one of the targets is the presynaptic voltage-gated calcium channels. That is initially why I became interested in calcium channels and their modulation by G protein activation.

Can you describe how the α2δ and β subunits modulate these channels?

Both α2δ and β subunits increase trafficking of the channels in different ways. β subunits appear to aid folding of the channels and protect them from endoplasmic reticulum-associated proteasomal degradation. Exactly how the α2δ subunits increase trafficking is still unknown. Both subunits also affect the channel voltage-dependent and kinetic properties in ways that depend somewhat on the isoform of the subunit, since there are four different β subunits, and the same number of α2δ subunits.

To what extent are the mechanisms of action of the drugs binding to α2δ and β subunits understood?

All our studies are consistent with the hypothesis that gabapentin binds to α2δ-1 (and α2δ-2) and reduces the trafficking of α2δ-1 and the associated channel. Some of our data indicates an interference of forward trafficking at the level of recycling endosomes. Data from others have also put forward other mechanisms of action of gabapentin. It would be terrific to get a structure of a channel with gabapentin bound to α2δ-1.

Do you think that disrupting calcium channel trafficking by targeting the beta subunit has a therapeutic potential?

This has been tried in various studies, and I always thought it had promise. One problem is that the interaction between β and the I-II linker of the α1 subunit occurs in a binding groove on β, which would be difficult to disrupt with a small molecule. Another problem would be how to get specificity as the β subunit binding site is quite conserved between the different channel α1 subunits.

What are your future research plans in this field?

I am very interested in defining exactly how α2δ subunits work and how neuronal calcium channels are targeted to specific sites.

What do you think are the remaining issues to solve to create a Ca_v2.2 clinical compound?

Selectivity, affinity, access to dorsal horn terminals for the alleviation of chronic pain.

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

I don’t think I could have toed the line in industry, and I enjoy following a consistent line of research, driven by the previous findings of my own group and others. It is a great privilege to have been able to do this.

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

No, but the reverse is also true.

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

Yes, I think academic pursuits have been down-graded, as a result of universities turning into businesses. The importance of demonstrating immediate “impact” of research is over-stressed. In my view we should concentrate on getting basic research right.

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

It is fascinating that dihydropyridines and other calcium channel blockers are very useful drugs, but their target was discovered after the drugs were identified; similarly local anaesthetics. The same is true when we think of drugs targeting auxiliary subunits (like gabapentinoids binding to α2δ and sulphonylurea binding to SUR regulatory subunits of KATP channels). The α2δ subunits would never have been thought of ab initio as a potential drug target. So finding drugs that target ion channel function is not impossible, and should become easier with higher throughput techniques.

Acknowledgements

Metrion Biosciences’ External Speaker Series was established as a forum for leading academic researchers to present their latest research to our staff and staff from other companies in the Cambridge area. The Metrion team welcomes suggestions for future speakers and topics via: this link.

You can also sign up for Metrion biosciences update via: this link. You can alter your preferences or unsubscribe at any time. 

External Speaker Series presentation featuring Professor Mustafa Djamgoz

Professor Mustafa Djamgoz (Imperial College London), the sixth presenter in Metrion Biosciences External Speaker Series, provides some insight into his research into the role of ion channels in cancer and metastasis.

You first an published observation regarding the upregulation of voltage-gated sodium channels (VGSCs) in cancer cell lines in 1995. What was your initial rationale for taking this approach?

The primary rationale was our conviction (from decades of research on ‘excitable’ cells) that electrical signalling plays a major role in cellular functioning in health and disease. Added to this, was our sense of curiosity whether (i) cancer cells generated electrical signals and (ii) electrical signalling differed between metastatic (i.e. aggressive) vs. non-metastatic or benign tumours.

In the 1995 paper, we adopted two such isogenic cell lines from rat prostate cancer as a model and their direct electrophysiological comparison led to the discovery of the VGSC and its role in promoting cellular invasiveness/metastasis in vitro and in vivo. Since then, such functional VGSC expression has also been discovered in cancers of breast, lung (several forms), colon, ovary, cervix and stomach by various international groups.

Where studied, the VGSC upregulation was found to occur concurrently with downregulation of voltage-gated outward / potassium currents, thereby making the membranes of these metastatic cells electrically excitable. We called this the “Celex Hypothesis” of metastasis, stating that it is the membrane excitability that makes these cancer cells hyperactive, disruptive, invasive and, ultimately, metastatic.

Following on your early research relating to sodium channels, we now have evidence for altered expression of potassium, calcium, TRP family, hERG and P2X channels in a range of tumour tissues. Which of these do you believe has the most promise to develop a new therapeutic?

Indeed, yes, there is such evidence, increasing almost daily! So, we are only scratching the tip of an iceberg! Our vision is exactly like that for the brain (a ‘biological universe’), all the ion channels are also in cancer (a ‘pathological universe’). The big question is which ion channel is the most important. Since metastasis is by far the most common cause of death from cancer, we have put the spotlight on VGSCs.

Nevertheless, we need to understand the pathophysiological role of all other ion channels, so we can exploit them most effectively, individually or, more likely, in combinations. So, it is like an orchestra, the VGSC could be the lead violinist but to be able to create the full symphony we must understand the other players as well.

Do you see ion channel modulator-based therapies for cancer solely as adjuncts to other chemotherapy and biologics approaches or is there potential for a stand-alone ion channel modulator therapeutic?

The problems associated with the current therapies for cancer are well known. I see both potentials for ion channel modulator-based therapies. Evidence is very strong that silencing Na_v1.5 eliminates metastasis in breast cancer in vivo models. If this translates to the clinic, then VGSC blockers could serve as mono-therapeutic agents.

There is also evidence, for example, that the effect of epidermal growth factor (EGF) in promoting small-cell lung cancer (SCLC) invasiveness occurs substantially through VGSC activity. The EGF receptor is already a major target for SCLC but suffers from the fact that blocking one growth factor pathway often leads to another one taking over. So, a combination therapy may be more effective.

The identification of novel, highly selective ion channel modulators is challenging. The relative lack of novel external architecture means limited opportunities for antibodies and top ten pharma companies with large budgets have been frustrated by issues associated with small molecule selectivity. How would you solve this?

This is not easy to answer! All I can do is to offer our experience. The predominant VGSC (Na_v1.5) expressed in breast and colon cancer is clearly a neonatal splice variant (nNa_v1.5). The spliced region has a unique amino acid sequence and this can be targeted using an antibody with a selectivity of at least two orders of magnitude compared with its ‘nearest neighbour (adult Na_v1.5).

Furthermore, we have evidence that nNa_v1.5 and adult Na_v1.5 are pharmacologically distinguishable, so a high throughput screen could reveal small molecules selective for nNa_v1.5. Finally, we have exploited the fact that growing tumours are hypoxic and hypoxia leads to the VGSCs to develop a persistent current (INaP) which, in turn, promotes invasiveness. The beauty of INaP is the fact that it could occur in any VGSC so its inhibitors may be applicable to several carcinomas irrespective of the subtype of VGSC(s) expressed!

What are the ultimate goals of your current research activities?

Ultimately, of course, to cure cancer! Now, I know, that’s a rather flippant remark. For a start, I would prefer not to use the word “cure” since once cancer touches someone, although it may somehow be put to bed (i.e. the patient is in remission), there will always be a danger that it will return, and it does at least in some cases.

So, we advocate ‘living with cancer’, rather like we can live chronically with diabetes and the AIDS virus. Living with cancer means suppressing metastasis since this is the main cause of death in cancer patients. In the foreseeable future, we plan to do this by exploiting the unique properties of the culprit VGSC as I discussed above.

Given access to suitable funding what would be your priority future research plans?

There is so much to do! Currently, we are focused on cancers of breast and colon due to the significant role played by nNa_v1.5 in metastasis. Whilst developing a monoclonal antibody to nNa_v1.5, we would like to take INaP blockers, such as ranolazine, into clinical trials within a few years. Then, we would like to look at other even harder-to-treat cancers like pancreas and glioblastoma. Finally, we would like to evaluate the prognostic potential of VGSC since all the signs are that this occurs very early in the acquisition of metastatic potential.

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

I was born an academic (!) and I was particularly lucky to train in the ‘old’ British system where curiosity and freedom ruled the research waves. I also enjoy teaching and always provoke my students to be better than me, so we can make real progress.

Having said that, I now wear two hats since our research has led to the founding of a small company (Celex Oncology Limited). I did struggle at the beginning to think and act like a man from industry, but I now feel that I can. The hardest thing still, at times, is not being able to freely and instantly discuss our experimental results.

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

This is getting better since the needs of the two sides are mutually compatible, in fact potentially synergistic. Still, there is a long way to go and much more room for improvement. One important step would be for companies to share the long-term vision of academics and invest in what may seem like early-stage research. I have spoken to lots of organizations who claim to invest in early-stage research, but it was almost always not the case, at least from an academic’s point of view.

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

It’s changed a lot, and I cannot say that it has necessarily changed for the better. Much more now is money-driven and the ‘human element’ has been eroded, unfortunately! Still, as long as you manage within the four walls of the lab, it is ok!

Acknowledgements

Metrion Biosciences’ External Speaker Series was established as a forum for leading academic researchers to present their latest research to our staff and staff from other companies in the Cambridge area. The Metrion team welcomes suggestions for future speakers and topics via: this link. 

You can also sign up for Metrion Biosciences updates via: this link. You can alter your preferences or unsubscribe at any time.

Review written by the Editor

Metrion Biosciences and AstraZeneca joined forces on Tuesday 8^th May 2018 to co-host the fifth Cambridge Ion Channel Forum at Medimmune’s Milstein Building on Granta Park, Cambridge (UK). Established in 2011, with previous co-organisers including Neusentis, Medimmune and BioFocus, this afternoon session of ion channel focused presentations provides an opportunity for delegates to participate in networking, present a poster and listen to presentations from respected ion channel researchers. A recurring theme throughout the 2018 event was the importance of automated patch clamp (APC) electrophysiology in support of the optimisation of small molecules, biologics and in the early cardiac safety profiling of selected compound series.

Professor Peter McNaughton gave the keynote speech

King’s College London’s Professor Peter McNaughton, who was also a presenter at the 2011 meeting, gave the 2018 the Keynote Lecture summarising his team’s progress towards developing selective hyperpolarization-activated cyclic nucleotide-gated ion channel blockers as novel analgesics for neuropathic pain. This research being founded on the observation that specific deletion of HCN2 in nociceptive neurons leads to reduced neuropathic and inflammatory pain sensation, without effects on normal sensation of acute pain.

Peter outlined the evolution of his project to develop potent and selective HCN2 blockers for therapeutic use in the clinic, with a key project objective of minimising block of HCN4 channels in the heart. Peter also touched upon his research into tinnitus, via a collaboration with Mark Wallace and Deborah Hall from the University of Nottingham. The hypothesis behind this work being that tinnitus may be reduced by use of HCN2 blockers to reduce the abnormally high firing in unmyelinated auditory nerve fibres. Both of Peter’s research programmes has significant therapeutic value and we look forward to developments towards the clinic.

Assessing the hERG liability of small molecules

Matt Bridgland-Taylor then presented a case study combining electrophysiology with intracellular concentration analysis to assess the hERG liability of small molecules. In addition to assessing any link between the intracellular concentration and the kinetic profile of block, this also allowed to verify that the hERG inactive compounds were accessing the CHO cells used in the electrophysiology assay.

Iontas’ KnotBody™ technology

Moving away from the focus on small molecules, Damian Bell gave an overview of Iontas’ KnotBody™ technology, whereby knottin toxins (cysteine knot mini-proteins) are fused into peripheral complementarity-determining regions (CDRs) of the antibody VL domain. This approach offers the potential for retaining the ion channel blocking activity of the knottin, whilst gaining an extended half-life and additional specificity conferred by multiple contact surfaces of the antibody. Damian presented proof of concept data where phage display was used to engineer specificity into both antibody and peptide, with QPatch electrophysiology data presented for both K_v1.3 and ASIC1a.

Skeletal muscle channelopathies

Skeletal muscle channelopathies was the topic of choice for Roope Mannikko from University College London, who discussed myotonia and periodic paralysis and the effect of Na_v1.4 channelopathies in relation to infant sudden death syndrome. Roope’s group has also demonstrated the use of NMR techniques to probe the interactions of Voltage-Sensing Domain (VSD)-1 with HM-3, a crab spider toxin which is known to inhibit gating pore currents due to mutations found in patients with Hypokalemic Periodic Paralysis (HypoPP).

£200,000 funding to further optimise the preclinical properties of lead series compounds

The afternoon was concluded by Metrion Biosciences’ CSO Marc Rogers presenting an overview of Metrion’s use of QPatch APC assays to support identification of novel small molecule inhibitors of the K_v1.3 channel to treat auto-immune disorders. The programme has identified nM potency blockers with good gene family but no species selectivity issues, strong efficacy in native human T-cell assays, and superior drug-like properties compared to leading preclinical small molecules and biologics such as ShK-186 (Dalazatide, Kineta Therapeutics).

Metrion has secured £200,000 Innovate UK funding to further optimise the preclinical properties of lead series compounds, and plans to secure a collaboration partner to further develop immune-sparing K_v1.3 drug candidates for the treatment of autoimmune and neurodegenerative diseases in the near future.

Future events

The next Metrion Biosciences hosted event will be on 11^th July 2018. Professor Trevor Jones will be presenting “Disruptive Influences on Drug Discovery and Healthcare Delivery”.

Review by the Editor

Researchers gather for interactive forum

Metrion Biosciences and Axol Bioscience joined forces on Wednesday 23^rd May to co-host the 2018 “Interactive Stem Cell Forum” in Cambridge (UK). A meeting featuring a morning of informative and thought-provoking talks from leading academic and industry-based researchers working in the stem cell field, followed by an afternoon of laboratory demonstrations in Metrion’s Granta Park headquarters.

The meeting enabled researchers from across Cambridge and the surrounding area to attend an event focused solely upon recent developments in the field of stem cell research spanning neuroscience and cardiac topics. It was also an opportunity for attendees to network with each other and also see demonstrations utilising Axol’s Induced Pluripotent Stem Cells (iPSCs) in Metrion’s laboratories; where the Metrion team also showcased manual patch electrophysiology, QPatch 48 automated electrophysiology and microelectrode array (MEA) assay platforms.

Review of available techniques applied to iPSC research

The morning session, chaired by Metrion Biosciences’ CSO Marc Rogers, started with a presentation by Matthew Daniels, a Consultant Cardiologist based at the University of Oxford. Matthew described some limitations of technologies that have previously been applied to iPSC research. For example chemical dyes such as BAPTA-AM and Fura-2 have significant cellular toxicity,  evidenced by such dyes negatively effecting the contractility properties of iPSC-derived cardiomyocytes. 

Matthew is a strong advocate for the adoption of alternative fluorescent/luminescent tools and optogenetic-based stimulation for iPSC cardiomyocyte research, he provided a thorough review of the available techniques and described some of his research into non-invasive phenotyping and drug screening. Use of such technology has enabled the monitoring of cells for periods of up to 90 days in his laboratory. He also presented preliminary results using microarrays of single cells on a single assay plate that may have potential for scale up and use in drug discovery.

The objectives of the CiPA initiative

The co-hosting companies also gave data-led presentations, with Metrion’s Sarah Williams discussing the establishment of Axol’s cardiomyocytes as a model system using manual patch clamp electrophysiology in Metrion Biosciences laboratories. Sarah reviewed the objectives of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, a multi-agency strategic enterprise with the objective of improving cardiac safety screening of potential new drugs.

Whilst Metrion has a substantial validation dataset for three of the CiPA ‘pillars’ Sarah focused on recent work using Axol’s atrial and ventricular phenotype human iPSC derived cardiomyocytes. Sarah’s presentation can be found here and an accompanying poster, presented at the 2018 Select Biosciences Stem Cells In Drug Discovery meeting, is here. Thanks to all Axol and Metrion staff who contributed towards this work

The pathology of Amyotrophic Lateral Sclerosis (ALS)

The pathology of Amyotrophic Lateral Sclerosis (ALS) was the topic of choice for Gareth Miles from the University of St Andrews, who discussed the use of stem cell-based technology to investigate the interactions between astrocytes and motor neurons in a humanised ALS model. Historic suffers of this disease include American baseball player Lou Gehrig and the renowned physicist Professor Stephen Hawking.

Using iPSC derived co-culture of astrocytes derived from healthy individuals and ALS patients with the TARDPB or C9ORF72 mutations with motor neurones from healthy individuals, the Miles’ group has demonstrated hypo excitability in the ALS astrocyte co-culture motor neurones correlating with loss of sodium and potassium currents. This data, combined with CRISPR studies removing the C9ORF72 ALS-causing mutation, implicates astrocyte-neuron signalling as a promising target for ALS drug discovery.

The phenotypic and functional characterisation of human iPSC derived microglia

Zoe Nilsson from Axol Bioscience then discussed the phenotypic and functional characterisation of human iPSC derived microglia. These are innate immune cells found within the central nervous system which possess key roles in neurogenesis and immunity and which Axol have developed as a co-culture with human iPSC derived cortical neurons. Zoe presented the use of such tools in drug discovery and linked back to Gareth’s earlier talk by describing the dangers of the overactivation of microglia which can lead to neuroinflammation and can play a critical role in ALS and other neurodegenerative disorders.

Using tissue engineering techniques to model Alzheimer’s Disease

Zoe was followed by Eric Hill from Aston University, who described his work using tissue engineering techniques to model Alzheimer’s Disease. Eric spoke about the many difficulties associated with drug discovery in the Alzheimer’s field, attributing this largely to the lack of high quality predictive experimental models. Additionally, it is now widely accepted that the first stages of Alzheimer’s disease may occur around twenty to thirty years before initiation of memory loss – further complicating the situation for disease modelling.

In a quest to produce a high quality predictive in vitro model the Hill lab is pioneering an approach using Alzheimer’s Disease-derived iPSC and 3D culture techniques. As part of the MESO-BRAIN initiative Eric’s team have been 3D printing cultures of Alzheimer’s and healthy’ iPSC astrocytes to form neural networks with defined biological architecture in polymer scaffolds.  Conductive polymer scaffolds may be used, enabling monitoring of electrical activity within the organoid structure or, alternatively, real time imaging techniques can be applied. Ultimately the Hill lab aims to produce a model suitable for early discovery compound screening or to trial other novel treatments for Alzheimer’s Disease.

The use of iPSCs in both disease modelling and as a safety pharmacology platform

The final Speaker was Daniel Sinnecker, a cardiologist from the Technical University of Munich. Daniel discussed the use of iPSCs in both disease modelling and as a safety pharmacology platform. For example, use of iPSC as an integral component in CiPA which linked well to earlier content within Sarah Williams’ presentation. Daniel also discussed the use of lentiviral transduction to insert genetically encoded voltage sensors into iPSC cardiomyocytes from healthy and long-QT type 1 (LQT1) patents.  Using this technique the Sinnecker lab has been able to quantify cardiomyocyte action potential characteristics in healthy iPSC and also demonstrate early after depolarisations in the LQT1 mutants. This technique shows great promise for evaluating cardiomyocyte iPSC characteristics over an extended time period.

Laboratory demonstrations

After a networking lunch, which prompted further conversation around the various themes presented in the morning session, Metrion Biosciences hosted a series of laboratory demonstrations in our Granta Park facility. The demonstrations involved use of Axol’s iPSCs, with Sarah Williams showcasing Metrion’s “gold standard” conventional manual patch clamp electrophysiology capabilities.

Edd Humphries then demonstrated one of Metrion’s QPatch 48 automated electrophysiology platforms using clonal stable cell lines. The QPatch is a device that produces high quality electrophysiology data for both routine screening in support of medicinal chemistry activities and is the platform upon which Metrion has validated its suite of high quality CiPA-compliant safety profiling assays.

Finally, Said El Haou showcased Metrion’s Axion Biosystem’s Maestro MEA system, a versatile platform able to capture real-time, information rich, recordings from iPSC and cultured native neurons, with the facility to evaluate effects of novel compounds over extended time periods (days to weeks).

Acknowledgements

This event, closely followed the 2018 Cambridge Ion Channel Forum co-hosted by Metrion Biosciences and AstraZeneca on 8^th May, was a further example of Metrion Biosciences commitment to promoting and generating high quality science in the Cambridge bio cluster. We would like to thank the Axol team for being excellent co-hosts and we look forward to organising our next event. You can sign up for updates regarding Metrion sponsored events, Metrion’s external speaker presentations and services updates HERE and you can refine your topics of interest and opt out at any time.