Structural insights into small molecule activation of TRPC4/5 channels

Lead Research Organisation: University of Leeds
Department Name: School of Medicine

Abstract

The cell is the basic unit of all known living organisms, and humans consist of many different types of cells, the majority of which are highly specialised. In order to function properly, cells need to communicate with their environment and with neighbouring cells. This requires the transmission of information across the cell membrane, which separates the cellular content from the extracellular environment. One major mechanism of communication is the movement across cell membranes of ions - mainly sodium, potassium, calcium and chloride - through channel-forming proteins that are located within the membrane, so-called ion channels.

Many human diseases result from abnormalities in the function of ion channels, and many successful therapeutic drugs work by activating or blocking ion channels. Our research focuses on ion channels called TRPC4 and TRPC5 channels, which are increasingly recognised as potential drug targets in a variety of diseases - including cancer, heart failure, cardiovascular and metabolic disease, epilepsy and anxiety disorders - but for which the development of activators and blockers as drugs has proven difficult. For example, we previously discovered that Englerin A, a natural product isolated from an African tree used in traditional medicine, selectively kills renal cancer cells by the potent activation of TRPC4 channels. Englerin A is also a very potent activator of TRPC5 channels. However, Englerin A is too unstable and too toxic to be used as an anti-cancer drug.

In this project, we will study how Englerin A interacts with TRPC4 and TRPC5 channels . We will use a combination of experimental approaches, building on the specific expertise of the different team members. For example, we will use analogues of Englerin A that can chemically react with TRPC4/5 channels, and use mass spectrometry to identify where in the channels the reactions take place. In addition, we will use state-of-the-art electron microscopes - part of a recent £17m investment by the University of Leeds and the Wellcome Trust - to determine the three-dimensional structures of TRPC4/5 channels and their complexes with Englerin A.

These results will reveal how Englerin A works on the molecular level, and how the activity of TRPC4/5 channels can be regulated by small molecules. This will enable future development of drugs that targets specific TRPC4 or TRPC5 channels, which may lead to the development of the first drugs that target these channels. We will ensure the future use of our results in the drug discovery process through our ongoing collaboration with the Lead Discovery Center of the Max Planck Society, with the aim to develop drug-like molecules for clinical trials. In addition, we will publish our results in open access publications, and make our data and materials freely available through public repositories.

Technical Summary

Transient Receptor Potential Cannonical (TRPC) proteins form tetrameric, non-selective ion channels permeable by Na+ and Ca2+. TRPC channels have key roles in cellular signal transduction/integration, and specific TRPC multimers are increasingly being recognised as potential drug targets for the treatment of, for example, anxiety and other CNS disorders (C5, C1/C4), heart failure (C5), cancer (C5, C1/C4), and cardiovascular and metabolic disease (C5). However, both fundamental and translational studies on TRPC channels are hampered by the paucity of potent, specific chemical modulators, and the lack of understanding of TRPC-small molecule interactions currently prevents the rational design of such modulators. This project will significantly develop our understanding of the molecular interactions between TRPC4/5 channels and the natural product (-)-Englerin A ((-)EA), the most potent and efficacious TRPC4/5 agonist known to date. Based on previous studies, we hypothesise that (-)EA occupies a well-defined pocket of TRPC4/5 channels, where it may transfer its glycolate group to a key nucleophilic TRPC4/5 residue. To test this hypothesis and determine the mechanism-of-action of (-)EA, we will identify TRPC4 or TRPC5 residues and domains essential to (-)EA-induced TRPC4/5 activation (O1), use chemical crosslinking and mass spectrometry (MS) to map peptides around the (-)EA binding site of TRPC4/5 (O2) and use single-particle cryo-electron microscopy (EM) to determine the 3D structures of specific TRPC4 and/or TRPC5 channels and their complexes with (-)EA (O3). This multidisciplinary project will lead to new insights in the structure, molecular interactions, and regulation of TRPC4/5 channels, which would underpin structure-based design in TRPC drug discovery.

Planned Impact

This project will deliver structural insights into the regulation of TRPC4/5 ion channels, which are increasingly being recognised as potential drug targets for the treatment of, for example, anxiety and other CNS disorders, heart failure, cancer, and cardiovascular and metabolic disease. Through a multidisciplinary research programme, we will identify the binding site of the most potent activator of these channels, and characterise the molecular interactions within the protein:ligand complex. The results will underpin future structure-based discovery of specific TRPC modulators, either as valuable tools in fundamental studies of TRPC biology, or as drug leads.

In addition to the clinical relevance of TRPC4/5 inhibition, activation of TRPC4 (or rather heteromeric TRPC1/C4 channels) is relevant in the context of kidney cancer research. However, TRPC5 channels are significantly easier to study, and several essential TRPC5 constructs are already available in our labs. Therefore, we have decided to initially validate our approach using TRPC5 channels, before translating it to the highly similar TRPC4 channels. In the longer term, we aim to solve high-resolution structures of all known homo- and heteromeric TRPC1/4/5 channels and compare both their agonist and their antagonist binding sites. However, this aim is beyond the scope of the current proposal.

In addition to its impact on basic research, the main potential beneficiaries of this project are: (i) potential end-users in the pharmaceutical industry/biotechnology sector - and therefore ultimately (ii) the public/patients - because of the translational potential of the results in drug discovery.

To realise the potential of our research, we will disseminate our research widely through open access publications and presentations at conferences and meetings, and we will liaise directly with potential end-users in academia, technology transfer organisations and the pharmaceutical industry (with help of a dedicated research?novation development manager). We already have ongoing collaborations with the Lead Discovery Center of the Max Planck Society (LDC) and with AstraZeneca, and we are currently negotiating a full partnership with the LDC, with the aim to develop TRPC4/5 modulators into lead compounds with proven efficacy in at least one relevant animal model. If successful, we will then liaise with pharmaceutical companies and with clinicians to progress compounds to clinical trials, for example through Leeds' Clinical Trials Research Unit.

In order to make the public aware of our research, its impact on the drug discovery process, and more general issues of high societal interest related to this project (cancer research, properties and safety of natural vs synthetic molecules, toxicity, genetically modified organisms, and the drug discovery process), we will be pro-active in public engagement, for example through exhibits at public science events organised by the Astbury Centre/University of Leeds, the Royal Society, and through the production of a YouTube video.

Publications

10 25 50

 
Description The research team has purified and determined the structure of TRPC5 channels in the presence of several modulators, providing key insights into the modes-of-action of multiple small molecules. In addition, the team has developed and characterised several new chemical tools and protocols to study TRPC1/4/5 channels, including new modulators, new photoaffinity probes, and new photoaffinity labelling protocols. The team has contributed to scholarship in the area of TRPC channels by writing a review on TRPC1/4/5 channels, in which we critically analyse the evidence for the existence of different tetrameric TRPC1/4/5 channels, and the impact of pharmacological tools on the understanding of the role of TRPC1/4/5 channels in disease.
Exploitation Route Other researchers can read our papers and data sets and use the findings to progress the field of TRPC1/4/5 research. Our cryoEM pipeline is also a key technology in our new spinout company, CalTIC GmbH (www.cal-tic.com)
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Our data and methods have been integrated into a drug discovery pipeline that involves an academic-industrial partnership, including the formation of our spin-out company CalTIC (www.cal-tic.com).
First Year Of Impact 2021
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BioEmPiRe; Accessing uncharted but essential landscapes to biological machineries by pulse EPR
Amount £988,938 (GBP)
Funding ID BB/W019795/2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2023 
End 10/2023
 
Description MRC Confidence in Concept
Amount £25,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2021 
End 10/2021
 
Title AM237 - TRPC5 (partial) agonist and TRPC4 inhibitor 
Description This compound is a partial agonist of TRPC5:C5 channels and an inhibitor of other TRPC1/4/5 channels. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact This compound has helped us understand the mode-of-action of TRPC1/4/5 modulators 
 
Title Cryo-EM of TRPC5 channels in complex with small molecules 
Description We have developed a pipeline for the assessment of small molecule binding to TRPC5 channels by cryo-EM, providing detailed structural insight into the mode-of-action of TRPC1/4/5 modulators as well as physiologically relevant motifs of TRPC5 (such as binding sites of lipids and metal ions) 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact The technology is currently being implemented into our TRPC drug discovery pipeline (through MRC CiC funding). 
URL https://www.nature.com/articles/s42003-020-01437-8
 
Title Labelling of extracellular loop of TRPC channels 
Description In collaboration with the group of Mark Howarth (Oxford), we developed a method for the labelling of extracellular loop[s of TRPC proteins, using DogTag/DogCatcher technology 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact Upon request from other international research groups, we have shared our plasmids with new TPC constructs 
URL https://www.sciencedirect.com/science/article/pii/S2451945621003159
 
Title Xanthine-based photoaffinity probes 
Description We have developed xanthine-based photoaffinity probes for assessment of ligand engagement by TPC5 channels in human cells. These tools and our protocols will support future drug discovery programmes. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact We have shown for the first time that the most potent TRPC1/4/5 modulators work by direct binding to TRPC protein. We have also developed the first binding assay to assess TRPC5 modulators in cells. 
URL https://chemrxiv.org/articles/Xanthine-Based_Photoaffinity_Probes_Allow_Assessment_of_Ligand_Engagem...
 
Title EMDB files: Human TRPC5 
Description EMDB files for 3 different TRPC5 cryo-EM structures EMDB-10903 EMDB-10909 EMDB-10910 For details, see https://www.nature.com/articles/s42003-020-01437-8 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact Used in internal drug discovery pipeline (funding MRC CiC and VC funding - TBA) 
URL https://www.nature.com/articles/s42003-020-01437-8
 
Title Human TRPC5 in complex with Pico145 (HC-608) 
Description PDB file for TRPC5 structure in complex with Pico145 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact Used in our in-house drug discovery program (funding through MRC CiC and VC funding - TBA) 
URL https://www.rcsb.org/structure/6YSN
 
Description High-speed AFM to study TRPC channel dynamics 
Organisation University of Leeds
Department School of Physics and Astronomy
Country United Kingdom 
Sector Academic/University 
PI Contribution We have worked with Dr George Heath, a new academic in Leeds' Bragg Centre/School of Physics, to measure dynamics of TRPC5 with high-speed AFM. We have made the protein, exchanged it into liposomes and provided small molecules.
Collaborator Contribution The collaborators used HS-AFM to image TRPC5 in the absence and presence of a small molecule.
Impact The project is now being continued by a self-funded international PhD student jointly supervised by George Heath (Physics) and Robin Bon (Medicine).
Start Year 2019
 
Description TRPC4 in intestinal smooth muscle cells 
Organisation Taras Shevchenko National University of Kyiv
Country Ukraine 
Sector Academic/University 
PI Contribution Small molecules and associated knowledge and structural understanding of their mode-of-action
Collaborator Contribution Evaluation of small molecules (-)-englerin A and Pico145 in intestingal smooth muscle and in mouse studies.
Impact https://www.sciencedirect.com/science/article/pii/S0753332223014701?via%3Dihub
Start Year 2021
 
Description TRPC4/5 photopharmacology 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution Because of our success with TRPC5 structural biology/pharmacology, we were asked to join a consortium of 5 German groups working on photopharmacological probes of TRPC4/5 channels. We showed that the probes bind to TRPC5 in both photochemical states, thereby providing evidence that they work as efficacy switches. A paper is currently in preparation.
Collaborator Contribution The partners developed the probes, assessed their photochemistry and photopharmacology in cells and tissues.
Impact not yet - paper in preparation
Start Year 2022
 
Description TRPC5 channel structure-function studies 
Organisation Academy of Sciences of the Czech Republic
Country Czech Republic 
Sector Academic/University 
PI Contribution Structural insights into TRPC5 channel modulation by (-)-englerin A and lipids
Collaborator Contribution structure-function studies of TRPC5 (site-directed mutagenesis, patch-clamp, molecular dynamics)
Impact 1 paper under review at EMBO Reports 1 paper in preparation
Start Year 2022
 
Description Tagging extracellular loops of TRPC channels 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We worked with the group of Prof. Mark Howarth to apply DogTag/DogCatcher technology for the tagging of extracellular loops of TRPC channels. The initial results were published in Keeble et al., Cell Chem. Biol. 2021. We designed cloned the TRPC constructs, checked their expression and function, and developed Western blot and imaging protocols to determine extracellular labelling.
Collaborator Contribution The Howarth group developed the DogTag/DogCatcher technology, provided advice on the design of TRPC constructs and labelling protocols, and made the labelling reagents (functionalised DogCatchers)
Impact https://www.sciencedirect.com/science/article/pii/S2451945621003159 Multidisciplinary: molecular biology, chemical biology, cellular biochemistry
Start Year 2020
 
Description Astbury Conversation - 6th form demonstration 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We helped organise a practical course on structural biology for 6th form students, to help them understand the public lecture by Nobel Laureate Michael Levitt on structural biology of GPCRs
Year(s) Of Engagement Activity 2018
 
Description Invited lecture at GP2A 2021 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk on TRPC channel structural pharmacology
Year(s) Of Engagement Activity 2021
URL https://gp2a.org/index.php/gp2a-2021-annual-conference/
 
Description Invited lecture to RECI VIII Ion Channel Symposium 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The symposium was attended by >100 participants, mainly from Spain, Portugal, France and Italy
Year(s) Of Engagement Activity 2022
 
Description Invited talk at Imperial COllege London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact A research talk for 80-100 students, postdocs and staff
Year(s) Of Engagement Activity 2022
 
Description Invited talk at University of Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Research seminar for an audience of 60-80 students, postdocs and staff
Year(s) Of Engagement Activity 2022
 
Description Online seminar series of the German Transregional Collaborative Research Centre 152: TRiPs to Homeostasis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk within this seminar series on TRPC channel structural pharmacology
Year(s) Of Engagement Activity 2021
 
Description Plenary lecture at MolTag closing conference, Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited plenary lecture on TRPC channel chemical biology and structural pharmacology to >100 attendees of the closing conference of this Austrian PhD programme
Year(s) Of Engagement Activity 2023
URL https://moltag.univie.ac.at/archive/details/news/the-moltag-closing-conference/
 
Description Press release for paper in Communications Biology 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We wrote a press release based on our recent paper on cryo-EM structure determination of TRPC5 channels. For details, see:
https://medicinehealth.leeds.ac.uk/medicine/news/article/450/understanding-ion-channel-inhibition-to-open-doors-in-drug-discovery

This press release was picked up by at least 10 news outlets including EurekAlert and Phys.org. For details see; https://nature.altmetric.com/details/94794469/news
Year(s) Of Engagement Activity 2020
 
Description Talk at Nottingham Trent University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Invited talk on TRPC channel chemical biology and structural pharmacology to >50 academics and students
Year(s) Of Engagement Activity 2023
 
Description Talk at RSC Chemical Biology meets Drug Discovery 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact >200 participants from a broad international audience, from both academia and industry
Year(s) Of Engagement Activity 2022
 
Description plenary lecture at European Symposium of Bio-organic Chemistry 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary lecture to ca. 50 attendees of the main symposium in Wales. My talk was also one of 4 selected to feature at the pre-symposium in Manchester (for ca. 40 attendees who could not make it to Wales).
Year(s) Of Engagement Activity 2023
URL https://www.esboc.org.uk/?page_id=870