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.
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.
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.
Organisations
- University of Leeds (Lead Research Organisation)
- Academy of Sciences of the Czech Republic (Collaboration)
- UNIVERSITY OF OXFORD (Collaboration)
- Taras Shevchenko National University of Kyiv (Collaboration)
- Ludwig Maximilian University of Munich (LMU Munich) (Collaboration)
- UNIVERSITY OF LEEDS (Collaboration)
- Freie Universität Berlin (Project Partner)
Publications
![publication icon](/resources/img/placeholder-60x60.png)
Baradaran-Heravi A
(2022)
Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903.
in The Journal of biological chemistry
![publication icon](/resources/img/placeholder-60x60.png)
Bauer C
(2020)
Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels
in RSC Chemical Biology
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
Klebl DP
(2020)
Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.
in Structure (London, England : 1993)
![publication icon](/resources/img/placeholder-60x60.png)
Lepannetier S
(2018)
Activation of TRPC1 Channel by Metabotropic Glutamate Receptor mGluR5 Modulates Synaptic Plasticity and Spatial Working Memory.
in Frontiers in cellular neuroscience
![publication icon](/resources/img/placeholder-60x60.png)
Minard A
(2019)
Potent, selective, and subunit-dependent activation of TRPC5 channels by a xanthine derivative.
in British journal of pharmacology
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
Wright DJ
(2020)
Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.
in Communications biology
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 |