Structural insights into small molecule activation of TRPC4/5 channels

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.

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.

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.