Project Summary The goal of the proposed research is to understand the polymodal activation of TRPV1, specifically its activation by heat, protons, and chemical ligands. Understanding the molecular mechanisms that underlie TRPV1 function has significant implications in human health. TRPV1 is a polymodally regulated ion channel that is activated by many diverse stimuli, including heat, protons (low pH), and chemical ligands, like capsaicin, the pungent vanilloid from chili peppers. Over the past decade, there has been significant interest in developing TRPV1 antagonists to combat many types of pain and other relevant indications. One of the main complications in TRPV1 therapeutic intervention is that antagonists commonly dysregulate body temperature. Recent computational modeling and meta-analysis of human clinical trials suggest which modes of TRPV1 should be targeted for the development of analgesic antagonists that mitigate off-target effects. This proposal aims to dissect the independence, interdependence, and crosstalk between canonical TRPV1 activation modes and decipher the respective mechanisms. Nuclear magnetic resonance spectroscopy (NMR) and electrophysiology techniques will be used to achieve these goals. These data will be used to understand which TRPV1 regions underlie particular functions and illuminate allostery, cooperativity, and crosstalk between activation modes. To achieve these goals, two specific aims will be carried out. Aim 1 will focus on the characterization of a minimal TRPV1 construct inspired from natural TRPV1-isoforms that recapitulates the features of the full-length channel with electrophysiology and NMR studies. Additionally, this aim will provide the first structures of a human TRPV1 domain. A membrane domain that is responsible for ligand binding and involved in thermosensing. The structural studies will access non-cryogenic temperatures giving rise to information about the mechanism of thermosensing. These mechanistic and structural studies will be validated and contextualized with cellular studies. Aim 2 will focus on dissecting the allostery and crosstalk between TRPV1 heat, proton, and ligand modes of activation. One series of experiments will rely on validating computational predictions of human TRPV1 allosteric networks with whole- cell patch-clamp measurements. Another set of experiments will leverage chemical ligands from preclinical experiments and clinical trials that will be used with mutagenesis to identify ligand binding sites and how TRPV1 mode selectivity is achieved. The last sub-aim will employ an NMR-detected ligand screen of TRPV1 agonists and antagonists which will be subjected to emerging statistical analysis and learning techniques to generate methodologies capable of predicting which modes of activity TRPV1 modulators will activate. Significant preliminary electrophysiology and NMR data coupled with computational analysis indicate the feasibility of these aims during the timeframe of this proposal. The proposed biophysical and functional TRPV1 studies aim to better understand the molecular mechanisms that govern the function and complicate druggability and are anticipated to guide the development of the next generation of TRPV1 antagonists.

Project Narrative The TRPV1 ion channel protein is important in human physiology and has emerged as a promising drug target for diverse diseases, including many types of pain. This proposed research sets out to better understand the reasons that past TRPV1 drug discovery efforts have been generally unsuccessful and develop tools that will help guide future drug discovery efforts.