Understanding human TRPV1 polymodal activation

项目摘要 拟议研究的目标是了解TRPV1的多模式激活，特别是它的激活 通过热、质子和化学配体。了解TRPV1功能的分子机制 对人类健康有重大影响。TRPV1是一种多模调节的离子通道，由 许多不同的刺激，包括热、质子(低pH值)和化学配体，如辣椒素，辛辣的香草素 从辣椒中提取的。在过去的十年中，人们对开发TRPV1拮抗剂产生了极大的兴趣 对抗多种类型的疼痛和其他相关指征。TRPV1治疗的主要并发症之一 干预措施是，拮抗者通常会扰乱体温。最近的计算建模和 人体临床试验的荟萃分析建议TRPV1的哪些模式应该作为开发的目标 减轻非靶点效应的止痛拮抗剂。这项建议旨在剖析独立， 规范的TRPV1激活模式之间的相互依赖和串扰，并解密各自的 机械装置。将使用核磁共振波谱和电生理技术 来实现这些目标。这些数据将用于了解哪些TRPV1区域是特定功能的基础 并阐明了活化模式之间的变构、协作性和串扰。为了实现这些目标，有两个目标 具体目标将付诸实施。目标1将集中于最小TRPV1结构的特征 来自天然的TRPV1-异构体，它概括了全长通道的电生理和 核磁共振研究。此外，这一目标将提供人类TRPV1结构域的第一个结构。一种膜 负责配体结合并参与热敏的结构域。结构研究将进入 非低温温度引起了关于热敏机制的信息。这些 机械和结构研究将得到验证，并与细胞研究联系起来。《目标2》将专注于 剖析了TRPV1的热、质子和配体激活模式之间的变构和串扰。一个系列 的实验将依赖于验证人类TRPV1变构网络的计算预测 细胞膜片钳测量。另一组实验将利用临床前的化学配体 将用于突变的实验和临床试验，以确定配体结合部位以及TRPV1如何 实现了模式选择性。最后一个子目标将使用核磁共振检测的TRPV1激动剂的配体筛选 以及将受到新兴统计分析和学习技术影响的拮抗剂 能够预测TRPV1调节器将激活的活动模式的方法。意义重大 初步的电生理和核磁共振数据与计算分析相结合，表明了这些方法的可行性 在这项提案的时间框架内实现目标。拟议的生物物理和功能TRPV1研究旨在更好地 了解支配功能和复杂的可药性的分子机制，并预计 以指导下一代TRPV1拮抗剂的开发。

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.