Understanding human TRPV1 polymodal activation

项目摘要 这项研究的目的是了解TRPV 1的多模式激活，特别是它的激活 通过热量质子和化学配体了解TRPV 1功能的分子机制 对人类健康有重大影响。TRPV 1是一种多模态调节的离子通道，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 从红辣椒。在过去的十年中，人们对开发TRPV 1拮抗剂产生了极大的兴趣 以对抗多种类型的疼痛和其他相关适应症。TRPV 1治疗的主要并发症之一是 干预是拮抗剂通常失调体温。最近的计算模型和 人体临床试验的荟萃分析表明，TRPV 1的哪些模式应作为开发的目标 减轻脱靶效应的镇痛拮抗剂。该提案旨在剖析独立性， 相互依赖性和典型TRPV 1激活模式之间的串扰，并破译各自的 机制等将使用核磁共振光谱（NMR）和电生理学技术 来实现这些目标。这些数据将用于了解TRPV 1区域的特定功能 并阐明激活模式之间的变构性、协同性和串扰。为了实现这些目标，两个 将实现具体目标。目标1将集中在一个最小的TRPV 1结构的表征启发 来自天然TRPV 1-同种型，其用电生理学再现全长通道的特征， NMR研究。此外，这一目标将提供人类TRPV 1结构域的第一个结构。的膜 负责配体结合并参与热敏的结构域。结构研究将访问 非低温温度引起关于热敏机理的信息。这些 机制和结构研究将得到验证，并与细胞研究相结合。目标2将侧重于 剖析TRPV 1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TRPV 1变构网络的计算预测， 细胞膜片钳测量。另一组实验将利用来自临床前的化学配体， 实验和临床试验，将用于诱变，以确定配体结合位点和如何TRPV 1 实现了模式选择性。最后一个子目标将采用NMR检测的TRPV 1激动剂配体筛选 和拮抗剂，将受到新兴的统计分析和学习技术，以产生 能够预测TRPV 1调节剂将激活哪种活性模式的方法。显著 初步的电生理学和核磁共振数据加上计算分析表明，这些可行性， 在这个时间段内，我们的目标是。拟议的生物物理和功能TRPV 1研究旨在更好地 了解控制功能和复杂化可药用性的分子机制， 指导下一代TRPV 1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.