Understanding human TRPV1 polymodal activation

了解人类 TRPV1 多模式激活

• 批准号: 10521997
• 负责人: Wade D. Van Horn
• 金额: $ 38.31万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10521997
• 关键词: Adverse event; Agonist; Analgesics; Binding; Binding Sites; Biometry; Biophysics; Body Temperature; Capsaicin; Cells; Chemicals; Chili Pepper; Clinical Trials; Computer Analysis; Computer Models; Coupled; Coupling; Data; Dependence; Development; Disease; Drug Targeting; Electrophysiology (science); Future; Generations; Goals; Health; Human; Hyperthermia; Individual; Ion Channel; Ion Channel Protein; Length; Ligand Binding; Ligands; Measurement; Membrane; Membrane Proteins; Meta-Analysis; Methodology; Methods; Modeling; Molecular; Molecular Probes; Monitor; Mutagenesis; NMR Spectroscopy; Nature; Obesity; Orthologous Gene; Outcome; Pain; Pharmacology; Physiological; Physiology; Process; Protein Isoforms; Protons; Regulation; Research; Rodent; Role; Series; Spectrum Analysis; Statistical Data Interpretation; Stimulus; Structural Models; Structure; Study models; TRPV1 gene; Techniques; Technology; Temperature; Testing; Therapeutic; Therapeutic Intervention; Thermogenesis; Time; Transmembrane Domain; Vanilloid; antagonist; base; biophysical analysis; cancer type; combat; drug discovery; experimental study; human data; human model; interest; mathematical model; new technology; next generation; pain sensation; patch clamp; pre-clinical; screening; sensor; single molecule; statistical learning; success; targeted treatment; tool; tumor progression; voltage

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.

项目摘要 拟议的研究的目的是了解TRPV1的多峰激活，特别是其激活 通过热，质子和化学配体。了解TRPV1功能的分子机制 对人类健康具有重要意义。 TRPV1是一种由多发性调节的离子通道，被激活 许多多样的刺激，包括热，质子（低pH）和化学配体，例如辣椒素 来自辣椒。在过去的十年中，人们对开发TRPV1拮抗剂的兴趣很大 打击许多类型的疼痛和其他相关的适应症。 TRPV1治疗的主要并发症之一 干预是拮抗剂通常会失调体温。最近的计算建模和 人类临床试验的荟萃分析表明，哪种TRPV1的模式应针对发展 减轻靶向效果的镇痛拮抗剂。该提议旨在剖析独立性， 相互依存关系和规范TRPV1激活模式之间的串扰并破译相应的 机制。将使用核磁共振光谱（NMR）和电生理技术 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 并在激活模式之间阐明了变构，合作和串扰。为了实现这些目标，两个 将执行具体目标。 AIM 1将重点放在最小TRPV1构造的表征上 从自然trpv1-异型类型中，用电生理学和 NMR研究。此外，此目标将提供人类TRPV1域的第一个结构。膜 负责配体结合并参与热效的域。结构研究将访问 非晶体温度引起了有关热能机理的信息。这些 机械和结构研究将通过细胞研究验证和背景。 AIM 2将重点关注 在TRPV1热，质子和配体激活模式之间剖析变构和串扰。一个系列 实验将依赖于验证人类TRPV1变构网络的计算预测 细胞斑块钳测量。另一组实验将利用临床前的化学配体 实验和临床试验将与诱变一起用于鉴定配体结合位点以及如何TRPV1 实现模式选择性。最后一个子-IAM将采用TRPV1激动剂的NMR检测的配体屏幕 以及将受到新兴统计分析和学习技术的对抗者 能够预测哪种活性TRPV1调节器将激活的方法。重要的 初步电生理学和NMR数据与计算分析相结合表明这些可行性 目的是在此提案的时间范围内。拟议的生物物理和功能性TRPV1研究旨在更好 了解控制该功能并使吸毒性复杂的分子机制，并被预期 指导下一代TRPV1拮抗剂的发展。