Mechanistic studies of the menthol receptor TRPM8: a novel target for analgesic drugs

薄荷醇受体TRPM8的机制研究：镇痛药物的新靶点

基本信息

批准号：
10679072
负责人：
Melinda Diver
金额：
$ 24.32万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10679072
关键词：
Address Affect Agonist American Analgesics Animals Award Behavioral Binding Binding Sites Biochemical Biological Models Biophysics Calcium Cells Cellular Membrane Chemicals Complex Cryoelectron Microscopy Development Electrodes Electrophysiology (science)Environment Esthesia Eucalyptol Family Family member Functional disorder Goals Human Hypersensitivity Ion Channel Ions Irritants Ligand Binding Lipid Binding Lipids Liposomes Mass Spectrum Analysis Membrane Menthol Mentors Minor Modality Molecular Molecular Conformation Muscle Cramp Natural Compound Natural Products Neurons Neuropathy Nociceptors Pain Pain Disorder Pathway interactions Peptides Persistent pain Pharmacotherapy Phase Phosphatidylinositols Physiological Phytochemical Plants Population Post-Translational Protein Processing Preparation Property Proteins Regulation Research Role Scientist Sensory Signal Pathway Signal Transduction Somatosensory Receptor Sprain Stimulus Structure Syndrome TRP channel Therapeutic Toxin Training Venoms Work allodynia antagonist biophysical analysis biophysical properties biophysical techniques career career development chemotherapy chronic pain chronic painful condition cold temperature improved in vivo insight neurotransmission novel pain chronification pain receptor pain reduction pain sensation pain signal painful neuropathy particle pharmacologic programs protein reconstitution proteoliposomes rational design receptor receptor function reconstitution response sensor structural biology tool trait treatment strategy voltage voltage clamp

项目摘要

Project Summary Chronic pain sufferers, an estimated one third of the American population, struggle to identify ways to reduce pain and improve their daily lives. Transient receptor potential (TRP) channels detect a wide range of physical and chemical stimuli, and through their integration of and response to these stimuli have an essential role in the pathophysiology of many chronic pain disorders. Notably, many natural products from plants and venomous animals target TRP channels, and can therefore be used to identify and characterize these important contributors to pain sensation. For instance, TRP melastatin 8 (TRPM8), the somatosensory receptor gated by cold temperatures, is also activated by natural cooling agents, such as menthol and eucalyptol, commonly used topical analgesic agents. Furthermore, TRPM8 is essential for the development of cold allodynia, a debilitating hypersensitivity to cold resulting from chemotherapy or other neuropathic insults. Despite the importance of TRPM8 and other TRP channels to the transition from acute to chronic pain states, the molecular basis for ligand binding, channel gating, and ion permeation remain incompletely understood. The objective of this proposal is to determine the mechanisms whereby TRPM8 conducts ions across cellular membranes in response to diverse signals, including those from plant-derived compounds that produce a cold sensation. The specific aims are to: 1) determine atomic structures of TRPM8 in different conformational states (K99 phase), 2) study the electrophysiological properties of TRPM8 (K99 phase), and 3) probe mechanisms of TRPM8 modulation by phosphatidylinositol lipids (R00 phase). Single-particle electron cryo-microscopy (cryo- EM) structures will be determined of TRPM8 alone and in complex with agonists, antagonists, or natural toxins. In particular, toxins from animal venoms are powerful tools for elucidating the structural mechanisms underlying channel gating and modulation. Structure-function analyses aimed at determining gating mechanisms and validating ligand binding sites will be conducted, as will biophysical studies of purified, reconstituted protein to functionally characterize intrinsic gating of TRPM8. Modulation by bioactive lipids is a unifying functional trait of TRP channels, including TRPM8, and thus effects of phosphatidylinositol lipids on TRPM8 will be explored and non-covalently bound lipids will be identified using native mass spectrometry. These goals are significant because they will enhance the biophysical and molecular understanding of pain sensation and, specifically, how TRPM8 modulation contributes to chronic pain. Ultimately, the aim is to assist in the rational design of novel TRPM8-based analgesic drugs. My mentor, Dr. Julius, as well as my expert advisors in cryo-EM (Dr. Cheng), electrophysiology (Dr. Kirichok), protein-lipid interactions (Dr. Marty), and pain signaling (Dr. von Zastrow), will provide training, in preparation for my career as an independent scientist.

项目概要慢性疼痛患者（估计占美国人口的三分之一）正在努力寻找减轻疼痛的方法疼痛并改善他们的日常生活。瞬时受体电位 (TRP) 通道可检测多种物理量和化学刺激，并通过它们的整合和对这些刺激的反应在许多慢性疼痛疾病的病理生理学。值得注意的是，许多来自植物和有毒动物以 TRP 通道为目标，因此可用于识别和表征这些通道痛觉的重要贡献者。例如，TRP melastatin 8 (TRPM8)，体感受体受低温控制，也被天然清凉剂激活，例如薄荷醇和桉油精，常用的外用镇痛药。此外，TRPM8对于感冒的发生至关重要。异常性疼痛，化疗或其他神经性损伤引起的对寒冷的过敏。尽管 TRPM8 和其他 TRP 通道对于急性疼痛状态向慢性疼痛状态的转变非常重要，配体结合、通道门控和离子渗透的分子基础仍未完全了解。该提案的目的是确定 TRPM8 在细胞间传导离子的机制膜响应不同的信号，包括来自植物源性化合物的信号，这些化合物会产生寒冷感觉。具体目标是：1）确定不同构象状态下TRPM8的原子结构（K99相），2）研究TRPM8（K99相）的电生理特性，3）探针机制 TRPM8 通过磷脂酰肌醇脂质调节（R00 相）。单粒子电子冷冻显微镜（cryo- 单独的TRPM8以及与激动剂、拮抗剂或天然毒素的复合物将确定TRPM8的结构。特别是，来自动物毒液的毒素是阐明结构机制的有力工具底层通道选通和调制。旨在确定门控的结构功能分析将进行机制和验证配体结合位点，以及纯化的生物物理学研究，重组蛋白以功能表征 TRPM8 的内在门控。生物活性脂质的调节是统一 TRP 通道（包括 TRPM8）的功能特征，以及磷脂酰肌醇脂质对将探索 TRPM8，并使用天然质谱法鉴定非共价结合的脂质。这些目标意义重大，因为它们将增强对疼痛的生物物理和分子理解感觉，特别是 TRPM8 调节如何导致慢性疼痛。最终的目的是帮助合理设计基于 TRPM8 的新型镇痛药物。我的导师朱利叶斯博士，也是我的专家冷冻电镜（Cheng 博士）、电生理学（Kirichok 博士）、蛋白质-脂质相互作用（Marty 博士）等领域的顾问疼痛信号传导（冯·扎斯特罗博士）将提供培训，为我作为独立科学家的职业生涯做好准备。