Cardiovascular drug target, TRPV2

心血管药物靶点TRPV2

• 批准号: 10420467
• 负责人: THEODORE G WENSEL
• 金额: $ 43.75万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10420467
• 关键词: Agonist; Animal Model; Binding; Biological Assay; Cardiac Myocytes; Cardiovascular Agents; Cardiovascular Physiology; Cardiovascular system; Cations; Cellular Assay; Cellular Membrane; Clinical; Clinical Research; Complement; Cryoelectron Microscopy; Cultured Cells; Defect; Development; Diabetes Mellitus; Disease; Drug Targeting; Dyes; Electrophysiology (science); Engineering; Enzymes; Face; Family; Future; Goals; Ion Channel; Ion Channel Protein; Knock-out; Ligands; Lipids; Malignant Neoplasms; Measures; Mechanical Stress; Mediating; Membrane; Monitor; Muscular Dystrophies; Mutagenesis; Mutation; Pathologic; Pharmaceutical Preparations; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiology; Play; Process; Property; Proteins; Recombinants; Regulation; Reporting; Role; Specificity; Structure; Symptoms; TRPV channel; Testing; Therapeutic; Vesicle; antagonist; base; cardiovascular health; design; experimental study; extracellular; inhibitor; mechanical stimulus; mutant; nanodisk; novel therapeutics; patch clamp; reconstitution; response; voltage

The cation channel TRPV2 is important in development and function of the cardiovascular system. Multiple studies suggest it is gated in response to mechanical stimulus, but the mechanism appears to be indirect and is not yet known. Drugs targeting either activation or inhibition of TRPV2 have been reported to have beneficial effects on pathological cardiovascular symptoms in both animal models and clinical studies, but the available drugs have poor specificity and potency. The goal of this project is to develop a better understanding of the structural and cellular basis of TRPV2 regulation, and to begin the process of developing more specific and efficacious inhibitors and activators. Specific Aim 1 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in a purified and reconstituted state. Phosphoinositide composition will be manipulated and channel activity with and without added agonists and inhibitors, or in response to mechanical stimulus, will be measured using Ca2+-indicator dyes or with patch-clamp electrophysiology. These experiments will test the hypothesis that TRPV2 is a constitutively active channel and subject to negative regulation by phosphatidylinositol (4,5) bisphosphate (PIP2), as well as the counter hypotheses that PIP2 is stimulatory or alters channel selectivity or that effects of phosphoinositides are indirect. They will also test the hypothesis that TRPV2 is directly mechanosensitive. Specific Aim 2 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in the context of cellular membranes. TRPV2 will be expressed in cultured cells and assayed using Ca2+-flux assays and patch clamp experiments. Expression of recombinant enzymes engineered to respond to extracellular ligands, or to membrane voltage, as well as fluorescently tagged phosphoinositide binding domains and inhibitors, will be used to manipulate and monitor phosphoinositides on the cytoplasmic face of the membrane. These experiments will test the hypothesis that TRPV2 is regulated by intracellular phosphoinositides and membrane tension in a cellular context. Specific Aim 3: Determine the structural basis for regulation of TRPV2. The structure of TRPV2 in lipid nanodics will be determined in the presence of phosphoinositides as well as agonists and inhibitors using cryo-electron microscopy. Mutagenesis experiments will target residues proposed to play important roles in regulation by phosphoinositides or other effectors. Mutations will be tested for their effects on function and structure. These experiments will further test the hypothesis that direct interactions mediate the effects of these regulatory molecules and identify the structural basis for those which do so. Accomplishment of these aims will make a major contribution to our understanding of the general principles governing the regulation of ion channels of the TRPV family, clarify the mechanistic basis for the roles of TRPV2 in normal physiology and disease, and help to guide future therapeutic efforts relying on TRPV2 regulation as a target.

阳离子通道 TRPV2 对于心血管系统的发育和功能很重要。多种的 研究表明它是响应机械刺激而门控的，但该机制似乎是间接的并且 目前尚不清楚。据报道，针对 TRPV2 激活或抑制的药物具有有益效果。 在动物模型和临床研究中对病理性心血管症状的影响，但现有的 药物的特异性和效力较差。该项目的目标是更好地了解 TRPV2 调节的结构和细胞基础，并开始开发更具体和更具体的过程 有效的抑制剂和激活剂。具体目标 1 是确定磷酸肌醇和 纯化和重构状态下机械应力对 TRPV2 活性的影响。磷酸肌醇组合物将是 添加或不添加激动剂和抑制剂或响应机械作用的操纵和通道活动 刺激，将使用 Ca2+ 指示剂染料或膜片钳电生理学进行测量。这些 实验将检验 TRPV2 是一个组成型活跃通道并受到负面影响的假设 磷脂酰肌醇 (4,5) 二磷酸 (PIP2) 的调节，以及 PIP2 的反假设 刺激或改变通道选择性或磷酸肌醇的作用是间接的。他们还将测试 TRPV2 直接机械敏感的假设。具体目标 2 是确定以下效果： 磷酸肌醇和机械应力对细胞膜中 TRPV2 活性的影响。 TRPV2 将 在培养细胞中表达并使用 Ca2+-通量测定和膜片钳实验进行测定。表达 重组酶被设计为响应细胞外配体或膜电压，以及 荧光标记的磷酸肌醇结合域和抑制剂，将用于操作和监测 膜细胞质面上的磷酸肌醇。这些实验将检验以下假设： TRPV2 受细胞内磷酸肌醇和细胞膜张力的调节。具体的 目标 3：确定 TRPV2 调控的结构基础。脂质纳米颗粒中 TRPV2 的结构将是 使用冷冻电子在磷酸肌醇以及激动剂和抑制剂存在下测定 显微镜。诱变实验将针对在调节中发挥重要作用的残基 磷酸肌醇或其他效应物。将测试突变对功能和结构的影响。这些 实验将进一步检验直接相互作用介导这些调节作用的假设 分子并确定其结构基础。这些目标的实现将使 对我们理解离子通道调节的一般原则做出了重大贡献 TRPV家族，阐明TRPV2在正常生理和疾病中作用的机制基础，帮助 指导未来以 TRPV2 调节为目标的治疗工作。