Structure/Function Studies on the Mechanisms of Purinergic Receptor Activation and Antagonism

嘌呤受体激活和拮抗机制的结构/功能研究

• 批准号: 10199000
• 负责人: Steven Elias Mansoor
• 金额: $ 24.88万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10199000
• 关键词: Affect; Affinity; Agonist; Area; Binding; Biological Assay; Biological Process; Cardiovascular system; Cations; Coronary Arteriosclerosis; Cryoelectron Microscopy; Cytoplasmic Tail; Data; Development; Disabled Persons; Disease; Disulfides; Drug Design; Electrophysiology (science); Event; Family; Fluorescence; Foundations; G-Protein-Coupled Receptors; Grant; Growth; Health Sciences; Hemorrhage; Hemostatic function; Homo; Human; Hypertension; Immune system; Inflammation; Inflammatory; Institutes; Ion Channel; Ion Channel Gating; Ions; Kinetics; Lateral; Ligand Binding; Ligands; Mediating; Membrane Proteins; Mentorship; Modeling; Molecular; Molecular Conformation; Nervous system structure; Neurons; Oregon; P2X-receptor; Patients; Pharmacology; Phospholipids; Physiology; Platelet Activation; Platelet aggregation; Play; Postdoctoral Fellow; Process; Property; Protein Biochemistry; Publications; Publishing; Purinoceptor; Receptor Activation; Refractory; Research; Research Personnel; Resolution; Rest; Risk; Role; Site; Site-Directed Mutagenesis; Specificity; Structure; Synaptic Transmission; Testing; Tissues; Training; Universities; Work; X-Ray Crystallography; base; cardiovascular risk factor; crosslink; desensitization; design; experimental study; extracellular; in silico; insight; novel; receptor; receptor function; response; small molecule; structural biology; targeted treatment; virtual screening

PROJECT SUMMARY Purinergic (P2X) receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, the molecular mechanisms for P2X receptor antagonism are unclear, especially for non-competitive antagonists where almost nothing is known. The study of P2X receptors has been handicapped by a paucity of small molecules that serve as selective high-affinity agonists and antagonists against the various receptor subtypes. Very recently, the applicant published atomic resolution structures of human P2X3 (hP2X3) in an apo/resting state, an agonist-bound/open-pore state, an agonist- bound/closed-pore/desensitized state and two competitive antagonist-bound states, identifying a novel cytoplasmic domain that he hypothesizes plays a pivotal and unique structural role in receptor activation and desensitization. The aims of this grant are designed to build off that work in order to firmly establish the principles of P2X receptor antagonism, determine the structural role of key cytoplasmic residues involved in P2X receptor activation and desensitization, and identify novel small molecule competitive and non-competitive antagonists directed at homo-trimeric P2X1 and P2X3 receptor subtypes and hetero-trimeric P2X2,3 receptors, utilizing ligand binding assays and electrophysiology to study function and X-ray crystallography and cryo electron microscopy to study structure. This research will be carried out by an applicant with excellent training in membrane protein biochemistry and a strong publication record. The training for this proposal will occur at the Vollum Institute of Oregon Health and Science University under the mentorship of Dr. Eric Gouaux, a world leader in the structural biology of ion channels and transporters who has previously transitioned numerous post-doctoral trainees to independence. To prepare the applicant for a successful transition to independence, Dr. Gouaux will oversee the candidate's structural biology training in cryo electron microscopy and continued growth in X-ray crystallography. In addition to Dr. Gouaux, an institute of established investigators supports the applicant with expertise in membrane protein biochemistry, ligand-receptor interactions/binding assays, and the electrophysiology of ion channels. This training will be essential for the candidate to become an independent investigator focused on studying the structure and function of purinergic receptors in order to develop pharmacological agents for the treatment of cardiovascular conditions such as angina, hypertension, and platelet aggregation.

项目概要 嘌呤能 (P2X) 受体是三聚体、非选择性阳离子通道，由 ATP 激活，发挥着重要作用 在心血管、神经和免疫系统中发挥作用。尽管它们在人类生理学中具有核心功能， 作为治疗药物的潜在靶点，P2X受体拮抗的分子机制尚不清楚， 特别是对于几乎一无所知的非竞争性对手。 P2X受体的研究 由于缺乏作为选择性高亲和力激动剂的小分子而受到限制 针对各种受体亚型的拮抗剂。最近，申请人发表了原子分辨率 人 P2X3 (hP2X3) 在 apo/静息状态、激动剂结合/开孔状态、激动剂- 结合/闭孔/脱敏状态和两种竞争性拮抗剂结合状态，确定了一种新颖的 他假设细胞质结构域在受体激活和 脱敏。这笔赠款的目的是为了巩固这项工作，从而牢固地建立 P2X受体拮抗原理，确定参与的关键细胞质残基的结构作用 P2X受体激活和脱敏，并识别新型小分子竞争性和非竞争性 [0100] 针对同源三聚体P2X1和P2X3受体亚型以及异源三聚体P2X2,3受体的拮抗剂， 利用配体结合测定和电生理学来研究功能以及 X 射线晶体学和冷冻 电子显微镜研究结构。这项研究将由受过良好培训的申请人进行 在膜蛋白生物化学方面拥有丰富的研究成果，并拥有良好的发表记录。该提案的培训将在 俄勒冈健康与科学大学 Vollum 研究所，在 Eric Gouaux 博士的指导下， 离子通道和转运蛋白结构生物学领域的领导者，之前已转变了许多 博士后培养人员独立。为了让申请人为成功过渡到独立做好准备， Gouaux 博士将监督候选人在冷冻电子显微镜方面的结构生物学培训，并继续 X射线晶体学中的生长。除了 Gouaux 博士之外，一个由知名研究人员组成的研究所也支持 申请人具有膜蛋白生物化学、配体-受体相互作用/结合测定方面的专业知识，以及 离子通道的电生理学。该培训对于候选人成为一名 独立研究者专注于研究嘌呤能受体的结构和功能，以便 开发治疗心绞痛、高血压等心血管疾病的药物 和血小板聚集。