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: 10438783
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10438783
• 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; antagonist; base; cardiovascular risk factor; crosslink; desensitization; design; experimental study; extracellular; in silico; insight; novel; rational design; 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.

项目总结 嘌呤能(P2X)受体是三聚体的非选择性阳离子通道，由ATP激活，发挥重要作用 在心血管、神经和免疫系统中的作用。尽管它们在人类生理学和 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。 尤其是对于几乎一无所知的非竞争性对手。对P2X受体的研究已经 由于缺乏作为选择性高亲和力激动剂的小分子而受到阻碍 针对各种受体亚型的拮抗剂。最近，申请者发表了原子决议 人P2X3(HP2X3)在脱脂/静息状态、激动剂结合/开孔状态、激动剂- 结合/闭孔/钝化态和两个竞争性拮抗剂结合态，鉴定了一种新的 他假设的细胞质结构域在受体激活和释放中起着关键和独特的结构作用 脱敏。这笔赠款的目的是为了在这项工作的基础上再接再厉，以便牢固地确立 P2X受体拮抗原理，确定关键胞质残基参与的结构作用 P2X受体的激活和脱敏，并鉴定新的竞争性和非竞争性小分子 针对同三聚体的P2X1和P2X3受体亚型以及异三聚体的P2X2，3受体的拮抗剂， 利用配基结合试验和电生理学研究功能、X射线结晶学和低温 用电子显微镜研究结构。这项研究将由一位训练有素的申请者进行 在膜蛋白生物化学方面有很强的发表记录。针对该提案的培训将在 在Eric Gouaux博士的指导下，俄勒冈健康与科学大学Vollum研究所，一个世界 离子通道和转运蛋白结构生物学领域的领军人物 博士后实习生走向独立。为使申请者为成功过渡到独立做好准备， Gouaux博士将监督候选人在低温电子显微镜方面的结构生物学培训，并继续 X射线结晶学中的生长。除了Gouaux博士，一个由老牌调查人员组成的研究所还支持 具有膜蛋白生物化学、配体-受体相互作用/结合分析专业知识的应聘者，以及 离子通道的电生理。这一培训对于应聘者成为 独立研究人员专注于研究嘌呤能受体的结构和功能，以便 开发治疗心血管疾病的药物，如心绞痛、高血压、 和血小板聚集。