Structure-Guided Studied of GPCRs of RAS

RAS GPCR 的结构引导研究

基本信息

批准号：
9246190
负责人：
Sadashiva S Karnik
金额：
$ 55.87万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9246190
关键词：
Address Adhesions Affect Agonist Allosteric Site Angiotensin II Angiotensin Receptor Antihypertensive Agents Atherosclerosis Autoantibodies Binding Biology Biophysics Blood Vessels Cardiac Cardiovascular Diseases Cardiovascular Physiology Cell Adhesion Cell physiology Cells Cellular biology Chemosensitization Chronic Clinic Clinical Data Coupling Crystallography Cyclic GMP Cytoskeleton Development Dimensions Disease Drug Targeting Endothelial Cells Essential Hypertension Exhibits Functional disorder G-Protein-Coupled Receptors Gene Expression Generations Goals Growth Heart Heart failure Heterodimerization Hormones Human Inflammatory Integrin-mediated Cell Adhesion Pathway Kidney Kidney Diseases Kidney Failure Knowledge Laboratories Lead Ligands Mediating Methodology Modeling Molecular Molecular Conformation Molecular Models Movement Mus Muscle Cells Muscle Contraction Mutagenesis Organ Pathogenicity Pathology Pharmacology Phenotype Phosphorylation Physiological Population Process PubMed Receptor Signaling Receptor, Angiotensin, Type 1 Regulation Renin-Angiotensin-Aldosterone System Research Rodent Role Signal Transduction Site Site-Directed Mutagenesis Structure Study models Technical Expertise Techniques Testing Transgenic Mice Vascular Smooth Muscle analog base cardiovascular disorder risk cell growth regulation clinical practice combat desensitization design drug development drug discovery filamin hypertension control hypertension treatment in vivo inhibitor/antagonist migration molecular modeling mouse model novel novel therapeutics preclinical study prevent public health relevance receptor receptor binding small molecule inhibitor three dimensional structure three-dimensional modeling tool trafficking

项目摘要

Abstract The AngII type 1 receptor (AT1R) is widely known to be the master regulator of normal cardiovascular physiology. In a variety of diseases chronic stimulation of AT1R causes organ damage due to AngII-induced abnormal growth, adhesion, migration and inflammatory gene expression in cells. AT1R blockers (ARBs) effectively control hypertension but their efficacy in preventing organ damage varies widely due to unknown mechanism. Efforts have been made in several laboratories to elucidate the molecular basis of pleotropic AT1R signaling process. We have focused our research on structure, conformation and pharmacological mechanisms governing AT1R. We have elucidated mechanisms governing AT1R pharmacology by using site- directed mutagenesis, cell signaling, design of AngII-analogs, and transgenic mouse models. We were the first to show ligand-independent and ligand-biased signaling in AT1R, AT2R and MAS. We have recently elucidated the first 3D-structure of ARB-bound human AT1R, as an important step for beginning structure-based studies of this antihypertensive drug-target. This knowledge is the primer to extend the structure determination approach to AT2R and the modeling approach to AT2R and MAS leading to structure based drug discovery (SBDD) for these receptors. AT1R structure has revealed the presence of a filamin binding motif (FBM) suggesting that AT1R may directly activate cell adhesion signaling through Filamin A (FLNa). Molecular dynamic studies of AT1R reveal allosteric pockets in the receptor that interface functional aspects of AT1R. The AT1R pocket 1 separates the auto-antibody binding ECL2 from orthosteric ligand pocket. Pocket 2 is distinct from trans-membrane functional sites and it may be responsible for AT1R heterodimerization with other GPCRs. Allosteric ligands could intervene with pathologies caused by autoantibodies and heterodimers targeting AT1R. Hence, extending structure-based studies (i) to AT2R and MAS, (ii) to target AT1R-FLNa coupling and (iii) to discover allosteric ligands has the potential to generate new tools targeting GPCRs of RAAS more effectively than at present. We propose following three aims: Aim 1. Define ligand-receptor atomic contacts for AT2R by crystallography and for MAS by molecular modeling. Validate ligand-receptor contacts by functional tests. We will elucidate 3D-structure of AT2R and target residues in AT2R and MAS for structure-function analysis to provide basis for drug development. Aim 2. Determine the mechanism by which AT1R-FLNa interaction regulates integrin-mediated cell adhesion/movement signaling. We will validate FBM of AT1R by mutagenesis and structural analysis to develop an inhibitor of this interaction. We will study the effects of inhibiting AT1R-FLNa coupling on AT1R induced FLNa phosphorylation and adhesion-based phenotypic modulation of cells. Aim 3. Discover chemotypes targeting allosteric sites of AT1R and characterize allosteric ligand pharmacology and functions. We will disrupt coupling between allosteric and orthosteric sites by small molecule inhibitors. Effect of disruption on AT1R signaling in cells and mice will be evaluated. We will use state-of-the-art molecular, biophysical, cell biology and in vivo techniques in our preclinical studies to advance our understanding of long unresolved issues in AT1R biology. Our findings are easily translatable to the clinic and may facilitate the development of novel therapeutics.

抽象的众所周知，AngII 1 型受体 (AT1R) 是正常心血管的主要调节因子生理。在多种疾病中，AT1R 的慢性刺激会导致 AngII 诱导的器官损伤细胞的异常生长、粘附、迁移和炎症基因表达。 AT1R 阻滞剂 (ARB) 有效控制高血压，但由于未知因素，其预防器官损伤的功效差异很大机制。多个实验室已做出努力来阐明多效性 AT1R 的分子基础信令过程。我们的研究重点是结构、构象和药理学 AT1R 的调控机制。我们通过使用位点阐明了 AT1R 药理学的控制机制定向诱变、细胞信号传导、AngII 类似物的设计和转基因小鼠模型。我们是第一个显示 AT1R、AT2R 和 MAS 中配体独立和配体偏向的信号传导。我们最近阐明了第一个结合 ARB 的人类 AT1R 3D 结构，这是开始对该抗高血压药物靶点进行基于结构的研究。这些知识是扩展的基础 AT2R 的结构确定方法以及 AT2R 和 MAS 的建模方法导致这些受体的基于结构的药物发现（SBDD）。 AT1R结构揭示了一个细丝蛋白结合基序 (FBM) 表明 AT1R 可能通过细丝蛋白直接激活细胞粘附信号 A (FLNa)。 AT1R 的分子动力学研究揭示了受体中与功能接口连接的变构袋 AT1R 的各个方面。 AT1R 口袋 1 将结合 ECL2 的自身抗体与正位配体口袋分开。 Pocket 2 与跨膜功能位点不同，它可能负责 AT1R 与其他 GPCR 异二聚化。变构配体可以干预由以下原因引起的病理：针对 AT1R 的自身抗体和异二聚体。因此，将基于结构的研究 (i) 扩展到 AT2R 和 MAS，(ii) 靶向 AT1R-FLNa 偶联，以及 (iii) 发现变构配体有可能产生新的针对 RAAS GPCR 的工具比目前更有效。我们提出以下三个目标：目标 1. 通过晶体学定义 AT2R 的配体-受体原子接触，通过分子建模定义 MAS 的配体-受体原子接触。通过功能测试验证配体-受体接触。我们将阐明 AT2R 的 3D 结构和目标 AT2R和MAS中的残基进行结构功能分析，为药物开发提供依据。目标 2. 确定 AT1R-FLNa 相互作用调节整合素介导的细胞的机制粘附/运动信号。我们将通过诱变和结构分析来验证 AT1R 的 FBM 开发这种相互作用的抑制剂。我们将研究抑制AT1R-FLNa偶联对AT1R的影响诱导 FLNa 磷酸化和基于粘附的细胞表型调节。目标 3. 发现针对 AT1R 变构位点的化学型并表征变构配体药理学和功能。我们将通过小分子抑制剂破坏变构和正构位点之间的耦合。将评估破坏对细胞和小鼠中 AT1R 信号传导的影响。我们将在临床前研究中使用最先进的分子、生物物理、细胞生物学和体内技术增进我们对 AT1R 生物学中长期未解决问题的理解。我们的发现很容易转化为临床并可能促进新疗法的开发。