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

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

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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 Angii 1型受体（AT1R）广为人知是正常心血管的主调节剂生理。在多种疾病中，慢性刺激AT1R会导致Angii引起的器官损害细胞中异常生长，粘附，迁移和炎症基因表达。 AT1R阻滞剂（ARB）有效控制高血压，但由于未知而导致器官损伤的功效差异很大机制。已经在几个实验室中努力阐明了多骨AT1R的分子基础信号过程。我们将研究重点放在结构，构象和药理上管理AT1R的机制。我们采用了站点 - 定向诱变，细胞信号，Angii Analogs的设计和转基因小鼠模型。我们是第一个在AT1R，AT2R和MAS中显示与配体无关和配体偏置信号传导。我们最近阐明了ARB结合的人AT1R的第一个3D结构，作为重要步骤对这种降压药目标的初始结构研究。这些知识是扩展的入门 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结构揭示了存在丝蛋白结合基序（FBM）表明AT1R可以直接激活细胞粘附信号传导 a（flna）。 AT1R的分子动力学研究揭示了受体中的变构袋，即界面功能 AT1R的各个方面。 AT1R口袋1将自动抗体结合的ECL2与正常配体袋分开。口袋2与跨膜功能部位不同，可能是AT1R的原因与其他GPCR的异二聚化。变构配体可以干预由靶向AT1R的自身抗体和异二聚体。因此，将基于结构的研究扩展到AT2R和 MAS，（ii）靶向AT1R-FLNA耦合和（iii）发现变构配体有可能生成新的针对RAA的GPCR的工具比目前更有效。我们提出以下三个目标： AIM1。通过晶体学定义AT2R的配体原子接触，通过分子建模定义MAS。通过功能测试验证配体受体接触。我们将阐明AT2R和目标的3D结构 AT2R和MAS中的残留物进行结构功能分析，以提供药物开发的基础。 AIM 2。确定AT1R-FLNA相互作用调节整联蛋白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耦合对AT1R的影响诱导细胞的FLNA磷酸化和基于粘附的表型调节。 AIM 3。发现针对AT1R的变构位点的化学型并表征了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直角位点的耦合。将评估破坏对细胞和小鼠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促进我们对AT1R生物学长期未解决的问题的理解。我们的发现很容易翻译成该诊所并可能促进新型治疗剂的发展。