Structure-Guided Studied of GPCRs of RAS

摘要 血管紧张素转换酶1型受体(AT1R)被认为是正常心血管的主要调节因子 生理学。在多种疾病中，由于血管紧张素转换酶Ⅱ诱导的器官损伤，AT1R的慢性刺激引起 细胞生长、黏附、迁移和炎性基因表达异常。AT1R阻滞剂(ARB) 有效控制高血压，但由于未知，它们在预防器官损伤方面的效果差别很大 机制。几个实验室已经做出努力来阐明多效性AT1R的分子基础 信令过程。我们的研究主要集中在结构、构象和药理作用上。 管理AT1R的机制。我们已经通过使用Site-Site来阐明AT1R药理学的机制- 定向突变、细胞信号传递、血管紧张素Ⅱ类似物的设计和转基因小鼠模型。我们是第一个 在AT1R、AT2R和MAS中显示非配体依赖和配体偏向的信号转导。 我们最近已经阐明了第一个与ARB结合的人AT1R的三维结构，这是 开始对这种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的结构确定方法和导致AT2R和MAS的建模方法 这些受体的基于结构的药物发现(SBDD)。AT1R结构揭示了一种 微丝结合基序(FBM)提示AT1R可能通过微丝直接激活细胞黏附信号 A(外研社)。AT1R的分子动力学研究揭示了与功能相互作用的受体中的变构口袋 AT1R的各个方面。AT1R口袋1将自身抗体结合ECL2与正构体配体口袋分开。 Pocket 2与跨膜功能位点不同，它可能与AT1R有关 与其他GPCRs的异二聚化。变构配体可干预黄曲霉毒素引起的病理 针对AT1R的自身抗体和异源二聚体。因此，将基于结构的研究(I)扩展到AT2R和AT2R MAS，(Ii)靶向AT1R-Flna偶联和(Iii)发现变构配体有可能产生新的 以RAAS的GPCRs为目标的工具比目前更有效。我们提出以下三个目标： 目的1.用结晶学方法确定AT2R的配体-受体原子间的接触关系，用分子模拟方法确定MAS的配体-受体原子接触关系。 通过功能测试验证配体-受体接触。我们将阐明AT2R和靶的3D结构 AT2R和MAS中的残基用于结构功能分析，为药物开发提供依据。 目的2.确定AT1R-Flna相互作用调节整合素介导的细胞的机制 粘着/移动信号。我们将通过诱变和结构分析来验证AT1R的FBM 开发一种抑制这种相互作用的药物。我们将研究抑制AT1R-Flna偶联对AT1R的影响 诱导Flna的磷酸化和基于黏附的细胞表型改变。 目的3.发现针对AT1R变构位点的化学类型并表征变构配体的药理作用 和功能。我们将用小分子抑制剂破坏变构和正构之间的偶联。 将评估干扰对细胞和小鼠AT1R信号的影响。 我们将在临床前研究中使用最先进的分子、生物物理、细胞生物学和活体技术。 以促进我们对AT1R生物学中长期悬而未决的问题的理解。我们的发现很容易被翻译成 可用于临床，并可促进新疗法的发展。

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.