Structure-Guided Studied of GPCRs of RAS

摘要 血管紧张素Ⅱ 1型受体（AT 1 R）是众所周知的正常心血管系统的主要调节因子， physiology.在多种疾病中，AT 1 R的慢性刺激由于血管紧张素II诱导的血管紧张素II受体活化而引起器官损伤。 细胞的异常生长、粘附、迁移和炎性基因表达。AT 1 R阻滞剂（ARB） 有效控制高血压，但由于未知原因，它们在预防器官损伤方面的功效差异很大 机制几个实验室已经努力阐明多效性AT 1 R的分子基础 信号过程。我们的研究主要集中在结构、构象和药理学方面 控制AT 1 R的机制我们已经阐明了AT 1 R药理学的机制，通过使用网站- 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 以显示AT 1 R、AT 2 R和MAS中的配体非依赖性和配体偏向性信号传导。 我们最近阐明了ARB结合的人AT 1 R的第一个3D结构，这是研究ARB结合的重要一步。 开始对这种降压药物靶点进行基于结构的研究。这些知识是延伸到 AT 2 R的结构确定方法以及AT 2 R和MAS的建模方法， 基于结构的药物发现（SBDD）。AT 1 R结构揭示了一种 细丝蛋白结合基序（FBM）表明AT 1 R可能通过细丝蛋白直接激活细胞粘附信号传导 A（FLNa）。AT 1 R的分子动力学研究揭示了受体中的变构口袋， 关于AT 1 R AT 1 R口袋1将自身抗体结合ECL 2与正构配体口袋分开。 口袋2不同于跨膜功能位点，它可能负责AT 1 R 与其他GPCR异二聚化。变构配体可以干预由 靶向AT 1 R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AT 2 R， MAS，（ii）靶向AT 1 R-FLNa偶联和（iii）发现变构配体具有产生新的 比目前更有效地针对RAAS的GPCR工具。我们提出以下三个目标： 目标1。通过晶体学定义AT 2 R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。 通过功能测试确定配体-受体接触。我们将阐明AT 2 R和靶点的三维结构， AT 2 R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AT 1 R-FLNa相互作用调节整合素介导的细胞凋亡的机制 粘附/运动信号传导。我们将通过诱变和结构分析验证AT 1 R的FBM， 开发这种相互作用的抑制剂。我们将研究抑制AT 1 R-FLNa偶联对AT 1 R的影响 诱导FLNa磷酸化和基于粘附的细胞表型调节。 目标3。发现靶向AT 1 R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偶联。 将评价破坏对细胞和小鼠中AT 1 R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 以促进我们对AT 1 R生物学中长期未解决问题的理解。我们的研究结果很容易转化为 临床，并可能促进新疗法的发展。

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.