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.
Complications from essential hypertension include dysfunction affecting heart, kidney, and vasculature as well as the pathology of heart failure, renal failure and atherosclerosis. We have recently solved the three- dimensional structure of AT1R bound to two different antihypertensive drugs. With this knowledge and novel technical expertise, we propose to elucidate the structural basis for: (i) molecular pharmacology of the other two angiotensin receptors, AT2R and MAS; (ii) a novel mode of AT1R signaling to cytoskeleton through filamin; (iii) novel allosteric modulators of AT1R, AT2R and MAS. These studies are pertinent for understanding normal and pathogenic functions of three GPCRs for RAS hormones in vivo, and may lead to new generation of drugs that target allosteric sites on these receptors which influence a broad spectrum of cardiovascular diseases.
