Angiotensin (AT) receptor antagonism shows promise as a strategy for therapeutic control of some forms of hypertension. Many of the effective, antihypertensive non-peptide AT1 receptor antagonists developed over the past several years have complex mechanisms of action. A need still exists to understand better the properties of these compounds as a need for more safe and selective drugs may arise in the future. One means of achieving this is to develop a model of AT1 receptor structure. Until unforeseen technological advances are made in purifying and crystallizing integral membrane receptor proteins, models of receptor structure can still be developed by combining site- directed mutagenesis with structure-activity-relationship (SAR) analysis. The fundamental goal of this research is to develop such a model by identifying molecular interactions of currently available AT peptides and non- peptide compounds with the AT receptors. The longer term benefit and ultimate test of this research would be the production of novel drug structures designed rationally from predictions of the spatial arrangement of AT receptor domains. The outcomes may be even more broadly generalized as a test for the utility of structural predictions for drug design and development for this broad class of proteins. Hypotheses are proposed to: 1) test the possibility that residues in extracellular domains of AT receptors are involved in AT peptide interactions, unlike for G-protein coupled receptors for biogenic amines; 2) to identify the receptor binding sites for the non-peptide phenylimidazole antagonist ligands and to understand the forces of interactions between the receptor and these compounds and 3) to determine if insurmountable antagonism of AT receptors by peptide and non-peptide antagonists results from a pharmacologic disequilibrium and not complex allostericism and to understand the structural and molecular basis for insurmountable antagonism. To begin to address these issues, the pharmacological, functional and molecular diversity of AT receptor species isoforms will be exploited to identify domains and specific amino acid residues in the receptors associated with their divergent phenotypes. Mutagenic strategies that include the exchange of divergent amino acid residues among differing AT receptor isoforms, are proposed as a first step in identifying these binding domains. Once sites of ligand contact have been established by this comparative approach, an empirical approach will be employed to refine the model, to reveal other residues that contact ligand which are common to all AT receptor isoforms, and to determine the specificity and forces dictating these receptor-ligand interactions. To achieve this, the effects of mutations at sites neighboring those identified by the comparative approach will be analyzed. The effects of a series of point mutations at any single of these sites will then be analyzed using radioligand binding and functional SAR studies, employing a diverse array of peptide and non- peptide ligand derivatives. Similar approaches will be employed to reveal the mechanisms of insurmountable antagonism of AT receptors, and to establish the molecular determinants that differentiate it from surmountable antagonism.
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