TETRAZOLE AND CARBOXYLATE GROUPS OF ANGIOTENSIN RECEPTOR ANTAGONISTS BIND TO THE SAME SUBSITE BY DIFFERENT MECHANISMS

To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT(1) receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys(102) and Arg(167) mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar(1),Ile(8)]angiotensin II, and several carboxylate analogues of losartan. An Ala(199) substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan, Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys(199) is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT(1) receptor involves multiple contacts with residues such as Lys(199) and His(256) that constitute the same subsite of the ligand binding pocket, However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.