TY - JOUR T1 - Mapping the Binding Site of Six Nonpeptide Antagonists to the Human V<sub>2</sub>-Renal Vasopressin Receptor JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 564 LP - 571 DO - 10.1124/jpet.105.095554 VL - 316 IS - 2 AU - Rosemarie Macion-Dazard AU - Nicholas Callahan AU - Zhen Xu AU - Nan Wu AU - Marc Thibonnier AU - Menachem Shoham Y1 - 2006/02/01 UR - http://jpet.aspetjournals.org/content/316/2/564.abstract N2 - Whereas arginine vasopressin binds to its receptor subtypes V1R and V2R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V2R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V2 R and V1R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(±)-4′-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(±)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a Ki of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V2 R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V1R and V2R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor. The American Society for Pharmacology and Experimental Therapeutics ER -