Small-molecule vasopressin-2 receptor antagonist identified by a G-protein coupled receptor "pathway" screen

Abstract

G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V2R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V2R. Increased cell Cl- conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I- addition. The Z′ factor for the assay was ∼0.7 with the V2R agonist [deamino-Cys1, Val4, D-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V2R antagonists that are unrelated structurally to known V 2R antagonists. The most potent compound, V2R inh-02, which was identified by screening 35 structural analogs, competitively inhibited V2R-induced cAMP elevation with Ki value of ∼70 nM and fully displaced radiolabeled vasopressin in binding experiments. V2Rinh-02 did not inhibit forskolin or β2-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V2R than for V1aR. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics.