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

G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V(2)R) 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 V(2)R. 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 approximately 0.7 with the V(2)R 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 V(2)R antagonists that are unrelated structurally to known V(2)R antagonists. The most potent compound, V(2)R(inh)-02, which was identified by screening 35 structural analogs, competitively inhibited V(2)R-induced cAMP elevation with K(i) value of approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(2)-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V(2)R than for V(1a)R. 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.