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Small-Molecule Vasopressin-2 Receptor Antagonist Identified by a G-Protein Coupled Receptor "Pathway" Screen

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California (B.Y., A.M., L.T., A.S.V.); and Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand (B.Y., V.C.)

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 V₂R antagonists. The most potent compound, V₂R_inh-02, which was identified by screening 35 structural analogs, competitively inhibited V₂R-induced cAMP elevation with K_i value of 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V₂R_inh-02 did not inhibit forskolin or ₂-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V₂R than for V_1aR. 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.

Address correspondence to: Dr. Alan S. Verkman, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. E-mail: alan.verkman{at}ucsf.edu

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