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 V2R antagonists. The most potent compound, V2Rinh-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.
Footnotes
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This work was supported by grants DK72517, DK35124, HL59198, EY13574, EB00415, and HL73856 from the National Institutes of Health, and Research Development Program (R613) and Drug Discovery grants from the Cystic Fibrosis Foundation (to A.S.V.). B.Y. is an MD-PhD student at Mahidol University, Bangkok, whose tenure in the Verkman laboratory was supported in part by a Royal Golden Jubilee grant PHD/0292/2545 from the Thailand research fund and by National Center for Genetic Engineering and Biotechnology (BIOTEC) Grant 3-2548, National Science and Technology Agency, Thailand.
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; CFTR, cystic fibrosis transmembrane conductance regulator; YFP, yellow fluorescent protein; AVP, arginine vasopressin; dDAVP, [deamino-Cys1, Val4, d-Arg8]-vasopressin; V1aR, vasopressin-1a receptor; V2R, vasopressin-2 receptor; V1bR, vasopressin-1b receptor; FST, Fischer rat thyroid; HA, hemagglutinin; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; w, wild type; m, mutant; SR 49059, (2S)-1-(((2R,3S)-5-chloro-3-(o-chlorophenyl)-1-((3,4-dimethoxyphenyl)sulfonyl)-3-hydroxy-2-indolinyl)carbonyl)-2-pyrrolidinecarboxamide; SR 121463B, benzamide, N-(1,1-dimethylethyl)-4-((cis-5′-ethoxy-4-(2-(4-morpholinyl)ethoxy)-2′-oxospiro(cyclohexane-1,3′-(3H)indol)-1′ (2′H)-yl)sulfonyl)-3-methoxy-, phosphate; OPC 21268, 1-(1-(4-(3-acetylaminopropoxy)benzoyl)-4-piperidyl)-3,4-dihydro-2(1H)-quinolinone; YM 087, (1,1′-biphenyl)-2-carboxamide, N-(4-((4,5-dihydro-2-methylimidazo(4,5-d)(1)benzazepin-6(1H)-yl)carbonyl)-phenyl)-, monohydrochloride; OPC 41061, (-)-4′-((7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl)-o-tolu-m-toluidide; VPA 985, N-(3-chloro-4-(5H-pyrrolo(2,1-c)(1,4)benzodiazepin-10(11H)-ylcarbonyl)phenyl)-5-fluoro-2-methyl-benzamide.
- Received January 25, 2007.
- Accepted April 13, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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