VPAC1 and VPAC2 receptor agonist

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 ∼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)-2 H-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 ∼70 nM and fully displaced radiolabeled vasopressin in binding experiments. V 2 R inh-02 did not inhibit forskolin or β 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.

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Materials and Methods

Plasmids. Plasmids encoding human wild-type and mutant (W164S) V 2 Rs were kindly provided by Dr. Daniel Bichet (University of Montreal, Montreal, QC, Canada). N-terminal c-myc-tagged V 2 Rs were created by polymerase chain reaction introduction of a c-myc sequence after the methionine initiation sequence. Polymerase chain reaction products were subcloned into plasmid pCDNA3.1Hyg+ (Invitrogen, Carlsbad, CA) at XbaI and HindIII restriction sites to give plasmids wV 2 R-Hyg (wild type) and mV 2 R-Hyg

Results

Expression of the Wild-Type and the Mutant V 2 Rs in FRT Cells. Stably transfected FRT cell lines were generated that coexpress human wild-type CFTR, YFP-H148Q/I152L, and c-myc-tagged wild-type V 2 R or the mutant V 2 R-W164S. The c-myc-tag was inserted at the external-facing V 2 R N terminus. Wild-type V 2 R showed a plasma membrane distribution by c-myc staining (Fig. 1B), whereas no membrane staining was seen in nontransfected cells or cells expressing V 2 R-W164S that has a defect in cellular

Discussion

We report a novel, cell-based functional assay of G s-or G i-coupled GPCR modulators based on cAMP-dependent activation of CFTR Cl- channels. The assay is technically simple, inexpensive, and readily adaptable to fluorescence-based, high-throughput screening formats. The assay used an epithelial cell line suitable for both fluorescence and electrophysiological measurements. The cells have low basal halide conductance and cAMP concentration, excellent transfection efficiency, and rapid growth on

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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.

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; V 1a R, vasopressin-1a receptor; V 2 R, vasopressin-2 receptor; V 1b R, vasopressin-1b receptor; FST, Fischer rat thyroid; HA, hemagglutinin; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; w, wild type; m, mutant; SR 49059, (2 S)-1-(((2 R,3 S)-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′-(3 H)indol)-1′ (2′H)-yl)sulfonyl)-3-methoxy-, phosphate; OPC 21268, 1-(1-(4-(3-acetylaminopropoxy)benzoyl)-4-piperidyl)-3,4-dihydro-2(1 H)-quinolinone; YM 087, (1,1′-biphenyl)-2-carboxamide, N-(4-((4,5-dihydro-2-methylimidazo(4,5-d)(1)benzazepin-6(1 H)-yl)carbonyl)-phenyl)-, monohydrochloride; OPC 41061, (-)-4′-((7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1 H-1-benzazepin-1-yl)carbonyl)-o-tolu-m-toluidide; VPA 985, N-(3-chloro-4-(5 H-pyrrolo(2,1-c)(1,4)benzodiazepin-10(11 H)-ylcarbonyl)phenyl)-5-fluoro-2-methyl-benzamide.