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First published on April 13, 2007; DOI: 10.1124/mol.107.034496

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Buranee Yangthara
Aaron Mills
Varanuj Chatsudthipong
Lukmanee Tradtrantip
A. S Verkman
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Received for publication January 26, 2007.
Revised April 13, 2007.
Accepted for publication April 13, 2007.

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

Buranee Yangthara 1, Aaron Mills 2, Varanuj Chatsudthipong 1, Lukmanee Tradtrantip 2, A. S Verkman 2*

1 Mahidol University 2 University of California, San Francisco

* Address correspondence to: E-mail: alan.verkman{at}ucsf.edu

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 (YFP-H148Q/I152L) were transfected with V2R. Increased cell Cl- conductance following agonist-induced cAMP elevation was assayed using a platereader from cell fluorescence following solution I- addition. The Z'-factor for the assay was ~0.7 with the V2R agonist dDAVP (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 of 35 structural analogs, competitively inhibited V2R-induced cAMP elevation with Ki ~ 70 nM, and fully displaced radiolabeled vasopressin in binding experiments. V2Rinh-02 did not inhibit forskolin or beta2-adrenergic receptor-induced cAMP production, and was >50-times more potent for V2R vs. V1aR. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential utility in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs.


Key words: cAMP, Ion transporters (SERCA, Na/K ATPase, CFTR), Fluorescence techniques, Receptor binding studies, Peptide hormones


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R. De La Fuente, W. Namkung, A. Mills, and A. S. Verkman
Small-Molecule Screen Identifies Inhibitors of a Human Intestinal Calcium-Activated Chloride Channel
Mol. Pharmacol., March 1, 2008; 73(3): 758 - 768.
[Abstract] [Full Text] [PDF]


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