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High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based ß-Arrestin2 Recruitment Assay

Martin Audet

University of Montreal, Department of Biochemistry, Montreal, Quebec, Canada.

Philippe Garneau

Jerry Pelletier

McGill University, Department of Biochemistry and McGill High-Throughput Screening Facility, Montreal, Quebec, Canada.

Michel Bouvier

University of Montreal, Department of Biochemistry, Montreal, Quebec, Canada.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of ß-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with ß-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- ß-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and ß-arrestin2- Renillaluciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. Atotal of 26,000 compounds were screened for inhibition of the agonist-promoted ß-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced ß-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1- ßarrestin recruitment assay in stablemammalian cells and its successful application in HTS for GPCRs antagonists.

Key Words: BRET • ß-arrestin • GPCR • CCR5 • high-throughput screening • luciferase reporter assay • fluorescence reporter assay

Journal of Biomolecular Screening, Vol. 10, No. 5, 463-475 (2005)
DOI: 10.1177/1087057105275344


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