Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 10, Issue 5, Pages 463-475Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057105275344
Keywords
BRET; beta-arrestin; GPCR; CCR5; high-throughput screening; luciferase reporter assay; fluorescence reporter assay
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In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-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 beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1-beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2-Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted P-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced P-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 BRETI-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.
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