High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay
2005; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.1177/1087057105275344
ISSN2472-5560
AutoresFadi F. Hamdan, Martin Audet, Philippe Garneau, Jerry Pelletier, Michel Bouvier,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoIn 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 beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-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-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.
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