title: High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.
abstract: 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 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.
authors.affiliation: University of Montreal, Department of Biochemistry, Montreal, Quebec, Canada.
authors.forename: Fadi F
authors.initials: FF
authors.lastname: Hamdan
authors.forename: Martin
authors.initials: M
authors.lastname: Audet
authors.forename: Philippe
authors.initials: P
authors.lastname: Garneau
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D001331:Automation; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004735:Energy Transfer; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006678:HIV / Q000378:metabolism; D006801:Humans; D049410:Luciferases, Renilla / Q000378:metabolism; D008163:Luminescent Measurements; D046911:Macromolecular Substances / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D019713:Receptors, CCR5 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors*; D048790:Renilla; D013997:Time Factors; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D046911:Macromolecular Substances; D019713:Receptors, CCR5; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; D002118:Calcium