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

Abstract

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of β-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 β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-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 β-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced β-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.