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-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 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-β-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists. (Journal of Biomolecular Screening 2005:463-475)

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

“…Analysis of ␤-Arrestin2 Binding to the ␤ 2 AR Using Bioluminescence Resonance Energy Transfer (BRET)-␤-Arrestin2 recruitment was monitored following the protocol of Hamdan et al (28). HEK 293 cells were grown in 6-well plates to 80% confluence in DMEM with 10% FBS.…”

“…This assay involved treating HEK 293 cells co-expressing a ␤ 2 AR-Renilla reniformis luciferase II fusion (␤ 2 AR-RLucII) and GFP10-tagged ␤-arrestin2 with the various pepducins ( Fig. 2B) (28). Several ICL1-derived pepducins effectively promoted ␤-arrestin2 interaction with the ␤ 2 AR, whereas ICL2 and ICL3 pepducins had no effect ( Fig.…”

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

“…This phenomenon, also referred to as biased agonism, functional selectivity or signal trafficking, has an important impact on GPCR drug discovery because it raises the possibility to design signaling pathway-specific therapeutics. Activation of downstream signaling events of GPCRs is traditionally recorded with assays based on quantification of distinct intracellular second messengers such as Ca 2+ , IP1 and cyclic AMP 5,9-11 and/or translocation of β-arrestin proteins 5,[12][13][14][15][16] . Since GPCRs from different coupling classes typically produce one or more specific second messenger, and may additionally engage non-G protein effectors [17][18][19][20][21] , several assays are needed to obtain quantitative information about each signaling event.…”

“…We therefore anticipate that DMR as a holistic read-out of cell 13 function will advance the emerging areas of systems biology and systems pharmacology and thereby promote the discovery of mechanistically novel therapeutics. …”

Antagonism of dopamine D2 receptor/β-arrestin 2 interaction is a common property of clinically effective antipsychotics

“…The use of ␤-arrestin recruitment to activated receptors has been developed to follow activation of multiple GPCRs and even used as a highthroughput screening tool to identify ligands (20,21).…”