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Updated: Aug 16, 2026
Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
Published on: June 28, 2019
High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer
Fadi F Hamdan1, Martin Audet, Philippe Garneau
1University of Montreal, Department of Biochemistry, Montreal, Quebec, Canada.
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.
Insights
Researchers developed a novel bioluminescence resonance energy transfer (BRET) assay using stable cell lines to screen for G protein-coupled receptor (GPCR) antagonists. This method successfully identified potential CCR5 antagonists for HIV research.
Area of Science:
- Biochemistry and Molecular Pharmacology
- Cellular and Molecular Neuroscience
- Drug Discovery and Development
Background:
- G protein-coupled receptors (GPCRs) are crucial drug targets, mediating cellular responses to diverse stimuli.
- Beta-arrestin recruitment is a key signaling event for many GPCRs, influencing receptor desensitization and internalization.
- Developing robust assays for screening GPCR modulators is essential for identifying novel therapeutics.
Purpose of the Study:
- To develop stable mammalian cell lines coexpressing beta-arrestin2 and VENUS-fused GPCRs for BRET-based assays.
- To establish and validate a BRET1-beta-arrestin2 translocation assay for quantifying GPCR activation and inhibition.
- To apply this assay in a high-throughput screening (HTS) campaign for antagonists of the CCR5 receptor.
Main Methods:
- Generation of HEK293 cell lines stably coexpressing beta-arrestin2-Renilla luciferase and VENUS-fused GPCRs (Class A and B).
- Utilized bioluminescence resonance energy transfer (BRET1) to measure beta-arrestin2 translocation to GPCRs upon activation.
- Performed HTS of 26,000 compounds using a CCR5-VENUS and beta-arrestin2-Rluc coexpressing cell line to identify CCR5 antagonists.
Main Results:
- Successfully developed stable cell lines for BRET-based GPCR assays, accommodating transient and stable beta-arrestin interactors.
- Identified 12 compounds that specifically inhibited agonist-induced beta-arrestin2 recruitment to CCR5 in an HTS campaign.
- Confirmed the antagonist activity of three hit compounds in further functional assays, demonstrating inhibition of CCR5 signaling.
Conclusions:
- This study presents the first BRET1-beta-arrestin recruitment assay in stable mammalian cells for GPCR antagonist screening.
- The developed assay is effective for HTS, enabling the discovery of novel GPCR-targeted drug candidates.
- The identified CCR5 antagonists represent potential leads for therapeutic strategies against HIV infection.
