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β-Arrestin-Based Bret² Screening Assay for the "Non"-β-Arrestin Binding CB1 Receptor

Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia, milka.vrecl{at}vf.uni-lj.si

Pia Karina Nørregaard

7TM Pharma, Hørsholm, Denmar

Dorthe L.C. Almholt

7TM Pharma, Hørsholm, Denmar

Lisbeth Elster

7TM Pharma, Hørsholm, Denmar

Azra Poganik

Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia

Anders Heding

7TM Pharma, Hørsholm, Denmar

CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a β-arrestin 2—based screening assay for the CB1R using BRET² technology. When the existing BRET² screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with β-arrestin 2, resulting in unsatisfactory assay performance. To enhance the β-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with β-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC₅₀ and EC₅₀ values ranging from 0.3 nM to 7.5 µM. Both primary and secondary screening were performed with Z' > 0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays. (Journal of Biomolecular Screening 2009:371-380)

Key Words: 7TM receptors • CB1R • β-arrestin 2 • BRET2 • screening

Journal of Biomolecular Screening, Vol. 14, No. 4, 371-380 (2009)
DOI: 10.1177/1087057109333101


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