Beta-arrestin-based Bret2 screening assay for the "non"-beta-arrestin binding CB1 receptor.
CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a beta-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 beta-arrestin 2, resulting in unsatisfactory assay performance. To enhance the beta-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 beta-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 microM. 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.
Vrecl
Milka
M
Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia. milka.vrecl@vf.uni-lj.si
Nørregaard
Pia Karina
PK
Almholt
Dorthe L C
DL
Elster
Lisbeth
L
Pogacnik
Azra
A
Heding
Anders
A
D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D018004:Receptors, Bombesin / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000032:analysis* / Q000494:pharmacology*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D043884:Receptor, Cannabinoid, CB1; D018004:Receptors, Bombesin; D054852:Small Molecule Libraries; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C107120:bombesin receptor subtype 3