β-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter™ assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of β-galactosidase to measure receptor-β-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter™ β-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed β-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter™ cell line at the level of β-arrestin recruitment, Gi/o protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of β-arrestin recruitment was largely unaffected upon blockade of Gi/o protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of β-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully Gi/o protein regulated. The data indicate that the β-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of “biased ligands” (i.e., compounds that differ in G-protein coupling and β-arrestin-mediated cellular effects). (Journal of Biomolecular Screening 2008:986-998)
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