Abstract
Assays for high-throughput screening of G-protein-coupled receptors (GPCRs) have typically revolved around receptor binding, guanine nucleotide binding, and second-messenger assays measuring intracellular cAMP and calcium levels. New assay development has been directed toward G-protein-independent signaling pathways, including protein redistribution in response to activated receptors. β-arrestin recruitment to agonist-stimulated GPCRs is the basis for the Transfluor®, PathHunter®, and Tango™ GPCR screening platforms. In the Tango™ GPCR technology, receptor activation results in the recruitment of a TEV protease:β-arrestin fusion protein to the activated receptor where the TEV protease releases the GAL4-VP16 tethered to the target GPCR by a 7—amino acid TEV protease site. The release of the transcription factor results in expression of the β-lactamase (bla) reporter gene. The authors performed a small library screen with a Tango™ cell line expressing the κ opioid receptor and identified a series of compounds with a similar core chemical structure that were selective agonists for the κ opioid receptor over the µ and δ opioid receptors. These compounds were validated in additional second-messenger assays, confirming the agonist activity of the identified compounds. These results provide insight into the value of screening compounds in multiple assay technologies to better characterize the compound's potency and efficacy. (Journal of Biomolecular Screening 2009:381-394)