Multiplexed assays by high-content imaging for assessment of GPCR activity.

G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor® imaging assay is based on redistribution of cytosolic β-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor® alone has limitations: relying on a single mechanistic step of β-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multifplexed the Transfluor® assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor...