Parallel High-Throughput Screening with the TANGO GPCR-Ome β-Arrestin Recruitment Assay.

Parallel TANGO high-throughput screening for agonist activity was performed as previously described (Kroeze et al., 2015). Human embryonic kidney 293–derived cells containing a stable tetracycline-controlled transcriptional activator (tTA)–dependent luciferase reporter and a β-arrestin 2–tobacco etch virus (TEV) fusion gene were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 2 μg/ml puromycin (Sigma-Aldrich), and 100 μg/ml hygromycin B (DiscoverX, Fremont, CA) at 37°C in 5% CO₂ at 90% humidity. For transfection of 315 synthetic TANGO-ized GPCRs, cells were plated at 10⁶ cells per 150-mm cell culture dish on day 1. On day 2, cells in each dish were transfected with a synthetic TANGO-ized GPCR (a total of 315 transfected cell lines) using a calcium phosphate method (Kroeze et al., 2015). On day 3, transfected cells were transferred at 20,000 cells/well in 40 μl medium into poly(l-Lys)– coated, 384-well white clear-bottomed plates (Greiner Bio-One, Kremsmünster, Austria) in quadruplicate. Control cells transfected with DRD2 were seeded as 16 replicates in columns 1 and 2. On day 4, four single drug stimulation solutions (of either MTP, TAM, DOX, or BRM) and six combined drug stimulation solutions (of either 1:1 MTP + DOX, 1:1 MTP + TAM, 1:1 MTP + BRM, 1:1 DOX + BRM, 1:1 TAM + BRM, 1:1:1 MTP + DOX + BRM, and 1:1:1 MTP + TAM + BRM) were prepared in filter-sterilized assay buffer and 20 μl were added to each well to achieve a final concentration of 10 µM. For the control containing 16 replicates in column 1, 100 nM quinpirole drug stimulation solution was added, whereas no ligand was added in column 2. On day 5, drug-containing medium was removed from the wells and 20 μl Bright-Glo solution (Promega, Madison, WI) diluted 20-fold with assay buffer was added to each well. After incubation for 20 minutes at room temperature, luminescence was measured with Trilux (Trilux, Arnsberg, Germany). GraphPad Prism software (GraphPad Inc., La Jolla, CA) was used for data analyses.