Functional GPCR Receptor Assay

Tested and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Serial dilutions were prepared in a 96-well microplate in an assay buffer, and 8 to 10 concentrations were tested. An intrinsic activity assay was performed according to the manufacturer of the ready-to-use CHO-K1 cells with the stable expression of the human serotonin 5-HT_1A, 5-HT_2A, and D₂ receptors, human GPCR, the promiscuous G protein Gαqi/5 for the D₂ receptor, and α₁₆ for 5-HT_1A and 5-HT_2A (Perkin Elmer, Boston, MA, USA). The assay was executed according to a previously described protocol [81 (link)]. After thawing, cells were transferred to the assay buffer (DMEM/HAM’s F12 with 0.1% protease-free BSA) and centrifuged. The cell pellet was resuspended in the assay buffer, and coelenterazine h was added at a final concentration of 5 µM. The cell suspension was incubated at 16 °C (or 21 °C), protected from light with constant agitation for 16 h (or 4 h), and then diluted with the assay buffer to the concentration of 100,000 cells/mL (or 250,000 cells/mL). After 1 h of incubation, 50 µL of the cell’s suspension was dispensed using automatic injectors built into the radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, Boston, MA, USA) into white opaque 96-well microplates preloaded with test compounds. The immediate light emission generated following calcium mobilization was recorded for 30 s. In antagonist mode, after 25–30 min of incubation, the reference agonist was added to the above assay mix, and the light emission was recorded again. The final concentration of the reference agonist (100 nM serotonin for the 5-HT_1A receptor, 30 nM α-methylserotonin for the 5-HT_2A receptor, and 30 nM apomorphine for the D₂ receptor) was equal to EC₈₀. IC₅₀ and EC₅₀ values were calculated.