| Assay Method Information | |
| | Measurement of beta-arrestin Recruitment to Human Dopamine D2L Receptors |
| Description: | PathHunter CHO-K1 cells expressing tagged human D2L receptors and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded into 96-well white-walled clear bottom tissue culture plates in 90 μL/cell AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) at a density of 20,000 cells/well. The plates were incubated overnight in a humidified atmosphere with 5% CO2 at 37° C. Twenty to twenty-four hours later, 20 μL of test compound or vehicle in CP2 reagent and containing 2.2% DMSO was added to the cells, and they were incubated for 90 minutes at 37° C. Then, 55 μL PathHunter Detection Reagent (Eurofins DiscoverX) was added per well and plates were incubated for 60 minutes at 25° C., followed by luminescence detection using a PHERAstar FS multimode plate reader (BMG Labtech, Ortenberg, Germany). Raw data were converted to percent stimulation above basal values. Values were further converted to percent of maximal stimulation of β-arrestin recruitment by 30 μM dopamine. EC50 values were calculated from concentration-response curves of at least six concentrations run in duplicates by sigmoidal fitting using Origin 7.5 software (OriginLab Corporation, Northampton, MA, USA) and were defined as the concentration of the agonist with half-maximal stimulation. The pEC50 values were calculated as the negative logarithm of the EC50 value expressed in mol/liter and is shown in Table 6. The results indicate that the examples of the present disclosure are potent agonists of the G-protein-independent signaling pathway of human recombinant D2L receptors. |
| Affinity data for this assay | |
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