cAMP Inhibition Assay

D₄R- and D₂R -mediated inhibition of forskolin-stimulated cAMP production was assayed using the PerkinElmer LANCE UltracAMP assay kit (PerkinElmer, Inc., Waltham, Massachusetts). CHO-K1 cells stably expressing the human D₂R long isoform or D₄R were maintained in Ham’s F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 800 μg/mL G418, and 300 μg/mL hygromycin at 37 °C, 5% CO₂, and 90% humidity. Cells were seeded in 5 μL of Hank’s Balanced Salt Solution (with CaCl and MgCl₂) with 5 mM HEPES buffer and 0.2 μM sodium metabisulfite at a density of 5000 cells/well in 384-well white plates. Compounds and forskolin were made in the same buffer. Immediately after plating, cells were treated with 2.5 μL of compound (at various concentrations) and 2.5 μL of forskolin and incubated at room temperature for 30 min. The final concentration of forskolin was 10 μM. When running the assay in antagonist mode, the EC₈₀ of dopamine (10 nM) was added to the forskolin solution. Eu-cAMP tracer and ULight-anti-cAMP solutions were added as directed by the manufacturer, and cells were incubated for 2 h in the dark at room temperature, after which a TR-FRET signal was measured using a BMG Labtech PHERAstar FS (BMG Labtech, North Carolina). Values were normalized to a percentage of the control TR-FRET signal seen with a maximum concentration of dopamine for agonist mode assays and the EC₈₀ of dopamine for antagonist mode assays. Data were collected in triplicate from at least three independent experiments. Data analysis and normalization were performed in GraphPad Prism 9 (GraphPad Software, California). First, raw data was fit using a log(agonist/antagonist) vs response – variable slope (four parameters) curve fit. The data were normalized to the percent maximum dopamine response (agonist mode) or the EC₈₀ of dopamine (antagonist mode). The Hill coefficients of the concentration–response curves did not significantly differ from unity with the data fitting to a single-site model. Graphs are meaned concentration response curves from at least three independent experiments. Data in Table 2 was extracted from the meaned curves where E_max/I_max are expressed as mean ± SEM and the potencies are expressed as mean [95% confidence interval]. Fold selectivities for D₄R over the were also calculated and are presented in Table 2.