cAMP accumulation assay

Assays were performed with HTRF cAMP kit (Cisbio, 62AM4PEJ). A series dilution of compound (11 concentrations of compound, starting at 10 µM, 4-fold dilutions) was added into 384-well plates prior to cell seeding and DMSO was added to the control well. CHO-A2aR and CHO-A2bR cells were seeded in the plates at a density of 5,000 cells/well in F-12 medium containing 10% FBS. Plates were incubated at 37˚C for 30 min, then DMSO (control), adenosine (1, 10, and 100 µM), or NECA (0.1, 1, and 10 µM) were added to cells and further incubated at 37˚C 30 min. cAMP signal was measured on Envision (Perkin Elmer). The concentration of compound producing 50% inhibition of the cAMP (IC₅₀) was calculated using four-parameter logistic fit with XLFIT. The dose-response curves were fitted by nonlinear regression using GraphPad Prism.

NECA is a stable analogue of adenosine commonly used to measure adenosine pathway activity. Consistent with the trend in binding affinity to A2aR [31], NECA was also more potent than adenosine in stimulating cAMP accumulation via A2aR, approximately 10-fold stronger (Supplemental Fig. S1).