Two representative hit compounds per cluster were selected to further validate the inhibitory effects of each structural scaffold on AC1 activity and counter-screened for AC8mediated Ca 2+ /CaM-stimulated cAMP production. Dose-response curves were generated for each compound in HEK-AC1 cells and HEK-AC8 cells to quantify potency and AC8 selectivity. Hits were also assessed for cell viability using CellTiter-Glo to eliminate potential false-positives as a result of cell toxicity. Four clusters appeared to be false positives and were removed from further consideration. An additional cluster was excluded from further analysis because the compounds interfered with the fluorescence emission at 620 nm of the cAMP HTRF detection technology. The compounds from the remaining four scaffolds showed a dose-dependent inhibitory effect on AC1mediated Ca 2+ /CaM-stimulated cAMP production with IC50 values in the low micromolar range.