To discover additional potent and selective inhibitors of AC1 activity, our team designed and carried out a high-throughput screen of 10,240 compounds from the Life Chemical diversity library. Our primary screen sought to identify molecules that inhibit AC1 mediated Ca 2+ /CaMstimulated cAMP production in HEK cells stably expressing AC1 (HEK-AC1). HEK-AC1 cells were stimulated with the Ca 2+ ionophore A23187 and the accumulation of cellular cAMP levels was quantified using a homogenous time resolved fluorescence (HTRF) assay with cAMP detection reagents as described previously. The AC1 inhibitor ST034307 served as a positive control and DMSO as negative control, and the Z'-value for the screening assay was determined to be 0.6. The fluorescence values were normalized for cAMP levels of the DMSO (0% inhibition) and ST034307 (100% inhibition) controls on each plate, and hits were defined as those compounds that exhibited ≥ 90% inhibition of AC1-mediated Ca 2+ /CaM-stimulated cAMP production at a single dose of 10 µM. A total of 480 compounds met this criterion, representing approximately 5% of the library screened. The 480 compounds were filtered for pan-assay interference compounds (PAINS) of which 200 molecules were identified to contain PAINS-like substructures and were triaged. The remaining 280 hits were clustered into scaffolds with a Tanimoto cutoff of 70% similarity. This resulted in nine representative clusters containing, each having at least 8 compounds.