The compound screen of the Life Chemicals diversity library was completed at the Biomolecular Screening and Drug Discovery Core Facility (BSDD), Purdue University (West Lafayette, IN). Cryopreserved HEK293 cells stably expressing AC1 (HEK-AC1) were washed with pre-warmed Opti-MEM from Thermo Fisher Scientific (Waltham) and centrifuged for 5 minutes at 150 x g. Supernatant was discarded and the cells were resuspended in Opti-MEM, counted, and plated into white opaque 384-well plates from Perkin Elmer (Waltham, MA). The plates were incubated for 1-hour in a 37°C incubator supplemented with 5% CO2 to let the cells adhere to the plate prior compound addition. Subsequently, DMSO (negative control), the AC1 inhibitor ST034307 (positive control), or the Life Chemicals library compounds were added at a screening concentration of 10 μM using a pin tool liquid handling system. Following a 30-minute incubation at room temperature with DMSO or the compounds, the calcium ionophore, A23187, was added to all the wells to a final concentration of 3 μM in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). After 1-hour incubation with A23187 at room temperature, cAMP accumulation was measured using the HTRF cAMP kit from Cisbio (Bedford, MA). The screen was carried out on three separate days and a total of 10,240 compounds were screened. The percentage inhibition (%) was calculated by normalizing the cAMP levels of each of the screen compounds to the mean cAMP levels of the positive control, ST034307, (100% inhibition) and DMSO (0% inhibition) in each plate.