In vitro activity: CaCCinh-A01 (also known as TMEM16 Blocker I) is an inhibitor of TMEM16A and calcium-activated chloride channel (CaCC) with IC50s of 2.1 and 10 μM, respectively. TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). CaCCs are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. CaCCinh-A01 is a non-selective inhibitor of CaCCs that blocks ATP-stimulated chloride conductance in human salivary gland, intestinal, and bronchial epithelium (mean IC50 = 10 μM). As a TMEM16A/CaCC inhibitor, CaCCinh-A01 is a potential development candidate for drug therapy of hypertension, pain, diarrhea, and excessive mucus production. 3

Kinase Assay: CaCCinh-A01 (also known as TMEM16 Blocker I) is an inhibitor of TMEM16A and calcium-activated chloride channel (CaCC) with IC50s of 2.1 and 10 μM, respectively. 0 μM CaCCinh-A01 and 100 μM tannic acid strongly inhibit CaCC current following ATP stimulation. Calcium-dependent chloride current is reduced by 38±14, 66±10, and 91±1% by 0.1, 1, and 10 μM CaCCinh-A01, respectively. ATP-induced short-circuit currents are reduced by 38±7 and 78±3% at 10 and 30 μM CaCCinh-A01, respectively.

Cell Assay: Each well of a 96-well plate is washed three times with PBS (200 μL/wash), leaving 50 μL of PBS. Test compounds (including CaCCinh-A01) (0.5 μL) are added to each well at 25 μM final concentration. After 10 min, 96-well plates are transferred to a plate reader for fluorescence assay. Each well is assayed individually for TMEM16A-mediated I- influx by recording fluorescence continuously (400 ms/point) for 2 s (base line), then 50 μL of a 140 mM I- solution containing 200 μM ATP is added. The initial rate of I- influx is computed from fluorescence data by nonlinear regression.