Calcium-activated chloride channels (CaCCs) are always expressed in epithelial and nonepithelial cells. They are involved in epithelial fluid secretion, sensory signal transduction, smooth muscle contraction, oocyte fertilization, and other functions. CaCCs are promising drug targets for hypertension, asthma, secretory diarrheas, and pain

TMEM16A (anoctamin-1, ANO1) gene encodes a CaCC and exhibits calcium-activated Cl currents following heterologous expression.

TMEM16A is also widely spread in various tumors.

And it plays a role in tumor cell proliferation. TMEM16A knock-out mice die soon after birth due to tracheomalacia.
In an analysis of structure-activity relationships (SARs), to identify the most active TMEM16A inhibitor, researchers screened more than 800 chemical analogs.
T16Ainh-A01, A02, and A03 exhibit the most potent and active inhibitory effects, with IC50 values of 1.5-1.8 μM. T16Ainh-A01 and Digallic acid fully inhibit an ATP-induced short circuit current. The IC50 values are 1.1 and 3.6 μM, respectively.

In a Fluo-4 fluorescence measurement of ATP and ionomycin-stimulated cytoplasmic calcium elevation. T16Ainh-A01 can not alter Cytoplasmic calcium. Additionally, T16Ainh-A01 has little effect on CFTR Cl conductance (inhibited by <10%).
T16Ainh-A01 inhibits the TMEM16 isoform TMEM16B. In order to determine the inhibition mechanisms of T16Ainh-A01. Researchers develop a whole-cell patch-clamp analysis. T16Ainh-A01 inhibits nearly completely TMEM16A chloride current at all voltages. As a result, this indicates a voltage-independent block mechanism.

TMEM16A Inhibitors block calcium-activated chloride conductance in salivary gland cells.
Finally, to confirm the effect of T16Ainh-A01 on apical CaCC current, researchers measure apical membrane chloride conductance in CF bronchial epithelial cells following basolateral membrane permeabilization by 360 μg/ml nystatin and in the presence of a basolateral-to-apical chloride gradient. T16Ainh-A01 reduces UTP-stimulated peak current by ~25%.

In conclusion, T16Ainh-A01, an aminophenylthiazole, is a potent transmembrane protein 16A (TMEM16A) inhibitor. And it inhibits TMEM16A-mediated chloride currents with an IC50 value of ~1 µM.


[1]. Namkung W, et al. J Biol Chem. 2011 Jan 21;286(3):2365-74.