Abstract
Anoctamin1 (ANO1)/transmembrane protein 16A (TMEM16A), a calcium-activated chloride channel (CaCC), is involved in many physiological functions such as fluid secretion, smooth muscle contraction, nociception and cancer progression. To date, only a few ANO1 inhibitors have been described, and these have low potency and selectivity for ANO1. Here, we performed a high-throughput screening to identify highly potent and selective small molecule inhibitors of ANO1. Three novel ANO1 inhibitors were discovered from screening of 54,400 synthetic small molecules, and they were found to fully block ANO1 channel activity with an IC50 < 3 μM. Electrophysiological analysis revealed that the most potent inhibitor, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9), completely inhibited ANO1 chloride current with submicromolar potency. Notably, unlike previous small-molecule ANO1 inhibitors identified to date, Ani9 displayed high selectivity for ANO1 as compared to ANO2, which shares a high amino acid homology to ANO1. In addition, Ani9 did not affect the intracellular calcium signaling and CFTR chloride channel activity. Our results suggest that Ani9 may be a useful pharmacological tool for studying ANO1 and a potential development candidate for drug therapy of cancer, hypertension, pain, diarrhea and asthma.
Highlights
ANO1/TMEM16A, a calcium-activated chloride channel (CaCC), plays an important role in fluid secretion in various cell types including airway and intestinal epithelial cells, smooth muscle cells, intestinal pacemaker cells, sensory neurons, and several tumors [1, 2]
For the screening to identify ANO1 inhibitors, the Fisher rat thyroid (FRT) cells were pre-incubated with test compounds for 10 minutes prior to addition of an iodide and ATP, an agonist of P2 purinergic receptor which induces an increase in intracellular calcium concentration and activation of ANO1, containing solution
To investigate the effect of the three inhibitors on ANO1 channel activity, apical membrane current was measured in FRT-ANO1 cells (Fig 1B)
Summary
ANO1/TMEM16A, a calcium-activated chloride channel (CaCC), plays an important role in fluid secretion in various cell types including airway and intestinal epithelial cells, smooth muscle cells, intestinal pacemaker cells, sensory neurons, and several tumors [1, 2]. Evidence has been reported for ANO1 involvement in cell proliferation, cell migration, and cancer progression [3,4,5,6]. Emerging evidence suggested that pharmacological inhibition of ANO1 may be beneficial in treatment of diseases associated with ANO1 such as asthma, hypertension, diarrhea, pain and cancer. ANO1 is strongly expressed in airway mucin-secreting cells and airway smooth muscle in ovalbumin (OVA)-induced asthma mouse model. Pharmacological inhibition of ANO1 inhibits mucus secretion of airway epithelium and airway smooth.
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