Identification And Chracterization Of TMEM16A Type Calcium-activated Chloride Channel Inhibitors

Calcium-activated chloride channel?CaCCs?are widely expressed in non-excited cells such as endothelial cells and epithelial cells,as well as excitatory cells such as cardiomyocytes,nerve cells and vascular smooth muscle cells,where to play numerous important physiological functions.In 2008,the first CaCC?TMEM16A?was cloned,and subsequent studies showed that TMEM16A was widely expressed in respiratory epithelial cells,salivary gland epithelial cells,arterial smooth muscle,interstitial Cajal cells,sensory neurons and a variety of tumor cells.Selective modulators are the most important pharmacological tools for studying ion channels.High affinity,highly selective TMEM16A inhibitors not only can be used as chemical probes to reveal the mechanisms and pathophysiological functions of TMEM16A,but also provide lead compounds for the treatment of CaCCs-related secretory diarrhea.In this study,we identified several TMEM16A inhibitors from more than 400 natural small molecules by using the Fischer Rat Thyroid epithelia?FRT?stably co-transfected with human TMEM16A and the halide-sensitive green fluorescent protein YFP-H148Q/I152L.Selectivity,mechanisms of activation and and molecular pharmacology of the compounds were analyzed on the FRT cells,mice clonic mucosa and mice model of diarrhea.Results:1.Identification of three naphthoquinone compounds?plumbagin,swertiamarin and shikonin?as TMEM16A chloride channel inhibitors.Further studies indicated that these compounds could dose-dependently inhibite TMEM16A chloride ion channel activities,with IC₅₀ values of 12.46?M,14.83?M and 20.18?M,respectively.Inhibition of TMEM16A by these compounds showed fast and irreversible characteristics.2.Short-circuit current tests done on FRT-TMEM16A cells showed that apical application of plumbagin could dose-dependently inhibit TMEM16A-mediated currents stimulated by E_act,with the maximum inhibition rate of about 70%,IC₅₀ value about 5?M.3.Plumbagin had an inhibitory effect on the HT29 cell expression of enterocyte CaCC(CaCC_GI)and CFTR chloride channels.4.Plumbagin could inhibit CaCC_GI channel activity when applied on the colonic mucosal side with the maximal inhibition rate about 45.8%?p<0.01?;and itn had no significant effect on Na⁺-K⁺-ATPase and calcium-activated potassium channel5.Plumbagin could significantly increase ATP,CCh and ionomycin induced Ca²⁺concentration in HT-29 cells.6.In vivo activity studies,plumbagin could significantly inhibit intestinal peristalsis and intestinal fluid secretion in mice.Conclusion:In this study,we found a new structure of TMEM16A chloride ion channel inhibitor,which is of great theoretical significance for elucidating the role of TMEM16A in intestinal peristalsis and fluid secretion under both physiological and pathological conditions.