Discovery And Molecular Study Of Natural Small Molecule Inhibitors Targeting TMEM16A Ca²⁺-activated Chloride Channel

Ca²⁺-activated chloride channels?CaCCs?are anion channels widely expressed in various mammalian cells,play significant roles in multiple physiological functions such as epithelial fluid transport,gastrointestinal motility,smooth muscle contraction,neuronal excitation and nociception.Accumulated studies have reported that CaCCs function contributes to chronic constipation,hypertension,congenital megacolon,tumor and diarrhea.It has long been suggested by functional studies that there exist many types of CaCCs.Despite TMEM16A?alternative name ANO1?hasn't been cloned until 2008,molecular identities of other CaCCs remain unclear.Discovering new types of CaCCs can not only be of theoretical significance for the revelation of the exact physiological and pathological function,but also provide new therapeutic targets for the treatmemt of multiple diseases.CaCCs specific modulators are key tools to investigate various pharmacological and physiological functions of CaCCs at present,they can be used to seperately analyze konwn and unknown CaCCs chloride channel activities in cultured cells and ex-vivo tissue models.In addition,they can provide effective molecular probes in CaCCs physiological function and pathophysiological mechanisms researches.Current TMEM16A and intestinal epithelial CaCC?epithelial CaCC,CaCCGI?chloride channel modulators that have been screened from combinational chemical molecule library do much to help clarify CaCCs physiological functions.Therefore there is crying need for uncovering new samll molecule CaCCs modulators with high affinity,strong selectivity that can be used for ex-vivo and in-vivo activitiy studies.The present study aimed at screening new natrual small molecule inhibitors from natrual products that inhibit TMEM16A and CaCCs chloride channel activities mucosally thus enhance intestinal fluid secretion and intestinal motility.Methods:In the present study,we used FRT cells stalbly cotransfected with halide-sensitive yellow fluorescent proteins?YFP-H148Q/I152L?and human TMEM16A cDNA?FRT-TMEM16A-YFP-H148Q/I152L?as functional assay model for screening TMEM16A chloride channel inhibitors from natural molecule library.Next we used FRT cells stably cotransfected with YFP?YFP-H148Q/I152L?and human CFTR?FRT-CFTR-YFP-H148Q/I152L?and HT-29 cells endogenously expressed CFTR and CaCCGI chloride channels to analyze the selectivity of the above compounds;cell-based fluoresecnt assay and short-circuit current measurement were used to analyze the basic pharmacological properties of the active compounds.Furthermore,ex-vivo and in-vivo activities of TMEM16A inhibitors were investigated using mouse colonic epithelial short-circuit current measurement,mouse gastrointestinal peristalsis and neonatal rotaviral diarrheal mice model.Results:1.Identification of two lignan compounds kobusin and eudesmin,one naphthoquinone compound shikonin screened from natural molecule library as TMEM16A chloride channel inhibitors,with IC50 values of 20?M?kobusin?,50?M?eudesmin?and 6.5?M?shikonin?.2.Short-circuit current analysis in transfected cells showed that kobusin,eudesmin and shikonin inhibited TMEM16A-mediated short-circuit currents in FRT-TMEM16A cells apically,with IC50 values of 100?M?kobusin?,200?M?eudesmin?and 1?M?shikonin?;Kobusin,eudesmin or shikonin didn't inhibite TMEM16A-mediated Cl-current basolaterally.3.Kobusin and eudesmin activated CaCCGI chloride channel in human colnic carcinoma HT-29 cells and the effect was synergistic with ATP;shikonin inhibited CaCCGI-mediated Cl-current in HT-29 cells with IC50 values of 1.5?M;shikonin inhibted ATP-induced transient and sustained short-circuit currents,indicating the existence of different types of CaCCs induced by ATP;shikonin didn't inhibit cytoplasmic Ca²⁺concentration in HT-29 cells.The adverse effects of kobusin and eudesmin on TMEM16A and CaCCGI chloride channel activities suggest that TMEM16A and CaCCGI have different molecular characteristics.4.Functional assay results showed that koubusin and eudesmin activated CFTR chloride channel activity in FRT cells in a dose-dependent way with EC50 values of 30?M and 50?M,respectively;Kobusin and eudesmin manifested FSK-dependent,additive effects with IBMX,synergisitic effect with Gen on the activation of CFTR chloride channel acitivities.Acitvation of CFTR chloride channel activity by kobusin and eudesmin manifested fast and reversible characteristics,kobusin and eudesmin activated CFTR chlorde channel activity with maximal effect at 4 min and the effect was abolished 15 min after washout;Similar results comfirmed that kobusin and eudesmin activated CFTR-meidated short-circuit currents in HT-29 cells with a FSK-dependent manner.4.Ex-vivo short-circuit current measurements revealed that apical application of kobusin and eudesmin activated CFTR and CaCCGI-mediated Cl-currents in mouse clonic epithelia,and the effects could be completely abolished by specific inhibtors CFTRinh-172?CFTR?and CaCCinh-A01?CaCC?;shikonin inhibited carbachol-induced short-circuit current with maximal inhibiton rate of 48%?p<0.01?compared to that of CaCCinh-A01?34%,p<0.05?apically,had no inhibitory effect on CFTR chloride channel activity in mouse clonic epithelial either mucosally or serosally and no inhibitory effect on Na+/K+-ATPase activity in mouse clonic epithelia.5.In-vivo test results showed that orally administration of kobusin,eudesmin and shikonin mildly but significantly decreased mouse intentinal peristalsis;watery stools of rotaviral neonatal mice were reduced when orally administered shikonin the day before virus inoculation and the day with virus inoculation without affecting rotavirus infection,and the maximal inhibitory effect?0.692 mg/kg?was better than CaCCinh-A01?13.6 mg/kg?.Conclusion:1.The present study identified three new TMEM16A chloride channel nhibitors from different family,among which shikonin turned out to be the most high-affinity TMEM16A and CaCCGI inhibitor.2.The adverse effect of kobuisn and eudesmin on TMEM16A and CaCCGI chloride channel activities provides definite evidence that TMEM16A and CaCCGi are two different chloride channels with different molecular characterizations and properties.3.Activation of intestinal epithelial CaCCGI chloride channel to enhance subsequent fluid secretion to the lumen is one of the mechanisms underlying rotaviral secretory diarrhea,inhibition of luminal TMEM16A and CaCCGI chloride channels could reduce intestinal fluid secretion.4.Our study demonstrated the significance of TMEM16A in the gastrointestinal motility,inhibtion of TMEM16A reduced peristalsis of the intestine.CaCCs modulators identified in the present study have important theoretical significance in clarigying the functions of TMEM16A,CaCCGI and CFTR chloride channels in intestinal motility and fluid secretion under physiological and CaCCs chloride channel-related pathological?such as constipation and diarreha?conditions,they can be used to seperately analyze konwn and unknown CaCCs chloride channel activities.In addition,the present study provides new leading drugs and new strategies for the treatment of CaCCs-targeted rotaviral secretory diarrhea.