Discovery And Molecular Pharmacology Of Capsaicinoid TMEM16A Inhibitor

Calcium-activated chloride channels（CaCCs）are a class of anion channels that are widely expressed in mammalian cells.Participate in a variety of physiological processes such as transepithelial fluid secretion,smooth muscle contraction,and sensory signal transduction.TMEM16A（transmembrane protein 16A）is the first confirmed CaCC that is expressed in epithelial cells of the respiratory tract,salivary glands,and other tissues as well as arterial smooth muscle cells,intestinal pacemaker cells,sensory neurons,and various tumor cells.TMEM16A chloride channel modulator plays an important role in the study of its gating mechanism,pathophysiological function,etc.However,the regulators have been found to have a large number of deficiencies in affinity,selectivity and in vivo activity.Therefore,the discovery of TMEM16A modulator Still the focus of research in related fields.In this study,a fluorescence quenching function screening model（FRT/TMEM16A）was established using a Fischer rat thyroid epithelial（FRT）cell line stably transfected with human TMEM16A and a green fluorescent protein mutant YFP-H148Q/I152L highly sensitive to halogen elements./YFP-H148Q/I152L),using this model to screen compounds with inhibitory activity against the TMEM16A chloride channel from more than 500 natural small molecules.Fluorescence quenching experiments and short-circuit current experiments were used to analyze the basic molecular pharmacological properties of active compounds,using mouse colonic tissue short-circuit current experiments,mouse intestinal smooth muscle contraction experiments,mouse gastrointestinal peristalsis experiments,mouse total intestinal transport Time measurement experiments and a rat rotavirus diarrhea model were performed for in vitro and in vivo activity analysis.The experimental results are as follows:1.Using FRT/TMEM16A/YFP-H148Q/I152L assay model to screen four capsaicin-like TMEM16Achloridechannelinhibitors,namelycapsaicin,dihydrocapsaicin,nordihydrocapsaicin and homodihydrocapsaicin I,IC50 The values were 51.59μM,47.04μM,57.97μM,and 24.85μM,respectively.The inhibitory effects of capsaicin,nordihydrocapsaicin and homodihydrocapsaicin I on the chloride channel of TMEM16A are rapidly irreversible,while the above activities of dihydrocapsaicin show rapid and reversible characteristics.2.Short-circuit current results on FRT/TMEM16A/YFP-H148Q/I152L showed that capsaicin,dihydrocapsaicin,nordihydrocapsaicin and homodihydrocapsaicin I were all in the dose-dependent manner on the apical membrane side and the substrate.The maximum inhibition efficiencies on the apical membrane side were 87.3%,97.1%,84.8%,and 91.3%,respectively.The corresponding compound concentrations were 50μM,50μM,50μM,and20μM,respectively,and the IC₅₀0 values were approximately 27.34μM and 24.71μM,respectively.29.54μM,10.47μM,their affinity to TMEM16A from high to low is:capsaicin>homodihydrocapsaicin I>dihydrocapsaicin>nordihydrocapsaicin;the maximum inhibition efficiency of the basement membrane side is 85.7%,93.3%,88.8%,97.1%,the corresponding compound concentrations are 100μM,50μM,100μM and 50μM,respectively,IC₅₀0 values are about 56.94μM,27.82μM,97.65μM,28.43μM,respectively,their affinity for TMEM16A The order of high to low is:capsaicin>norhydrocapsaicin>dihy drocapsaicin>homodihydrocapsaicin I.The same compound has a lower inhibitory effect on the basement membrane side than the apical membrane side on the basement membrane side and the apical membrane side of TMEM16A.3.The results of short-circuit currents on FRT/CFTR/YFP-H148Q/I152L cells showed that capsaicin and dihydrocapsaicin had an activation effect on CFTR,and this activation had an additive effect on the activation of CFTR by FSK.Short-circuit current measurements on T84 cells showed that capsaicin,dihydrocapsaicin,dihydrocapsaicin and dihydrocapsaicin inhibited the current induced by FSK on T84.4.Short-circuit currents on HT-29 cells showed that capsaicin,dihydrocapsaicin,nordihydrocapsaicin and homodihydrocapsaicin I inhibited the expression of a Ca²⁺-activated chloride ion with unknown molecular identity on the cell.Current(CaCC_gie).The maximum inhibition rates were 78.9%,84.1%,75.4%,and 89.6%.The corresponding compound concentrations were 50μM,50μM,100μM,and 100μM,respectively.5.The results of selective analysis showed that the above four capsaicinoids did not inhibit Na⁺-K⁺-ATPase on the serosal side of mouse colonic epithelium at 200μM;the above four capsaicinoids significantly inhibited K⁺channels at 100μM.Inhibition rate was 51.2%,74.3%,57.2%,83.1%;50μM capsaicin,dihydrocapsaicin,dihydrocapsaicin and 20μM dihydrocapsaicin partially inhibited Ca²⁺concentration in HT-29 cells,inhibition rate It is82.3%,76.9%,64.1%,and 61.2%.6.The results of short-circuit current measurement of colonic mucosa in mice showed that capsaicin,dihydrocapsaicin,nordihydrocapsaicin and homodihydrocapsaicin I had no inhibitory effect on CFTR-mediated chloride current in mouse colonic mucosa.However,these compounds inhibited the chloride ion current induced by carbachol（Cch）in the mucosal epithelium of mice:50μM and 100μM capsaicin inhibited currents of 28.7%and 44.8%,respectively,50μM and 100μM of dihydrocapsaicin inhibited 32.2%and 68.9%of current,respectively,100μM and 200μM of nordihydrocapsaicin inhibited currents by 35%and 40%,respectively,and 100μM and 200μM of homodihydrocapsaicin I inhibited 53.3%,respectively.And 66.7%of the current.7.Immunohistochemical analysis showed that TMEM16A was highly expressed in the jejunum,ileum,distal colon and proximal colon smooth muscle of mice.8.The results of mouse intestinal smooth muscle contraction showed that the above four capsaicinoid compounds reduced the contractile strength of smooth muscle in different degrees at 100μM,and homodihydrocapsaicin I had no effect on the contraction frequency of smooth muscle,while capsaicin and dihydrocapsaicin And dihydrocapsaicin significantly reduced the frequency of smooth muscle contraction,and the above-mentioned contraction frequency was reduced by 27%,18%,and 23%compared with the control.9.The results of gastrointestinal peristalsis in mice showed that the above four capsaicinoid compounds inhibited gastrointestinal peristalsis in mice,and the peristaltic ratio was 30%,72.8%,58.1%,and 72.5%,respectively.10.The results of mouse total intestinal transit time experiments showed that the above four capsaicinoid compounds prolonged the total intestinal transit time of mice.Compared with the PBS control group,the total intestinal transit time was extended by 2.77h,0.77h,0.81h,and 2.43h,respectively.11.Rotavirus-induced model of diarrhea in young rats showed that dihydrocapsaicin and homodihydrocapsaicin I could significantly inhibit the occurrence of diarrhea caused by rotavirus.The inhibitory effect of high homodihydrocapsaicin I was better than that of dihydrocapsaicin.homodihydrocapsaicin I significantly inhibited gastrointestinal peristalsis in the test rats（inhibition rate 43.1%）,while dihydrocapsaicin had no significant effect on gastrointestinal motility in the young rats.In summary,this study found new activities of TMEM16A chloride channel inhibitors and capsaicinoids,and laid the foundation for further revealing the pharmacological activity of capsaicinoids,targeting TMEM16A.Lead compounds are provided for the treatment of diseases.