Discoveryand Molecular Pharmacology Studies Of Flavonoid TMEM16A Inhibitors

Calcium-activated chloride channel is an anion channel protein widely expressed in mammalian cells.It has important physiological functions in transepithelial transport,smooth muscle contraction,fertilization,olfactory conduction,neuronal excitation and repolarization of cardiac action potential.TMEM16A is the first confirmed CaCC expressed in vascular smooth muscle cells,respiratory and digestive tract epithelial cells,interstitial cells of cajal（ICC）,sensory neurons and various tumor cells.Selective modulators are important pharmacological tools for studying ion channels.Some CaCCs selective modulators（including inhibitors and activators）have been discovered,but due to low selectivity,chemical instability and poor activity in vivo,The need for in vivo functional studies of CaCCs is not fully met.In this study,a functional fluorescence assay cell model（FRT-TMEM16A-YFP-H148Q/I152L）was used to screen compounds with inhibitory activity against TMEM16A chloride channel from more than 500 natural small molecules,and activity was analyzed by fluorescence quenching experiments and short-circuit current experiments.The basic molecular pharmacological properties of the compounds were analyzed by in vitro and in vivo activities of the mouse colonic tissue short-circuit current test,mouse intestinal smooth muscle contraction test,mouse gastrointestinal peristalsis test and rat vascular ring tension test.Experimental results:1.Using FRT/TMEM16A/YFP-H148Q/I152L assay model,four flavonoids TMEM16A chloride channel inhibitors were obtained,which were rhododendrin,isorhamnetin,lycopene and baicalein,and the IC₅₀0 value was about 98.83μM.40.87μM,49.63μM,44.8μM.2.Short-circuit current measurements on FRT-TMEM16A-YFP-H148Q/I152L showed that rhododendrin,isorhamnetin,sarsin,and baicalein inhibited TMEM16A/CaCC-mediated chloride ion in a dose-dependent manner.The current and maximum inhibition efficiencies were 90%,73.9%,76.2%,and 91.3%,respectively,and IC₅₀0 values were approximately 49.7μM,20.83μM,24.5μM,27.3μM,and 20.1μM,respectively.3.Fluorescence quenching assay on FRT-CFTR-YFP-H148Q/I152L showed that rhododendrin,isorhamnetin,sarsin,and baicalein activated CFTR-mediated Cl^-current with EC₅₀0 values of 95.06μM,13.24μM,87.36μM,50.47μM.Short-circuit current measurement analysis showed that the activation of CFTR-mediated Cl^-current by rhododendrin and baicalein at low concentrations（2⁵0μM）,and the modulation of CFTR by rhododendrin and baicalein at high concentrations（100²00μM）The Cl^-current has an inhibitory effect,and isorhamnetin and sarsinol have an inhibitory effect on CFTR-mediated chloride current.On T84 cells,rhododendrin and sarcopidine were identical in results to FRT-CFTR cells.At low concentrations（2⁵0μM）,rhododendrin activates CFTR-mediated Cl^-current,while at high concentrations（100²00μM）,rhododendrin has an inhibitory effect on CFTR-mediated Cl^-current.It inhibits CFTR-mediated chloride current,and isorhamnetin（50²00μM）has an inhibitory effect on CFTR-mediated Cl^-current.The short-circuit current of colonic mucosa in mice showed that rhododendrin,isorhamnetin,sarcopine,and baicalein had no effect on CFTR-mediated chloride currents in mouse colonic epithelium.4.Fluorescence assay on HT-29 cells showed that isorhamnetin and high concentrations of rhododendrin inhibited CaCC-mediated Cl^-current with IC₅₀0 values of 29.57μM and 58.67μM,respectively.The sulphonin and baicalein have an activation effect on CaCC-mediated Cl^-current,and the EC₅₀0 values are 40.57μM and 50.16μM,respectively.Analysis of short-circuit currents on HT-29 and T84 cells showed that rhododendrin showed activation of CaCCs chloride channels at low concentrations（2²0μM）,while high concentrations（50²00μM）showed inhibition.Isorhamnetin showed an inhibitory effect in the concentration range of 10²00μM,and actin and xanthin showed activation in the concentration range of 5²00μM.5.The results of selective analysis showed that the above four flavonoids did not inhibit Na⁺-K⁺-ATPase on the serosal side of mice in the range of 10²00μM,but had significant inhibitory effect on K⁺channel,and the inhibition rate was 61.2.%,54.3%,57.6%,82.1%.6.The results of mouse colonic mucosal short-circuit current showed that 100μM and200μM of rhododendron could inhibit the chloride current induced by carbachol（CCh）in the colonic mucosa of mice,inhibiting 8.7%and 24.8%of current,respectively.100μM and 200μM isorhamnetin inhibited 32.2%and 48.9%,respectively,and there was almost no change in100μM and 200μM.The 100μM and 200μM baicalein inhibited 6%and 21.7%,respectively current.7.The results of the whole intestinal transit time of mice showed that the above flavonoids could prolong the transit time of the whole intestine,which was 70%,65.6%,56.4%and 64.5%,respectively,compared with the control group.8.The results of mouse intestinal smooth muscle contraction showed that 100μM rhododendrin,isorhamnetin,sarcopine and baicale significantly reduced the contraction frequency of smooth muscle and inhibited the contraction intensity.9.Rat vascular ring test results showed that isorhamnetin（50³00μM）can inhibit the increase of vascular smooth muscle contraction caused by norepinephrine（PE）.10.Tumor cell viability and migration experiments showed that rhododendron（1.5625²00μM）inhibited cell viability on both SW620 and NCM460 cells.Rhododendron（200μM）had the best inhibitory effect on NCM460,and it had the best inhibition effect on SW620 at 50μM.Isorhamnetin（12.5μM）had the best inhibitory effect on NCM460,and inhibited SW620 at 3.125μM.Both azadirachtin and isorhamnetin can inhibit the migration of SW620 cells.Conclusion:This study found that the four flavonoids of rhododendrin,isorhamnetin,sarcopine and baicalein can significantly inhibit the chloride channel activity of TMEM16A,which lays a foundation for further revealing the physiological pharmacological activity of flavonoids.Drug therapy for pulmonary hypertension,tumors,slow transit constipation and other related diseases provides new strategies and lead compounds.