Identification Of Resveratrol Oligomers As Inhibitors Of Cystic Fibrosis Transmembrane Conductance Regulator By High-throughput Screening Of Natural Products From Chinese Medicinal Plants

Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) have beenwidely used for characterizing CFTR function in epithelial fluid transport and in diseases suchas secretory diarrhea, polycystic kidney disease and cystic fibrosis. High potent CFTRspecified inhibitor is still the most important tool in CFTR function studies up to now. Due tolow specificity, poor tissue distribution property and/or bad in vivo activity, none of theinhibitors identified so far was proved to fully meet the needs of CFTR functional studies invivo. Big progress has been achieved in identification of CFTR selective small moleculemodulator, and still is one of the most highlighted task in the field.In the present study, we used a high-throughput screening (HTS)-based naturalcompound discovery strategy to systematically identify natural compounds that can inhibitCFTR activity from Chinese medicinal herbs. By screening40,000small molecule fractionsfrom500herbal plants, we identified42active fractions from5herbs and isolated twocompounds from Chinese wild grapevine (Vitis amurensis Rupr.) that inhibited CFTRconductance. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) studiesdetermined the two active compounds as trans-ε-viniferin (TV) and r-2-viniferin (RV),respectively. Both compounds dose-dependently inhibited CFTR-mediated iodide influx withIC50around20M (wt-CFTR),10μM (ΔF508-CFTR),10μM and3μM (G551D-CFTR).Further analysis by excised inside-out patch-clamp indicated that TV and RV significantlydecreased protein kinase A (PKA)-activated CFTR chloride currents. Representative traces ofCFTR from an excised inside-out patch showing reversible inhibition of the channel activityby TV and irreversible inhibition of the channel activity by RV. In ex vivo studies, TV andRV inhibited short-circuit currents in isolated rat colonic mucosa in a dose-dependent manner,and only mucosal applications of the compounds were effective, which suggested that thecompounds block CFTR Clˉconductance by occluding the CFTR anion pore at or near theexternal membrane surface. In a closed-loop model, intraluminal application of TV (2.5g)and RV (4.5g) significantly reduced cholera toxin–induced intestinal fluid secretion,suggesting their potential pharmacological use in the treatment of secretory diarrhea. TV andRV inhibited the fluid secretion in mouse trachea gland activated by pilocarpin or osthole. Theanalysis of protein species in trachea submucosal gland fluid secretions showed that there were merely differences among mucous secreted under different conditions. TV and RVincreased protein concentration in secreted fluid.In conclusion, inhibitory effects of TV and RV on CFTR Clˉcurrents and epithelial Clˉsecretion provided a new molecular mechanism for some of their health benefits includingantidiarrheal properties. CFTR channel inhibition adds to the list of resveratrol-derivedoligomers molecular targets and may account for some of its biological activities. The presentstudy validates our natural product high throughput screening method as an effective strategyto identify low abundance active compounds from natural products such as herbal plants.