Identification And Characterization Of Alkaloid CFTR Activators

CFTR(cystic fibrosis transmembrane conductance regulator) is a cAMP dependent chloride channel. It is extensively expressed by epithelial cells lined airway, gastrointestinal tract, pancreatic, sweat gland, testis,et al, and plays key roles in transport of electrolyte and fluid. The abnormal activity of CFTR chloride channel is related to pathogenesis of several diseases. Malfunction will result in cystic fibrosis, chronic pancreatitis, male sterility, habitual constipation, keratoconjunctivitis sicca (KCS, also called dry eye syndrome). Over activation of CFTR is related to diarrhea, polycystic kidney disease (ADPKD). High affinity CFTR regulators are important in studying CFTR-related diseases and mechanisms involved.The purpose of this study is to identify natural alkaloid compounds that can stimulate activation of wild-type human CFTR (wt-CFTR) chloride channel and molecular pharmacological mechanisms related. An FRT cell line stably coexpressing wt-CFTR and the high halide sensitive YFP (YFP-H148Q) cell based assay were used in the study. 14 alkaloids that can stimulate iodide transport mediated by wt-CFTR were identified from 46 natural alkaloids, among which dihydrocapsaicin, capsaicin, theophylline, and papaverine HCl exhibit the highest activities. In this paper molecular pharmacological properties of these compounds were systematically investigated. We found that (1) dihydrocapsaicin, capsaicin can dependently actived wt-CFTR, with affinities of 152±7.4μ?Μand 40.4±6.8, respectively; with maximal potencies of 0.4 mM/S and 0.5 mM/S, respectively. None pf the compounds stimulatedΔF508-CFTR mediated iodide influx or corrected its misprocessing defect. Further investigation manifested that the capsaicinoids might share common binding site in CFTR with genbistein, known CFTR activator. CFTR activation activity might account part of laxative effect of capsaicinoids. (2) Theophylline also activated CFTR choloride channel, with Kd and Vmax values were 130±1.27μ?M and 0.57±0.04 mM/S, respectively. Theophylline had no effect on the most common CFTR mutation forms of CFTR (ΔF508-CFTR and G551D-CFTR), and it could not correct the misprocessing defect ofΔF508-CFTR. The relationship between CFTR activation activity of theophylline and its multiphysiological effect is not clear. (3) Papaverine HCl could stimulate both wt CFTR andΔF508-CFTR chloride channels, but not G551D-CFTR chloride channel, with Vmax values are 0.85 mM/s and 0.08 mM/s, respectively; Kd values are 20μM and 40μM, respectively. Initial studies showed that papaverine HCl did not share common binding site in CFTR with genbistein.We identified several CFTR activators from natural alkaloid compounds and systematically investigated their pharmacological properties. Our results integrated (in part) and supplemented activities of alkaloid compounds. Molecular pharmacological studies of several alkaloids (dihydrocapsaicin, capsaicin, theophylline, and papaverine HCl) showed that CFTR protein might be one of the molecule drug targets of these compounds.