Discovery And Pharmacological Study Of Cystic Fibrosis Transmembrane Conductance Regulator Activators From Traditional Chinese Medicine

CFTR( cystic fibrosis transmembrane conductance regulator) is a chloride channel regulated by cAMP. It is expressed by epithelium lined airway, gastrointestinal tract, pancreatic, sweat gland, testis,et al, and involved in transport of electrolyte and fluid. The abnormal activity of CFTR chloride channel is related to pathogenesis of several diseases. Disfunction will result in cystic fibrosis, chronic pancreatitis, male sterility,constipation, et al. Overactivity has important role in the generation of diarrhea, ADPKD. So the modulators of this ion channel is valuable to the theraphy of these diseases.One purpose of this paper is to identify natural compounds from Chinese herbs that can stimulate the activity of wt-hCFTR chloride channel and to characterize their molecular pharmacological activity systematically. We use a FRT cell line stably coexpressing wt-hCFTR and a ultra-high halide sensitive (YFP-H148Q) EYFP and find 14 compounds which can stimulate iodide transport mediated by wt-CFTR from 386 natural compounds. They belong to coumarin, alkaloid, flavonoid and terpenoid and the most potential compounds are coumarins. Among them, EC50 value of osthole is only 100nM/L and is the most high affinity natural activator for wt-hCFTR at present. Osthole can stimulate the fluid secretion of submucosal gland at mouse pars laryngea pharyngis and elevate short circuit current across rat colonic mucosal epithelium. These results show the new pharmacological action of osthole and evaluate its potential therapeutic value for chronic pancreatitis and male sterility. We identified several anthraquinone compounds as wt-CFTR activators, as as Rhein, Aloe-emodin. They are also the effective constituents of cathartic herbs, but it is not clear for its definite target as catharsis. Our detailed study prove that the direct activation of Rhein to wt-CFTR is one mechanism of rhubarb as eliminate.Another purpose of this paper is to identify natural compounds from Chinese herbs that can correct defectiveΔF508-CFTR channel gating and membrane trafficking and also to evaluate their potential as therapeutic agents. CFTR mutations result in cystic fibrosis disease which influences functions of many organs. ?F508-CFTR is the most common mutation and is present in at least one allele in 90% of CF patients. Deletion of phenylalanine at 508 position causes two distinct defects in CFTR: (i) defect trafficking to plasma membrane and (ii) impaired intrinsic Cl- conductance (reduced open channel probability) after reaching cell surface.At present, several compounds correcting the function of ?F508-CFTR have been found,but most of them belong to synthetic compounds and are difficult to be developed as medicine. We use a FRT cell line stably coexpressingΔF508-CFTR and a ultra-high halide sensitive (YFP-H148Q/I152L) EYFP and find 14 compounds which can facility iodide transport mediated byΔF508-CFTR from 386 natural compounds. They belong to coumarin, alkaloid, flavonoid and terpenoid and the most potential compounds are coumarins. Pharmacological properties of these compounds were characterized systematically. Imperatorin is the most potential compound and EC50 is 15μM/L. Their action is dependent on forskolin, action quickly and reversibly. We suppose these potentiators activate ?F508-CFTR by directly binding, because they cannot elevate cAMP concentration and have additive effection with mixture of forskolin and IBMX. After structure and activity relationship analysis, we find the hydrophobicity of substitutions at six, seven, eight postion together decides the strong or weak activity. Additionally, we screen correctors of ?F508-CFTR and find Chrysin at higher concentration can promote ?F508-CFTR trafficking to plasma membrane at some extend. By larger screening or optimized the structure of Imperatorin and Chrysin, better activity and higher affinity potentiators and correctors should be found and as candidate for CFTR related disease therapy.We identify several CFTR potentiators from natural Chinese medicine compounds and identify their pharmacological activity. These compounds are very valuable to treat cystic fibrosis and other CFTR related diseases. Our study also affords a theory basis for the utilization of traditional Chinese medicine in modern drug discovery.