Activation of CFTR-mediated CI-Transport by Capsaicinoids in Cell Culture Model

Previous studies reported that capsaicin potentiates ΔF508 mutant cystic fibrosis transmembrane conductance regulator(CFTR) channel gating defect by transfected cell-based assays.It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients.We tried to reproduce and extend those pre-clinical data of previous studies.Cell-based fluorescence functional measurements in Fischer thyroid epithelial cells(FRT) expressing CFTR showed no effect of capsaicin on potentiating ΔF508-CFTR.while genistein showed a strongly positive activity.Studies show that capsaicin and dihydrocapsaicin activated cAMP-prestimulated wild-type CFTR in a dose-dependent manner with a maximal response of 70% of that activated by genistein,thus gave an apparent EC50 of (40.4±6.8)μmol/L and (150.2±7.4) μmol/L respectively.Preliminary study shows that the binding sites for capsaicin and dihydrocapsaicin may be probably partially overlapped with that for genistein because the maximal activation of wild-type CFTR with genistein is partially blocked by capsaicin and dihydrocapsaicin.     

Dictamine Stimulates Cystic Fibrosis Transmembrane Conductance Regulator CL Transport

Dictamine is a furoquinoline alkaloid isolated from Dictamus dasycarpus Turcz.In the present study,we found that dictamine is able to stimulate the chloride transport activity of wild-type and ΔF508 mutant CFTR.The activity is cAMP-dependent and can be completely reversed by specific CFTR inhibitor CFTRinh-172.In addition,dictamine can further increase the chloride transport activity when CFTR is maximally activated by the combination of cAMP stimulators forskolin(FSK)and IBMX,suggesting direct interaction of dictamine with CFTR.Dictamine may be useful for probing CFTR channel gating mechanisms and used as a lead compound to develop the pharmacological therapy of CFTR-related diseases such as idiopathic chronic pancreatitis and keratoconjunctivitis sicca and cystic fibrosis.     

Natural Compound Curcumin Corrects the Gating Defect of G551DMutant Cystic Fibrosis Transmembrane Conductance Regulator

In the present study, we identified the natural compound curcumin to be an effective G551D-CFTR activator by cell-based fluorescent assay and electrophysiological measurement. We demonstrated that curcumin can restore the impaired chloride conductance of G551D mutant CFTR. The activity is rapid, reversible, and cAMP-dependent. Our study identified a new natural lead compound for the pharmacological therapy of cystic fibrosis caused by G551D mutation of CFTR.     

20(S)-原人参二醇促进CFTR氯离子通道开放

利用氯离子通道细胞荧光测定模型对386种中药单体化合物进行筛选, 发现20(S)-原人参二醇对依赖于cAMP的CFTR氯离子通道具有激活作用. 20(S)-原人参二醇能够以剂量依赖的方式激活野生型CFTR氯离子通道, 其激活作用通过更为可靠的氯离子通道短路电流测定系统得到证实. 20(S)-原人参二醇对CFTR氯离子通道的激活效应具有作用迅速且可逆的特点. 其在发挥激活作用时依赖于腺苷环化酶激动剂Forskolin的存在, 单独与细胞孵育不提高细胞内cAMP的水平, 表明对CFTR氯离子通道的激活作用是通过与CFTR直接结合实现的. 该化合物对ΔF508-CFTR突变氯离子通道的开放也具有特征相似的激活作用.     

Activation of CFTR-mediated Cl- Transport by Capsaicinoids in Cell Culture Model

Previous studies reported that capsaicin potentiates F508 mutant cystic fibrosis transmembrane conductance regulator(CFTR) channel gating defect by transfected cell-based assays. It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients. We tried to reproduce and extend those pre-clinical data of previous studies. Cell-based fluorescence functional measurements in Fischer thyroid epithelial cells(FRT) e...     

Activation of G551D-CFTR by Bicyclooctane Compounds Is cAMP-dependent and Exhibits Low Sensitivity to Thiazolidinone CFTR Inhibitor CFTRinh-172

The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551 (G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane (BCO) G551D-CFTR activators(G551DBCOS) with Kd down to 1μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551DBCOS by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551DBCOS-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551DBCOS; (2) G551DBCOS and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.     

Natural Compound Curcumin-a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis(CF)is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator(CFTR)chloride channel.The most common point mutation △F508,which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90? patients.The natural compound curcumin was reported to correct the processing defect of △F508-CFTR and proposed as a potential therapeutic drug to cure CF.In the present study.we analyzed the efrect of curcumin on △F508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of △F508 mutant CFTR.The activity is rapid,reversible and cAMP-dependent.However,we couldn't reproduce the previously reported correction of the defective membrane trafficking of △F508-CFTR by curcumin.Therefore,curcumin may not be a superior lead compound for developing anti-CF drugs.     

Natural Compound Curcumin—— a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis（CF） is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator（CFTR） chloride channel. The most common point mutation AF508, which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90% CF patients. The natural compound curcumin was reported to correct the processing defect of AF508-CFTR and proposed as a potential therapeutic drug to cure CF. In the present study, we analyzed the effect of curcumin on AF508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of AF508 mutant CFTR. The activity is rapid, reversible and cAMP-dependent. However, we couldn＇t reproduce the previously reported correction of the defective membrane trafficking of AF508-CFTR by curcumin. Therefore, curcumin may not be a superior lead compound for developing anti-CF drugs.     

Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

A thiazolidinone CFTR inhibitor (CFTRinh-72) was synthesized by a three-step procedure with tri-fluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally bymeans of ^1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluo-rescent and electrophysiological methods. A large amount(100g) of high-quality small molecule thiazolidi-none CFTR chloride channel inhibitor, CFTRinh-72, can be produced with this simple three-step synthetic pro-cedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl )-5-[4-carboxyphenyl-methylene]-4-thiazolidinone was confirmed by ^1H NMR. The overall yield was 58% with a purity over 99%as analyzed by HPLC. The synthesized CFTRinh-72 specifically inhibited CFTR chloride channel function in acell-based fluorescence assay(Kd≈1.5μmol/L) and in a Ussing chamber-based short-circuit current assay(Kd≈0. 2μmol/L), indicating better quality than that of the commercial combinatorial compound. The syn-thesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The syn-thetic procedure developed here can be used to produce a large amount of the high-quality CFTRinh-72 suitablefor antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be usedto synthesize radiolabled CFTRinh-72 for in νiνo pharmacokinetics studies.     

Increased Sensitivity of Porcine ΔF508-CFTR Chloride Channel to Specific Small Molecule CFTR Inhibitors

Deletion of the codon encoding the phenylalanine residue at position 508(ΔF508) in the cystic fibrosis transmembrane conductance regulator(CFTR) is the most common mutation causing cystic fibrosis(CF).The human ΔF508 mutation results in a CFTR protein with impaired folding,trafficking,and gating in human and rodents.Recent studies suggest that pig ΔF508-CFTR can be efficiently processed to the plasma membrane as maturely glycosylated protein,indicating species difference of the molecular mechanisms of CFTR ...     

Visual DNA as a diagnostic tool

We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin‐coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE‐based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand‐held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off‐the‐shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point‐of‐care tests.     

Evaluation of Fused Pyrrolothiazole Systems as Correctors of Mutant CFTR Protein

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors.     

Identification of Herbal Compound Imperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

Calcium-activated chloride channels(CaCCs) are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A(ANO1 or anoctamin-1) is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epi...     

Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide–nanoparticle conjugates

Novel methods for application of oligonucleotide–gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide–gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for F 508, M470V, R74W and R75Q mutations.     

Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

Introduction Aloe,cascaraandsennaarewidelyusedasall purposelaxativemedicine.Itisgenerallyregardedthat thecatharticingredientsofaloe,cascaraandsennaare anthraquinoneandtheirderivatives[1].Ithasbeenwell definedthattheincreasedleakingofplasmaintointesti nall…     

Detection of the R553X DNA single point mutation related to cystic fibrosis by a "chiral box" D-lysine-peptide nucleic acid probe by capillary electrophoresis

In order to recognize the presence of the R553X point mutation of the cystic fibrosis (CF) gene in the human genome, a peptide nucleic acid (PNA) complementary to the mutated gene tract and bearing three adjacent chiral monomers based on D-lysine (chiral box) was synthesized and used as a probe in CE. Binding specificity was preliminarily studied with complementary and mismatched oligonucleotides by UV spectroscopy, electrospray MS, and electrophoresis, indicating a very high sequence selectivity. The chiral PNA probe was then hybridized to cyanine-5-labeled DNA samples (186 bp), obtained by PCR amplification, respectively, from: (a) normal homozygous subjects (wtDNA), (b) CF-affected homozygous subjects (mutDNA), (c) heterozygous subjects (healthy carriers) and denatured at low ionic strength. The PNA-DNA mixture was directly analyzed by CE with LIF detection: a new signal corresponding to the PNA-mutDNA duplex was observed, in the case of CF-affected homozygous subjects, whereas for the sample containing the mismatched sequence (normal homozygous wtDNA) only the signal corresponding to ssDNA (ss, single strand) was detected. In the case of heterozygous DNA, both PNA-mutDNA duplex and ssDNA were detected. With this simple assay, it was possible to discriminate in an easy way among the three cases (mutated homozygous, normal homozygous, and heterozygous subjects) with a total specificity, thus allowing a decisive advance for the diagnosis of CF.     

Influence of sialic acid and bacterial sialidase on differential adhesion of Pseudomonas aeruginosa to epithelial cells

Epithelial cell lines from several tissues show a differential sensitivity to Pseudomonas aeruginosa adherence. A549 (lung), HepG2 (liver) and Caco-2 (colon) cells presented an adhesion index of about 3, 1.5 and 5 CFU/cell, respectively, whereas Mz-Ch cell lines (gallbladder cholangiocytes) presented adhesion indexes up to 35. These variations could be associated with the variable amount of sialic acid in cell surface glycoconjugates. Moreover, the presence of free sialic acid in culture media induces the secretion by P. aeruginosa of a sialidase which is able to hydrolyze glycoconjugate-linked sialic acid. As shown with A549 cells, this specific hydrolysis increases bacterial adhesion, probably by unmasking new binding sites onto the cell surface.