A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmem-brane conductance regulator (CFTR) is one of the five most frequent cystic fibrosis (CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551DCFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.     

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.     

A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify smallmolecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation,     

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.     

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-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.     

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 Magnolin

Magnolin is a herbal compound from Magnolia biondii Pamp. It possesses numerous biological activities. Cystic fibrosis transmembrane conductance regulator（CFTR） is an epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs. In the present study, the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized. In CFTR stably transfected FRT cells, magnolin increases CFTR CI- currents in a concentration-dependent manner. The activation of magnolin on CFTR is rapid, reversible, and cAMP-dependent. Magnolin does not elevate cellular cAMP level, indicating that it activates CFTR by direct binding and interaction with CFTR protein. Magnolin selectively activates wildtype CFTR rather than mutant CFTR. Magnolin may present a novel class of therapeutic lead compound tbr the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca, idiopathic chronic pancreatiti, and chronic constipation.     

Evaluation of 1,2,3-Triazoles as Amide Bioisosteres In Cystic Fibrosis Transmembrane Conductance Regulator Modulators VX-770 and VX-809

The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11 , derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.     

Synthesis and characterization of a photoaffinity labelling probe based on the structure of the cystic fibrosis drug ivacaftor

Cystic Fibrosis (CF) is a genetic disorder caused by loss-of-function mutations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Ivacaftor (1) was the first therapeutic approved for the treatment of CF that is able to restore gating activity to certain CFTR variants although the mechanism of action is poorly understood. Herein we describe the synthesis of a photoaffinity labelling (PAL) probe (2) based on the structure of ivacaftor incorporating a photoreactive diazirine moiety for use in labelling studies designed to identify the binding site for ivacaftor on mutant CFTR. The PAL probe 2 retained potentiation activity, with a potency similar to 1, using a Fluorescent Imaging Plate Reader (FLIPR^®) assay measuring ion conductance potentiation of wild type (Wt)-CFTR. Photolabelling experiments with human serum albumin (HSA) as a model protein have shown that probe 2 can label HSA in a manner consistent with observed and predicted binding.     

Cystic fibrosis: CFTR correctors to the rescue

Cystic fibrosis transmembrane conductance regulator (CFTR) correctors are small molecules that target the most common cause of cystic fibrosis: misfolded F508del-CFTR. Using differential scanning fluorimetry, Sampson et?al. (2010) identify a CFTR corrector that interacts directly with the CFTR domain affected by the F508del mutation.     

Rational coupled dynamics network manipulation rescues disease-relevant mutant cystic fibrosis transmembrane conductance regulator

Many cellular functions necessary for life are tightly regulated by protein allosteric conformational change, and correlated dynamics between protein regions has been found to contribute to the function of proteins not previously considered allosteric. The ability to map and control such dynamic coupling would thus create opportunities for the extension of current therapeutic design strategy. Here, we present an approach to determine the networks of residues involved in the transfer of correlated motion across a protein, and apply our approach to rescue disease-causative mutant cystic fibrosis transmembrane regulator (CFTR) ion channels, ΔF508 and ΔI507, which together constitute over 90% of cystic fibrosis cases. We show that these mutations perturb dynamic coupling within the first nucleotide-binding domain (NBD1), and uncover a critical residue that mediates trans-domain coupled dynamics. By rationally designing a mutation to this residue, we improve aberrant dynamics of mutant CFTR as well as enhance surface expression and function of both mutants, demonstrating the rescue of a disease mutation by rational correction of aberrant protein dynamics.     

Electrochemical Immunosensors for the Rapid Screening of Cystic Fibrosis and Duchenne Muscular Dystrophy

Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well characterized progressive inherited diseases associated with significant morbidity and mortality. Therefore, the early, rapid and affordable diagnosis of these disorders through newborn screening is highly important for the appropriate management. Here, we report label‐free impedance immunosensors for the simple screening of CF and DMD through the detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein fragment and a peptide sequence for dystrophin (DMD). The biosensors were constructed by the covalent immobilization of specific antibodies for CFTR and DMD on standard gold (Au) electrodes. The immunosensors response was measured based on the change in the electrochemical impedance spectroscopy (EIS) signals after binding with the peptides. The specific recognition of the immunosensor surfaces to the target antigens leads to retardation of the access of ferri‐ferrocyanide redox molecules to the surface and thus, enhances the charge transfer resistance (R_ct). These impedimetric immunosensors enabled sensitive, fast, selective and accurate estimation of CFTR and DMD levels within a linear range from 1.0 pg/mL to 1 μg/mL and 1.0 pg/mL to 10 ng/mL with lower detection limits of 0.8 and 0.7 pg/mL for CFTR and DMD, respectively. Moreover, the immunosensors were tested for the detection of CFTR and DMD in human serum showing very good agreement with enzyme‐linked immunosorbent assays (ELISA). This work represents a novel low cost analytical method that aims to satisfy the unmet public health need in the early diagnosis of CF and DMD and can be extended to detect other hereditary disorders.     

Effect of Alpha-1 Antitrypsin on CFTR Levels in Primary Human Airway Epithelial Cells Grown at the Air-Liquid-Interface

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.     

Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell‐penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell‐permeable peptide substrate for real‐time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR‐associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis.     

Detection of CFTR mutations using temporal temperature gradient gel electrophoresis

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common autosomal recessive diseases with variable incidences and mutation spectra among different ethnic groups. Current commercially available mutation panels designed for the analysis of known recurrent mutations have a detection rate between 38 to 95%, depending upon the ethnic background of the patient. We describe the application of a novel mutation detection method, temporal temperature gradient gel electrophoresis (TTGE), to the study of the molecular genetics of Hispanic CF patients. TTGE effectively identified numerous rare and novel mutations and polymorphisms. One interesting observation is that the majority of the novel mutations are splice site, frame shift, or nonsense mutations that cause severe clinical phenotypes. Our data demonstrate that screening of the 27 exons and intron/exon junctions of the CFTR gene by TTGE greatly improves the molecular diagnosis of Hispanic CF patients.     

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.