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—— 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.     

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.     

Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.     

The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the DeltaF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of 'corrector' drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Solubilizing mutations used to crystallize one CFTR domain attenuate the trafficking and channel defects caused by the major cystic fibrosis mutation

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) Cl(-) channel. F508del, the most frequent CF-causing mutation, disrupts both the processing and function of CFTR. Recently, the crystal structure of the first nucleotide-binding domain of CFTR bearing F508del (F508del-NBD1) was elucidated. Although F508del-NBD1 shows only minor conformational changes relative to that of wild-type NBD1, additional mutations (F494N/Q637R or F429S/F494N/Q637R) were required for domain solubility and crystallization. Here we show that these solubilizing mutations in cis with F508del partially rescue the trafficking defect of full-length F508del-CFTR and attenuate its gating defect. We interpret these data to suggest that the solubilizing mutations utilized to facilitate F508del-NBD1 production also assist folding of full-length F508del-CFTR protein. Thus, the available crystal structure of F508del-NBD1 might correspond to a partially corrected conformation of this domain.     

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.     

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.     

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.     

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.     

Detection of the DeltaF508 mutation in the CFTR gene by means of time-resolved fluorescence methods.

A rapid recognition in the base sequence of nucleic acids is an important prerequisite toward the diagnosis of genetic diseases and their carrier states. We have developed a hybridisation method in which a fluorescently labeled oligonucleotide is used to detect point mutations in a target by a simple fluorescence lifetime analysis of the emission of the fluorescent label. We applied this method to detect the deltaF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a model system and with biologically derived PCR product and discuss the potential generality of this method.     

Identification of a NBD1-binding pharmacological chaperone that corrects the trafficking defect of F508del-CFTR

Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.     

Identification of the commonest cystic fibrosis transmembrane regulator gene DeltaF508 mutation: evaluation of PCR--single-strand conformational polymorphism and polyacrylamide gel electrophoresis

In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.     

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.     

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.     

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.     

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...