SLC26A9 stimulates CFTR expression and function in human bronchial cell lines

We investigated the possible functional‐ and physical protein‐interactions between two airway Cl^? channels, SLC26A9 and CFTR. Bronchial CFBE41o‐ cell lines expressing CFTR_WT or CFTR_ΔF508 were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole‐cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR_WT and CFTR_ΔF508 cell lines. In CFBE41o‐ cells and CFBE41o‐/CFTR_WT cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)‐stimulated currents were enhanced in SLC26A9‐transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR_(inh)‐172 or GlyH‐101 inhibition in both groups). In CFBE41o‐/CFTR_ΔF508 cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK‐stimulated currents and level of CFTR_(inh)‐172 inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK‐stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK‐stimulated currents was not found in oocytes co‐expressing SLC26A9 and CFTR_ΔF508. In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR_ΔF508. Our co‐immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9‐STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents. J. Cell. Physiol. 226: 212–223, 2010. © 2010 Wiley‐Liss, Inc.     

ΔF508-CFTR correctors: Synthesis and evaluation of thiazole-tethered imidazolones,oxazoles, oxadiazoles,and thiadiazoles

The most common mutation causing cystic fibrosis (CF) is deletion of phenylalanine residue 508 in the cystic fibrosis transmembrane regulator conductance (CFTR) protein. Small molecules that are able to correct the misfolding of defective ΔF508-CFTR have considerable promise for therapy. Reported here are the design, preparation, and evaluation of five more hydrophilic bisazole analogs of previously identified bithiazole CF corrector 1. Interestingly, bisazole ΔF508-CFTR corrector activity was not increased by incorporation of more H-bond acceptors (O or N), but correlated best with the overall bisazole molecular geometry. The structure activity data, together with molecular modeling, suggested that active bisazole correctors adopt a U-shaped conformation, and that corrector activity depends on the molecule’s ability to access this molecular geometry.     

Design and synthesis of a hybrid potentiator–corrector agonist of the cystic fibrosis mutant protein ΔF508-CFTR

A developing therapy of cystic fibrosis caused by the ΔF508 mutation in CFTR employs correction of defective CFTR chloride channel gating by a ‘potentiator’ and of defective CFTR protein folding by a ‘corrector’. Based on SAR data for phenylglycine-type potentiators and bithiazole correctors, we designed a hybrid molecule incorporating an enzymatic hydrolysable linker to deliver the potentiator (PG01) fragment 2 and the corrector (Corr-4a) fragment 13. The hybrid molecule 14 contained PG01-OH and Corr-4a–linker–CO₂H moieties, linked with an ethylene glycol spacer through an ester bond. The potentiator 2 and corrector 13 fragments (after cleavage) had low micromolar potency for restoration of ΔF508-CFTR channel gating and cellular processing, respectively. Cleavage of hybrid molecule 14 by intestinal enzymes under physiological conditions produced the active potentiator 2 and corrector fragments 13, providing proof-of-concept for small-molecule potentiator–corrector hybrids as a single drug therapy for CF caused by the ΔF508 mutation.     

Electrophysiological evidence for the presence of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse sperm

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/protein kinase A (PKA) pathway and involves increases in intracellular Ca²⁺, pH, Cl^?, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl^? channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole‐cell patch‐clamp recordings from testicular mouse sperm now reveal a Cl^? selective component to membrane current that is ATP‐dependent, stimulated by cAMP, cGMP, and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTR_inh‐172, two well‐known CFTR antagonists. Furthermore, the Cl^? current component activated by cAMP and inhibited by CFTR_inh‐172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss‐of‐function mutation, indicating that CFTR is responsible for this component. A Cl^? selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTR_inh‐172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. J. Cell. Physiol. 228: 590–601, 2013. © 2012 Wiley Periodicals, Inc.     

Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe(508) (DeltaF508). The DeltaF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 degrees C, we found that maturation could be restored if Val(510) was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of DeltaF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.     

Combination of Correctors Rescue ΔF508-CFTR by Reducing Its Association with Hsp40 and Hsp27

Correcting the processing of ΔF508-CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of cystic fibrosis transmembrane conductance regulator (CFTR). We transfected COS-7 cells with ΔF508, created HEK-293 stably expressing ΔF508, and utilized CFBE41o⁻ cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18 + C4 the mature C-band was expressed at the cell surface. After treatment with C18 + C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum-associated degradation, such as Hsp27 (HspB1) and Hsp40 (DnaJ). We saw a dramatic reduction in binding to heat shock proteins 27 and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4 + 18 treatment, indicating these correctors shared a common pathway for rescue involving a network of endoplasmic reticulum-associated degradation proteins.     

Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants

The most common cause of CF (cystic fibrosis) is the deletion of Phe(508) (DeltaF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with DeltaF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of DeltaF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of DeltaF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of DeltaF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of DeltaF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of DeltaF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on DeltaF508 CFTR maturation suggests that they directly interact at different sites of the protein.     

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.     

CFTR promotes malignant glioma development via up‐regulation of Akt/Bcl2‐mediated anti‐apoptosis pathway

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated Cl^‐ channel, is extensively expressed in the epithelial cells of various tissues and organs. Accumulating evidence indicates that aberrant expression or mutation of CFTR is related to carcinoma development. Malignant gliomas are the most common and aggressive intracranial tumours; however, the role of CFTR in the development of malignant gliomas is unclear. Here, we report that CFTR is expressed in malignant glioma cell lines. Suppression of CFTR channel function or knockdown of CFTR suppresses glioma cell viability whereas overexpression of CFTR promotes it. Additionally, overexpression of CFTR suppresses apoptosis and promotes glioma progression in both subcutaneous and orthotopic xenograft models. Cystic fibrosis transmembrane conductance regulator activates Akt/Bcl2 pathway, and suppression of PI3K/Akt pathway abolishes CFTR overexpression–induced up‐regulation of Bcl2 (MK‐2206 and LY294002) and cell viability (MK‐2206). More importantly, the protein expression level of CFTR is significantly increased in glioblastoma patient samples. Altogether, our study has revealed a mechanism by which CFTR promotes glioma progression via up‐regulation of Akt/Bcl2‐mediated anti‐apoptotic pathway, which warrants future studies into the potential of using CFTR as a therapeutic target for glioma treatment.     

A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.     

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR^?/?) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.     

Synthesis and biological evaluation of thiazole derivatives on basic defects underlying cystic fibrosis

Cystic fibrosis is a genetic disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator gene, encoding for CFTR protein. The most frequent mutation is the deletion of phenylalanine at position 508 (F508del), which leads to distinct defects in channel gating and cellular processing. In last years, several thiazole containing small molecules, endowed with dual F508del-CFTR modulator activity, proved to be able to target these defects. In search of new chemical entities able to restore CFTR function, we designed and synthesized a small series of sixteen thiazole derivatives. The designed compounds were studied as correctors and potentiators of F508del-CFTR. Although none of the molecules showed significant corrector activity, compounds 10 and 11 exhibited potentiator effects, thus allowing to determine some basic structural features which enable to obtain F508del-CFTR potentiator activity. In silico ADME studies showed that these derivatives obey Lipinski’s rule of five and are expected to be orally bioavailable. Therefore, these molecules may represent a good starting point for the design of analogues endowed with improved CFTR potentiator activity and a good pharmacokinetic profile.     

'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations

Cystic fibrosis is a genetic disease that is associated with abnormal sweat electrolytes, sino-pulmonary disease, exocrine pancreatic insufficiency, and male infertility. Insights into genotype/phenotype relations have recently been gained in this disorder. The strongest relationship exists between 'severe' mutations in the gene that encodes the cystic fibrosis transmembrane regulator (CFTR) and pancreatic insufficiency. The relationship between 'mild' mutations, associated with residual CFTR function, and expression of disease is less precise. Atypical 'mild' mutations in the CFTR gene have been linked to late-onset pulmonary disease, congenital bilateral absence of the vas deferens, and idiopathic pancreatitis. Less commonly, sinusitis, allergic bronchopulmonary aspergillosis, and possibly even asthma may also be associated with mutations in the CFTR gene, but those syndromes predominantly reflect non-CFTR gene modifiers and environmental influences.     

Integrative pan cancer analysis reveals the importance of CFTR in lung adenocarcinoma prognosis

Cystic fibrosis (CF) and cystic fibrosis transmembrane conductance regulator (CFTR) mutations have been shown to be associated with the risk of a variety of cancers. However, the clinical significance of aberrant CFTR gene expression in human tumors remains unknown. The expression profiles and prognostic landscapes of CFTR in human cancers were identified from the PubMed, OVID, CNKI, TCGA, ONCOMINE, PrognoScan, and GEPIA databases. Over 11, 000 cancer samples from the literature, GEPIA database, and PrognoScan database were included in this study. In general, CFTR has various expression and prognostic profiles in cancers, but the results from cross-database and meta-analyses revealed that CFTR is a robust biomarker for LUAD prognosis. Collectively, this study suggests that CFTR is an important prognostic biomarker for LUAD survival, implying that it could be used as a prognostic biomarker and therapeutic target for LUAD.     

Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes

Background

Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.

Methods

Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.

Results

We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTR_inh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.

Conclusions

We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.

General significance

Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.     

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTR^sev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTR^BD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTR^BD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTR^BD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTR^BD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTR^BD variants through multiple mechanisms. CFTR^BD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.     

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N‐terminal 25 amino acid deleted S. typhi native PilS protein (ΔPilS), which makes the pilus, was determined at 1.9 Å resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of ΔPilS and a target CFTR peptide, determined at 1.8 Å, confirms that residues 113–117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described. Received: 25 August 1997 / Accepted: 20 January 1998     

Drug–drug plasma protein binding interactions of ivacaftor

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR‐protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co‐administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co‐administered CF drugs for human serum albumin (HSA) and α₁‐acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site‐selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug–drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug–drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug–drug interactions of ivacaftor with co‐administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. Copyright © 2015 John Wiley & Sons, Ltd.