Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a
portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides
in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs,
each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in
ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue
at the center of the first predicted nucleotide-binding domain was detected in CF patients.
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"Cystic fibrosis (CF) is a common autosomal recessive disease afflicting ~1/3,200 Caucasians world-wide, which results from mutations in the gene encoding CFTR, the Cystic Fibrosis Transmembrane conductance Regulator (Riordan et al., 1989; Rommens et al., 1989). CFTR is a phosphorylation-stimulated anion channel expressed at the apical membrane of many secretory epithelia (Anderson et al., 1991; Kartner et al., 1991; Bear et al., 1992). "
[Show abstract][Hide abstract] ABSTRACT: Background and purpose:
The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR (CF Transmembrane conductance Regulator), a plasma membrane anion channel, therefore optimal pharmacotherapies will likely require both a "potentiator" to increase channel open probability and a "corrector" that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood.
CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin (Fsk)-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR.
The potentiated Isc response during Fsk stimulation was increased 6-fold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which when combined with previous measurements in patients receiving an oral dose of 250 mg twice daily suggests a peak free concentration in plasma of 1.5 - 8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809, however this inhibition did not occur in the presence of physiological protein levels (20-40 mg/ml). Chronic exposure to a low concentration of VX-770 (100 nM) in combination with VX-809 (1 μM) also did not reduce the Fsk stimulated Isc relative to cells that had been chronically exposed to VX-809 alone provided it was assayed acutely using the same, clinically relevant concentration of potentiator.
Conclusions and implications:
Chronic exposure to clinically relevant concentrations of VX-770 does not reduce F508del CFTR function when assayed at those concentrations. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by an inhibitory effect of VX-770.
British Journal of Pharmacology 10/2015; DOI:10.1111/bph.13365 · 4.84 Impact Factor
"The correlation between transepithelial fluid transport and mucociliary clearance is demonstrated by cystic fibrosis (CF) lung disease. The devastating disease is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) , a Cl − channel that is ubiquitously expressed at the luminal side of pulmonary epithelial cells  . The main function of CFTR is to facilitate Cl −   and HCO 3 −   secretion. "
International immunopharmacology 08/2015; 29(1). DOI:10.1016/j.intimp.2015.08.010 · 2.47 Impact Factor
"Cystic fibrosis (CF) is the most common severe autosomal recessive disease in Caucasians, caused by mutations in the gene (Riordan et al. 1989), that encodes for the CF transmembrane conductance regulator (CFTR) protein, a chloride (Cl À ) channel expressed at the apical membrane of epithelial cells and regulating salt and water transport "
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.