This preclinical, phase 2 report shows that VX-445, a CFTR potentiator when administered with tezacaftor and ivacaftor, improved lung function and reduced sweat chloride concentrations and symptoms ...in patients harboring one or two Phe508del alleles.
This companion article to the VX-445 report shows that VX-659, a new CFTR potentiator, when administered with tezacaftor and ivacaftor improved lung function, sweat chloride concentration, and ...symptoms in patients with cystic fibrosis who harbored one or two Phe508del alleles.
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.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of ...the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC50, 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.
Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis ...transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of ł0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In previous work, participants with a G970R mutation in cystic fibrosis transmembrane conductance regulator (CFTR) (c.2908G>C) had numerically lower sweat chloride responses during ivacaftor ...treatment than participants with other CFTR gating mutations. The objective of this substudy was to characterize the molecular defect of the G970R mutation in vitro and assess the benefit of ivacaftor in participants with this mutation. This substudy assessed sweat chloride, spirometry findings, and nasal potential difference on and off ivacaftor treatment in three participants with a G970R/F508del genotype. Intestinal organoids derived from rectal biopsy specimens were used to assess ivacaftor response ex vivo and conduct messenger RNA splice and protein analyses. No consistent or meaningful trends were observed between on‐treatment and off‐treatment clinical assessments. Organoids did not respond to ivacaftor in forskolin‐induced swelling assays; no mature CFTR protein was detected in Western blots. Organoid RNA analysis demonstrated that 3 novel splice variants were created by G970R‐CFTR: exon 17 truncation, exons 13–15 and 17 skipping, and intron 17 retention. Functional and molecular analyses indicated that the c.2908G>C mutation caused a cryptic splicing defect. Organoids lacked an ex vivo response with ivacaftor and supported identification of the mechanism underlying the CFTR defect caused by c.2908G>C. Analysis of CFTR mutations indicated that cryptic splicing was a rare cause of mutation misclassification in engineered cell lines. This substudy used organoids as an alternative in vitro model for mutations, such as cryptic splice mutations that cannot be fully assessed using cDNA expressed in recombinant cell systems.
Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel ...involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl⁻ secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by almost equal to10-fold, to almost equal to50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na⁺ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.
The use of human bronchial epithelial (HBE) cell cultures derived from the bronchi of CF patients offers the opportunity to study the effects of CFTR correctors and potentiators on CFTR function and ...epithelial cell biology in the native pathological environment. Cultured HBE cells derived from CF patients exhibit many of the morphological and functional characteristics believed to be associated with CF airway disease in vivo, including abnormal ion and fluid transport leading to dehydration of the airway surface and the loss of cilia beating. In addition, they can be generated in sufficient quantities to support routine lab testing of compound potency and efficacy and retain reproducible levels of CFTR function over time. Here we describe the development and validation of the CF HBE pharmacology model and its use to characterize, optimize, and select clinical candidates. It is expected that the pre-clinical testing of CFTR potentiators and correctors using epithelial cell cultures derived from CF patients will help to increase their likelihood of clinical efficacy.
Quinolinone-3-carboxamide 1, a novel CFTR potentiator, was discovered using high-throughput screening in NIH-3T3 cells expressing the F508del-CFTR mutation. Extensive medicinal chemistry and ...iterative structure–activity relationship (SAR) studies to evaluate potency, selectivity, and pharmacokinetic properties resulted in the identification of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, 48, ivacaftor), an investigational drug candidate approved by the FDA for the treatment of CF patients 6 years of age and older carrying the G551D mutation.
Abstract Background Ivacaftor (KALYDECO™, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the ...G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function. Methods The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface. Results Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance. Conclusions These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.
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