Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor.
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ABSTRACT:
SUBMITTER: Barbieri A
PROVIDER: S-EPMC8703531 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ivacaftor is a potentiator of the CFTR chloride channel and is in worldwide clinical use for the chronic treatment of cystic fibrosis in patients. There is evidence that the bioavailability of ivacaftor in the body may be influenced by the multi-drug exporter P-glycoprotein. Here we have employed purified and reconstituted P-glycoprotein to study its interaction with ivacaftor as well as the ability of the drug to compete with a known transported substrate of the protein. We find that ivacaftor stimulates the ATPase activity of the purified protein and can compete with the transport of the fluorescent substrate Hoechst 33342. These findings lead us to conclude that ivacaftor is very likely an efficiently transported substrate of P-glycoprotein. Evidence for state-dependent binding of ivacaftor was obtained using a fluorescent, cysteine-reactive reporter dye. The quiescent, nucleotide-free state in the P-glycoprotein transport cycle appears to bind ivacaftor strongly.
| S-EPMC5727471 | biostudies-literature
Project description:BackgroundIvacaftor increases CFTR channel activity and improves pulmonary function for individuals bearing a G551D mutation. Because ivacaftor structurally resembles quinolone antibiotics, we tested the hypothesis that ivacaftor possesses antibacterial properties.MethodsBioluminescence, colony forming unit, and minimal inhibitory concentration assays were used to assess viability of Staphylococcus aureus, Pseudomonas aeruginosa and multiple clinical microbial isolates.ResultsIvacaftor induced a dose-dependent reduction in bioluminescence of S. aureus and decreased the number of colony forming units. We observed a similar but less robust effect in P. aeruginosa following outer membrane permeabilization. Ivacaftor inhibited the growth of respiratory isolates of S. aureus and Streptococcus pneumoniae and exhibited positive interactions with antibiotics against lab and respiratory strains of S. aureus and S. pneumoniae.ConclusionThese data indicate that ivacaftor exhibits antibacterial properties and raise the intriguing possibility that ivacaftor might have an antibiotic effect in people with CF.
| S-EPMC4718582 | biostudies-literature
Project description:Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed "correctors" such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and "potentiators" such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ?F508. Thus, patients with ?F508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ?F508 homozygous cultures. This result reflected the destabilization of corrected ?F508 CFTR by VX-770, markedly increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining these drugs to maximize rescue of ?F508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.
| S-EPMC4272825 | biostudies-other
Project description:Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding ("correctors") and/or increase channel function ("potentiators") have proven to be highly effective in the therapeutic treatment of CF. Notably, incorporation of the next-generation CFTR corrector, elexacaftor, into a triple combination therapeutic (marketed as Trikafta) has shown tremendous clinical promise in treating CF caused by F508del-CFTR. Here, we report on a newly-described role of elexacaftor as a CFTR potentiator. We explore the acute and chronic actions, pharmacology, and efficacy of elexacaftor as a CFTR potentiator in restoring function to multiple classes of CFTR mutations. We demonstrate that the potentiating action of elexacaftor exhibits multiplicative synergy with the established CFTR potentiator ivacaftor in rescuing multiple CFTR class defects, indicating that a new combination therapeutic of ivacaftor and elexacaftor could have broad impact on CF therapies.
| S-EPMC8494914 | biostudies-literature
Project description:BackgroundAirway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF), an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR). Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function.Objective and methodsThe purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment.Results129 operational taxonomy units (OTUs), representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe Porphyromonas 1 increased (p<0.01) and that of Streptococcus 1 (S. mitis group) decreased (p<0.05), possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe Prevotella 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05). The study confirmed the presumed positive role of anaerobes in lung function.ConclusionSeveral airway microbiota components, notably anaerobes (obligate or facultative anaerobes), could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.
| S-EPMC4390299 | biostudies-literature
Project description:Background and purposeThe most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably 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.Experimental approachCF 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-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR.Key resultsThe potentiated Isc response during forskolin stimulation was increased sixfold 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 together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator.Conclusions and implicationsChronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770.
| S-EPMC4728415 | biostudies-literature
Project description:Over-production of reactive oxygen species (ROS) in the inner ear can be triggered by a variety of pathological events identified in animal models after traumatic noise exposure. Our previous research found that inhibition of the AMP-activated protein kinase alpha subunit (AMPKα) protects against noise-induced cochlear hair cell loss and hearing loss by reducing ROS accumulation. However, the molecular pathway through which AMPKα exerts its antioxidative effect is still unclear. In this study, we have investigated a potential target of AMPKα and ROS, cystic fibrosis transmembrane conductance regulator (CFTR), and the protective effect against noise-induced hair cell loss of an FDA-approved CFTR potentiator, ivacaftor, in FVB/NJ mice, mouse explant cultures, and HEI-OC1 cells. We found that noise exposure increases phosphorylation of CFTR at serine 737 (p-CFTR, S737), which reduces wildtype CFTR function, resulting in oxidative stress in cochlear sensory hair cells. Pretreatment with a single dose of ivacaftor maintains CFTR function by preventing noise-increased p-CFTR (S737). Furthermore, ivacaftor treatment increases nuclear factor E2-related factor 2 (Nrf2) expression, diminishes ROS formation, and attenuates noise-induced hair cell loss and hearing loss. Additionally, inhibition of noise-induced AMPKα activation by compound C also diminishes p-CFTR (S737) expression. In line with these in-vivo results, administration of hydrogen peroxide to cochlear explants or HEI-OC1 cells increases p-CFTR (S737) expression and induces sensory hair cell or HEI-OC1 cell damage, while application of ivacaftor halts these effects. Although ivacaftor increases Nrf2 expression and reduces ROS accumulation, cotreatment with ML385, an Nrf2 inhibitor, abolishes the protective effects of ivacaftor against hydrogen-peroxide-induced HEI-OC1 cell death. Our results indicate that noise-induced sensory hair cell damage is associated with p-CFTR. Ivacaftor has potential for treatment of noise-induced hearing loss by maintaining CFTR function and increasing Nrf2 expression for support of redox homeostasis in sensory hair cells.
| S-EPMC10528730 | biostudies-literature
Project description:The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporter superfamily that functions as an epithelial chloride channel. Gating of the CFTR ion conduction pore involves a conserved irreversible cyclic mechanism driven by ATP binding and hydrolysis at two cytosolic nucleotide-binding domains (NBDs): formation of an intramolecular NBD dimer that occludes two ATP molecules opens the pore, whereas dimer disruption after ATP hydrolysis closes it. CFTR dysfunction resulting from inherited mutations causes CF. The most common CF mutation, deletion of phenylalanine 508 (ΔF508), impairs both protein folding and processing and channel gating. Development of ΔF508 CFTR correctors (to increase cell surface expression) and potentiators (to enhance open probability, Po) is therefore a key focus of CF research. The practical utility of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), one of the most efficacious potentiators of ΔF508 CFTR identified to date, is limited by its pore-blocking side effect. NPPB-mediated stimulation of Po is unique in that it involves modulation of gating transition state stability. Although stabilization by NPPB of the transition state for pore opening enhances both the rate of channel opening and the very slow rate of nonhydrolytic closure, because of CFTR's cyclic gating mechanism, the net effect is Po stimulation. In addition, slowing of ATP hydrolysis by NPPB delays pore closure, further enhancing Po. Here we show that NPPB stimulates gating at a site outside the pore and that these individual actions of NPPB on CFTR are fully attributable to one or the other of its two complementary molecular parts, 3-nitrobenzoate (3NB) and 3-phenylpropylamine (3PP), both of which stimulate Po: the pore-blocking 3NB selectively stabilizes the transition state for opening, whereas the nonblocking 3PP selectively slows the ATP hydrolysis step. Understanding structure-activity relationships of NPPB might prove useful for designing potent, clinically relevant CFTR potentiators.
| S-EPMC4178936 | biostudies-literature
Project description:RationaleThe majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure.ObjectivesTo evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis.MethodsFerrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes.Measurements and main resultsFollowing cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy.ConclusionsThe pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.
| S-EPMC10079430 | biostudies-literature
Project description:BackgroundCystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis.MethodsSmall molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement.ResultsWe showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures.ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.
| S-EPMC11234710 | biostudies-literature