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Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

Farinha CM, Sousa M, Canato S, Schmidt A, Uliyakina I, Amaral MD - Pharmacol Res Perspect (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute Campo Grande-C8, 1749-016, Lisboa, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus

Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in polarized CFBE41o-cells stably expressing wt- or F508del-CFTR. Ussing chamber experiments were carried out in cells incubated at low temperature (26°C, 24 h) or at 37°C and under treatment with DMSO (control) or correctors C3 (25 μmol/L, 24 h), C4 (15 μmol/L, 24 h), VX-809 (3 μmol/L, 48 h), as indicated. Summary of the induced short-circuit currents (ΔIeq−sc) of polarized CFBE41o- cells upon luminal stimulation with forskolin (2 μmol/L) and genistein (50 μmol/L) (Fsk/Gen) and blocked with CFTR inh172 (30 μmol/L). Data are means ± SEM. *P < 0.05 relative to DMSO-treated cells.
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fig02: Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in polarized CFBE41o-cells stably expressing wt- or F508del-CFTR. Ussing chamber experiments were carried out in cells incubated at low temperature (26°C, 24 h) or at 37°C and under treatment with DMSO (control) or correctors C3 (25 μmol/L, 24 h), C4 (15 μmol/L, 24 h), VX-809 (3 μmol/L, 48 h), as indicated. Summary of the induced short-circuit currents (ΔIeq−sc) of polarized CFBE41o- cells upon luminal stimulation with forskolin (2 μmol/L) and genistein (50 μmol/L) (Fsk/Gen) and blocked with CFTR inh172 (30 μmol/L). Data are means ± SEM. *P < 0.05 relative to DMSO-treated cells.

Mentions: We then investigated how the three correctors restore activity of F508del-CFTR stably expressed in polarized CFBE41o- cells. Variation in equivalent short-circuit currents (ΔIeq−sc) in cells treated with either VX-809 (∼2.2-fold increase vs. DMSO) or C4 (∼1.5-fold increase vs. DMSO) showed an enhancement of CFTR-mediated Cl− currents, whereas cells exposed to C3 revealed a much smaller response, consistent with reported inhibitory effect of this compound at higher concentrations on channel activity (Kim Chiaw et al. 2010) (Fig.2). Although the response of cells treated with VX-809 showed the greatest efficacy among all three correctors, low temperature restored F508del-CFTR function to even higher values (∼67% of wt-CFTR, ∼12-fold increase vs. DMSO). This observation is in agreement with previous studies in these cells (Rowe et al. 2010; Moniz et al. 2013).


Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

Farinha CM, Sousa M, Canato S, Schmidt A, Uliyakina I, Amaral MD - Pharmacol Res Perspect (2015)

Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in polarized CFBE41o-cells stably expressing wt- or F508del-CFTR. Ussing chamber experiments were carried out in cells incubated at low temperature (26°C, 24 h) or at 37°C and under treatment with DMSO (control) or correctors C3 (25 μmol/L, 24 h), C4 (15 μmol/L, 24 h), VX-809 (3 μmol/L, 48 h), as indicated. Summary of the induced short-circuit currents (ΔIeq−sc) of polarized CFBE41o- cells upon luminal stimulation with forskolin (2 μmol/L) and genistein (50 μmol/L) (Fsk/Gen) and blocked with CFTR inh172 (30 μmol/L). Data are means ± SEM. *P < 0.05 relative to DMSO-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492728&req=5

fig02: Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in polarized CFBE41o-cells stably expressing wt- or F508del-CFTR. Ussing chamber experiments were carried out in cells incubated at low temperature (26°C, 24 h) or at 37°C and under treatment with DMSO (control) or correctors C3 (25 μmol/L, 24 h), C4 (15 μmol/L, 24 h), VX-809 (3 μmol/L, 48 h), as indicated. Summary of the induced short-circuit currents (ΔIeq−sc) of polarized CFBE41o- cells upon luminal stimulation with forskolin (2 μmol/L) and genistein (50 μmol/L) (Fsk/Gen) and blocked with CFTR inh172 (30 μmol/L). Data are means ± SEM. *P < 0.05 relative to DMSO-treated cells.
Mentions: We then investigated how the three correctors restore activity of F508del-CFTR stably expressed in polarized CFBE41o- cells. Variation in equivalent short-circuit currents (ΔIeq−sc) in cells treated with either VX-809 (∼2.2-fold increase vs. DMSO) or C4 (∼1.5-fold increase vs. DMSO) showed an enhancement of CFTR-mediated Cl− currents, whereas cells exposed to C3 revealed a much smaller response, consistent with reported inhibitory effect of this compound at higher concentrations on channel activity (Kim Chiaw et al. 2010) (Fig.2). Although the response of cells treated with VX-809 showed the greatest efficacy among all three correctors, low temperature restored F508del-CFTR function to even higher values (∼67% of wt-CFTR, ∼12-fold increase vs. DMSO). This observation is in agreement with previous studies in these cells (Rowe et al. 2010; Moniz et al. 2013).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute Campo Grande-C8, 1749-016, Lisboa, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus