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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 BHK cells. Iodide efflux from BHK cells stably expressing wt- or F508del-CFTR was measured directly or (F508del-CFTR cells) after low temperature (26°C, 48 h) incubation (A) or (F508del-CFTR cells) after treatment with 6.7 μmol/L C3, 10 μmol/L C4, 3 μmol/L VX-809 (B). Cells were stimulated with Forskolin (10 μmol/L) and Genistein (50 μmol/L) in the period indicated by the black solid line above graph of time course iodide efflux measurements. (C) Graph summarizing data of I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as a percentage of wt-CFTR activity. For F508del cells, the white bar corresponds to the efflux elicited by DMSO and the black bars to the increase promoted by each treatment. Data are mean ± SEM at each point (n = 4–6). Where error bars are not visible, the symbol has obscured them. *P < 0.05 relative to F508del-CFTR cells under DMSO (37°C).
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fig01: Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in BHK cells. Iodide efflux from BHK cells stably expressing wt- or F508del-CFTR was measured directly or (F508del-CFTR cells) after low temperature (26°C, 48 h) incubation (A) or (F508del-CFTR cells) after treatment with 6.7 μmol/L C3, 10 μmol/L C4, 3 μmol/L VX-809 (B). Cells were stimulated with Forskolin (10 μmol/L) and Genistein (50 μmol/L) in the period indicated by the black solid line above graph of time course iodide efflux measurements. (C) Graph summarizing data of I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as a percentage of wt-CFTR activity. For F508del cells, the white bar corresponds to the efflux elicited by DMSO and the black bars to the increase promoted by each treatment. Data are mean ± SEM at each point (n = 4–6). Where error bars are not visible, the symbol has obscured them. *P < 0.05 relative to F508del-CFTR cells under DMSO (37°C).

Mentions: We firstly functionally assessed the F508del-CFTR rescue by the three correctors in the three cellular models. To such end in BHK cells, since they do not polarize, we used the iodide efflux technique (instead of Ussing chamber measurements). BHK cells, although less relevant in the context of CFTR biology, are a simpler model that when transfected express high amounts of CFTR. This allows a more direct characterization of direct effects upon CFTR, independent of more sophisticated trafficking and regulated activity as in polarized cells. We were able to detect F508del-CFTR function after 12–24 h incubation with each of the three compounds plus potentiator genistein (Fig.1). Comparison of the respective activity peak levels with those of low-temperature rescued F508del-CFTR and of wt-CFTR, shows that for VX-809 the activity peak is ∼46% of wt-CFTR (Fig.1C), a value similar to that of low temperature rescued F508del-CFTR (∼55% of wt-CFTR), whereas C3 and C4 only restore the mutant function to ∼26% and ∼33% of wt-CFTR, respectively. Activity peaks for cells under all three correctors exhibit a delay of 2 min (VX-809) or 3 min (C3 and C4) compared to wt-CFTR (Fig.1B), whereas in low temperature rescue no delay is observed (Fig.1A).


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 BHK cells. Iodide efflux from BHK cells stably expressing wt- or F508del-CFTR was measured directly or (F508del-CFTR cells) after low temperature (26°C, 48 h) incubation (A) or (F508del-CFTR cells) after treatment with 6.7 μmol/L C3, 10 μmol/L C4, 3 μmol/L VX-809 (B). Cells were stimulated with Forskolin (10 μmol/L) and Genistein (50 μmol/L) in the period indicated by the black solid line above graph of time course iodide efflux measurements. (C) Graph summarizing data of I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as a percentage of wt-CFTR activity. For F508del cells, the white bar corresponds to the efflux elicited by DMSO and the black bars to the increase promoted by each treatment. Data are mean ± SEM at each point (n = 4–6). Where error bars are not visible, the symbol has obscured them. *P < 0.05 relative to F508del-CFTR cells under DMSO (37°C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig01: Functional assessment of F508del-CFTR rescue by C3, C4, and VX-809 in BHK cells. Iodide efflux from BHK cells stably expressing wt- or F508del-CFTR was measured directly or (F508del-CFTR cells) after low temperature (26°C, 48 h) incubation (A) or (F508del-CFTR cells) after treatment with 6.7 μmol/L C3, 10 μmol/L C4, 3 μmol/L VX-809 (B). Cells were stimulated with Forskolin (10 μmol/L) and Genistein (50 μmol/L) in the period indicated by the black solid line above graph of time course iodide efflux measurements. (C) Graph summarizing data of I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as a percentage of wt-CFTR activity. For F508del cells, the white bar corresponds to the efflux elicited by DMSO and the black bars to the increase promoted by each treatment. Data are mean ± SEM at each point (n = 4–6). Where error bars are not visible, the symbol has obscured them. *P < 0.05 relative to F508del-CFTR cells under DMSO (37°C).
Mentions: We firstly functionally assessed the F508del-CFTR rescue by the three correctors in the three cellular models. To such end in BHK cells, since they do not polarize, we used the iodide efflux technique (instead of Ussing chamber measurements). BHK cells, although less relevant in the context of CFTR biology, are a simpler model that when transfected express high amounts of CFTR. This allows a more direct characterization of direct effects upon CFTR, independent of more sophisticated trafficking and regulated activity as in polarized cells. We were able to detect F508del-CFTR function after 12–24 h incubation with each of the three compounds plus potentiator genistein (Fig.1). Comparison of the respective activity peak levels with those of low-temperature rescued F508del-CFTR and of wt-CFTR, shows that for VX-809 the activity peak is ∼46% of wt-CFTR (Fig.1C), a value similar to that of low temperature rescued F508del-CFTR (∼55% of wt-CFTR), whereas C3 and C4 only restore the mutant function to ∼26% and ∼33% of wt-CFTR, respectively. Activity peaks for cells under all three correctors exhibit a delay of 2 min (VX-809) or 3 min (C3 and C4) compared to wt-CFTR (Fig.1B), whereas in low temperature rescue no delay is observed (Fig.1A).

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