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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

Comparison of functional correction of F508del-CFTR in different cellular models. (A) Summary of data of either induced short-circuit currents (ΔIeq−sc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as fold change relative to DMSO control. (B) Summary of data of either induced short-circuit currents (ΔIsc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as percentage of wt-CFTR control. Data are means ± SEM. *P < 0.05 relative to DMSO.
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fig04: Comparison of functional correction of F508del-CFTR in different cellular models. (A) Summary of data of either induced short-circuit currents (ΔIeq−sc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as fold change relative to DMSO control. (B) Summary of data of either induced short-circuit currents (ΔIsc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as percentage of wt-CFTR control. Data are means ± SEM. *P < 0.05 relative to DMSO.

Mentions: To address the above-described cell specificity of correctors, we then compared the respective maximal effects (fold increase relatively to each DMSO-treated control). This analysis shows that VX-809 is the most effective of the three correctors (Fig.4A) in all cellular models tested. Importantly, VX-809 has its highest effect in primary cells (∼6-fold increase vs. DMSO), that is, significantly higher than observed in human bronchial epithelial (∼2.2-fold) or in hamster (∼3-fold) cell lines. The other correctors are not as effective in any of the three models tested. C3 is only effective in BHK cells whereas C4 has a modest effect both in CFBE and BHK cell lines. Curiously, neither of these laboratory correctors rescues F508del-CFTR in HBE primary cells (Figs. 3, 4A). When the rescuing efficacy of the three correctors was assessed by the percentage of wt-CFTR activity attained, once again VX-809 has the better results (Fig.4B). Interestingly, this analysis shows that the relative rescuing efficiencies for the three compounds are essentially maintained in all three cellular models tested (Fig.4B). Although the comparison shown joins together results from iodide efflux and Ussing chamber methods, both correspond, in the respective models (nonpolarized vs. epithelial polarized cells), to an assessment of global ion (Cl− or I−) transport (as opposed to a more thorough biophysical characterization of each CFTR channel), thus allowing a general inference on the overall effect of the compounds in the three models tested.


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)

Comparison of functional correction of F508del-CFTR in different cellular models. (A) Summary of data of either induced short-circuit currents (ΔIeq−sc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as fold change relative to DMSO control. (B) Summary of data of either induced short-circuit currents (ΔIsc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as percentage of wt-CFTR control. Data are means ± SEM. *P < 0.05 relative to DMSO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Comparison of functional correction of F508del-CFTR in different cellular models. (A) Summary of data of either induced short-circuit currents (ΔIeq−sc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as fold change relative to DMSO control. (B) Summary of data of either induced short-circuit currents (ΔIsc) upon stimulation with forskolin and genistein (for primary HBE cells or CFBE-F508del cell line) or I− efflux peak magnitude generated by the different treatments of BHK F508del-CFTR cells expressed as percentage of wt-CFTR control. Data are means ± SEM. *P < 0.05 relative to DMSO.
Mentions: To address the above-described cell specificity of correctors, we then compared the respective maximal effects (fold increase relatively to each DMSO-treated control). This analysis shows that VX-809 is the most effective of the three correctors (Fig.4A) in all cellular models tested. Importantly, VX-809 has its highest effect in primary cells (∼6-fold increase vs. DMSO), that is, significantly higher than observed in human bronchial epithelial (∼2.2-fold) or in hamster (∼3-fold) cell lines. The other correctors are not as effective in any of the three models tested. C3 is only effective in BHK cells whereas C4 has a modest effect both in CFBE and BHK cell lines. Curiously, neither of these laboratory correctors rescues F508del-CFTR in HBE primary cells (Figs. 3, 4A). When the rescuing efficacy of the three correctors was assessed by the percentage of wt-CFTR activity attained, once again VX-809 has the better results (Fig.4B). Interestingly, this analysis shows that the relative rescuing efficiencies for the three compounds are essentially maintained in all three cellular models tested (Fig.4B). Although the comparison shown joins together results from iodide efflux and Ussing chamber methods, both correspond, in the respective models (nonpolarized vs. epithelial polarized cells), to an assessment of global ion (Cl− or I−) transport (as opposed to a more thorough biophysical characterization of each CFTR channel), thus allowing a general inference on the overall effect of the compounds in the three models tested.

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