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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 primary cultures of CF (F508del/F508del) and non-CF human bronchial epithelial (HBE) polarized cells. (A) Original Ussing chamber recordings obtained for non-CF HBE cells (wt-CFTR) and (C) CF-HBE cells under treatments with DMSO (0.01%, 48 h), C3 (6.7 μmol/L, 24 h), C4 (10 μmol/L, 16 h) and VX-809 (3 μmol/L, 48 h). Note that, in primary F508del/F508del monolayers, VX-809 enhances the negative transepithelial voltage (Vte) deflection following the application of luminal forskolin (Fsk, 2 μmol/L), which is further potentiated by genistein (Gen, 50 μmol/L), and completely inhibited by CFTRinh-172 (inh, 30 μmol/L). (B) Summary of sensitive-ΔIeq−sc induced by CFTR agonists in wt-CFTR cells. (D) Summary of sensitive-ΔIeq−sc induced upon correctors and CFTR agonists exposure as referred in (C). Data are means ± SEM (triplicates from the same donor). *P < 0.05 relative to control (DMSO) cells.
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fig03: Functional assessment of F508del-CFTR Rescue by C3, C4, and VX-809 in primary cultures of CF (F508del/F508del) and non-CF human bronchial epithelial (HBE) polarized cells. (A) Original Ussing chamber recordings obtained for non-CF HBE cells (wt-CFTR) and (C) CF-HBE cells under treatments with DMSO (0.01%, 48 h), C3 (6.7 μmol/L, 24 h), C4 (10 μmol/L, 16 h) and VX-809 (3 μmol/L, 48 h). Note that, in primary F508del/F508del monolayers, VX-809 enhances the negative transepithelial voltage (Vte) deflection following the application of luminal forskolin (Fsk, 2 μmol/L), which is further potentiated by genistein (Gen, 50 μmol/L), and completely inhibited by CFTRinh-172 (inh, 30 μmol/L). (B) Summary of sensitive-ΔIeq−sc induced by CFTR agonists in wt-CFTR cells. (D) Summary of sensitive-ΔIeq−sc induced upon correctors and CFTR agonists exposure as referred in (C). Data are means ± SEM (triplicates from the same donor). *P < 0.05 relative to control (DMSO) cells.

Mentions: To test the rescuing effect of these CFTR correctors on patient-derived cells with endogenous F508del-CFTR, that is closer to in vivo conditions, we treated primary cultures of HBE cells derived from F508del-CFTR homozygous patients with compounds C3, C4, and VX-809. We used as a negative control equivalent cultures treated with the vehicle (DMSO). Response of DMSO-treated cultures cannot be distinguished from that of nontreated cultures, as we have shown previously (Moniz et al. 2013). Data from micro-Ussing chamber transepithelial voltage (Vte) measurements and respective ΔIeq−sc (Fig.3) show that VX-809 rescues F508del-CFTR function in response to either the cAMP agonist forskolin alone or in combination with potentiator genistein (Fig.3D). In contrast, the efficacy of both C3 and C4 is very modest, as previously shown (Van Goor et al. 2011). Importantly, these effects were specifically blocked by CFTR Inh172 (Fig.3C).


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 primary cultures of CF (F508del/F508del) and non-CF human bronchial epithelial (HBE) polarized cells. (A) Original Ussing chamber recordings obtained for non-CF HBE cells (wt-CFTR) and (C) CF-HBE cells under treatments with DMSO (0.01%, 48 h), C3 (6.7 μmol/L, 24 h), C4 (10 μmol/L, 16 h) and VX-809 (3 μmol/L, 48 h). Note that, in primary F508del/F508del monolayers, VX-809 enhances the negative transepithelial voltage (Vte) deflection following the application of luminal forskolin (Fsk, 2 μmol/L), which is further potentiated by genistein (Gen, 50 μmol/L), and completely inhibited by CFTRinh-172 (inh, 30 μmol/L). (B) Summary of sensitive-ΔIeq−sc induced by CFTR agonists in wt-CFTR cells. (D) Summary of sensitive-ΔIeq−sc induced upon correctors and CFTR agonists exposure as referred in (C). Data are means ± SEM (triplicates from the same donor). *P < 0.05 relative to control (DMSO) cells.
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fig03: Functional assessment of F508del-CFTR Rescue by C3, C4, and VX-809 in primary cultures of CF (F508del/F508del) and non-CF human bronchial epithelial (HBE) polarized cells. (A) Original Ussing chamber recordings obtained for non-CF HBE cells (wt-CFTR) and (C) CF-HBE cells under treatments with DMSO (0.01%, 48 h), C3 (6.7 μmol/L, 24 h), C4 (10 μmol/L, 16 h) and VX-809 (3 μmol/L, 48 h). Note that, in primary F508del/F508del monolayers, VX-809 enhances the negative transepithelial voltage (Vte) deflection following the application of luminal forskolin (Fsk, 2 μmol/L), which is further potentiated by genistein (Gen, 50 μmol/L), and completely inhibited by CFTRinh-172 (inh, 30 μmol/L). (B) Summary of sensitive-ΔIeq−sc induced by CFTR agonists in wt-CFTR cells. (D) Summary of sensitive-ΔIeq−sc induced upon correctors and CFTR agonists exposure as referred in (C). Data are means ± SEM (triplicates from the same donor). *P < 0.05 relative to control (DMSO) cells.
Mentions: To test the rescuing effect of these CFTR correctors on patient-derived cells with endogenous F508del-CFTR, that is closer to in vivo conditions, we treated primary cultures of HBE cells derived from F508del-CFTR homozygous patients with compounds C3, C4, and VX-809. We used as a negative control equivalent cultures treated with the vehicle (DMSO). Response of DMSO-treated cultures cannot be distinguished from that of nontreated cultures, as we have shown previously (Moniz et al. 2013). Data from micro-Ussing chamber transepithelial voltage (Vte) measurements and respective ΔIeq−sc (Fig.3) show that VX-809 rescues F508del-CFTR function in response to either the cAMP agonist forskolin alone or in combination with potentiator genistein (Fig.3D). In contrast, the efficacy of both C3 and C4 is very modest, as previously shown (Van Goor et al. 2011). Importantly, these effects were specifically blocked by CFTR Inh172 (Fig.3C).

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