Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis

Abstract

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, ΔF508. Thus, patients with ΔF508 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 ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 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 ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.

Figure 1. VX-770 treatment restores G551D function

Electrophysiological properties of G551D/ΔF508 cultures analyzed in Ussing chambers treated chronically with VX-770 (cVX770, 5 µM for 48 hrs) or with vehicle (0.1% DMSO). (A) Representative recording of I_SC measured in Ussing chambers. Quantification of response to treatment with (B) forskolin (significant difference between vehicle and cVX770, *P = 0.0009), (C) acute VX-770 (aVX770) (significant difference between vehicle and cVX770, *P = 0.0054), (D) forskolin + aVX770, (E) CFTR_inh-172. Primary CF HBE cultures (G551D/ΔF508) were derived from 3 different patients, 3–4 replicates were performed per patient for a total of 10 measurements per treatment.

Figure 2. Chronic VX-770 treatment inhibits functional rescue of ΔF508

(A) Representative I_SC traces of CF HBE cells recorded in Ussing chambers. Primary CF HBE cells (ΔF508/ΔF508) were treated with vehicle (DMSO) or VX-809 +/−VX-770 for 48 hrs at 5 µM each. (B) ΔI_SC response to forskolin observed in VX-809-treated CF HBE cells (*P = 0.0033, VX809 vs. vehicle) was prevented by chronic VX-770 treatment and significantly different from VX-809-treated cells (#P = 0.0147, VX809 vs. VX809+VX770). (C) CF HBE cells treated with VX-809 responded to acute VX-770 exposure (*P = 0.0177, VX809 vs. vehicle). This response was significantly abrogated in VX-809 + VX-770-treated cells (#P = 0.0031, VX809 vs. VX809+VX770). (D) The response to CFTR_inh-172 observed in VX-809-treated cells (*P = 0.0209, VX809 vs. vehicle) was significantly decreased in VX809+VX770-treated cells (#P = 0.0006, VX809 vs. VX809+VX770). Primary HBE cultures (ΔF508/ΔF508) were derived from 6 different patients, 2–4 replicates were performed per patient for a total of 15 measurements per condition.

Figure 3. VX-770 diminishes biochemical correction by increasing turnover of corrected ΔF508 CFTR

(A) CFTR Western blot of normal (NL) and CF HBE cultures treated with VX-809 (5 µM) +/−VX-770 (5 µM) for 48 hrs. * indicates the mature, complex glycosylated form of CFTR, band C; ^● indicates the immature band B. (B) Turnover of rescued ΔF508 in BHK-21 cells. ΔF508 was rescued at 27°C in the presence of VX-809 +/− VX-770 for 24 hrs. After adding cycloheximide (200 µg/ml, 37°C) cells were lysed at the indicated times and analyzed by Western blotting. (C) Quantification of remaining band C over time, normalized to actin (n = 3).

Figure 4. VX-770-induced hindrance of ΔF508 correction is dose-dependent

(A) I_SC traces of CF HBE cells (ΔF508/ΔF508) recorded in Ussing chambers. CF HBE cells were treated as indicated (VX-809: 5 µM, VX-770: 1 or 5 µM) for 48 hrs. (B) CFTR function in VX-809-treated cells decreased as chronic VX-770 concentrations increased. Significant reduction of the area under the curve (AUC)/min calculated from the time period between CFTR stimulation by forskolin and CFTR inhibition by CFTR_inh-172 (yields average ΔI_sc (µA/cm²)) was observed in CF cells chronically treated for 48 hrs with VX-809 when compared to VX-809 and 1 µM VX-770, (*P = 0.0352). A further reduction was detected when the chronic VX-770 concentration was increased to 5 µM (#P = 0.0049, VX-809 + 1 µM VX-770 vs. VX-809 + 5 µM VX-770). Primary CF HBE cultures were derived from at least 4 different CF patients; 2–5 replicates were performed per patient for a total of at least 14 measurements per condition. (C) In VX-809-corrected CF HBE cultures (ΔF508/ΔF508), the presence of chronic VX-770 at 50 nM caused a significant decline of the slope after forskolin treatment (*P = 0.0353, VX-809 vs. VX-809 + 50 nM VX-770). Primary CF HBE cultures were derived from 4 different patients; 3–5 replicates were performed per patient for a total of at least 15 measurements per condition. (D) Quantification of C:B band ratio in CF HBE cultures (ΔF508/ΔF508). CFTR C:B band ratio decreased in CF HBE cells as chronic VX-770 concentrations were increased. The C:B band ratio was significantly reduced in CF cells chronically treated for 48 hrs with VX-809 and 1 µM VX-770 compared to VX-809 alone (*P = 0.0181), and a further reduction was detected when the chronic VX-770 concentration was increased from 1 µM to 5 µM (#P = 0.0151, VX-809 + 1 µM VX-770 vs. VX-809 + 5 µM VX-770). Primary CF HBE cultures (ΔF508/ΔF508) from 4 different patients were analyzed. (E) Representative Western blot of CF HBE cells (ΔF508/ΔF508) showing decrease of VX-809-corrected ΔF508 as chronic VX-770 concentrations were increased.

Figure 5. Chronic VX-770 treatment decreases function of wild-type CFTR

(A) Representative I_SC traces of NL HBE cells recorded in Ussing chambers. Cultures were treated with vehicle (DMSO) or 5 µM VX-770 for 48 hrs. HBE cells that were chronically treated with VX-770 showed significantly reduced response to (B,C) forskolin (*P = 0.0198 for forskolin peak and *P = 0.0008 for forskolin plateau) and (D) CFTR_inh-172 (*P = 0.0014). Primary HBE cultures were derived from 6 different individuals, 2–4 replicates were performed per individual for a total of 17 measurements per condition. (E) Western blot of HBE cultures treated with VX-809 (5 µM) +/−VX-770 (5 µM) for 48 hrs. Mature CFTR was diminished in HBE cells that were chronically treated with VX770.

Figure 6. Key physiological properties were not altered in chronically VX-770-treated HBE cultures

(A) Microscopy after hematoxylin and eosin (H&E) staining of HBE cultures did not reveal a detectable difference between VX-770- or vehicle-treated cells (bar = 10 m). (B) Transepithelial resistance (Rt) of primary HBE cultures was not altered after chronic treatment with VX-770. (C) Nystatin responses were not significantly different in primary HBE cultures that were treated with vehicle or VX-770 (48 hrs, 5 µM). Nystatin was added to the apical side in Ussing chambers. Primary HBE cultures were derived from 5 different individuals, and 2–4 replicates per individual were performed.

Figure 7. VX-770 reduces stability of CFTR

(A) The amount of mature CFTR was reduced when NL HBE cells were chronically treated with VX-770 (48 hrs, 5 µM). G551D is more stable than NL CFTR and the amount of mature G551D protein in CF cultures (G551D/ΔF508) was not significantly reduced by 48 hrs treatment with 5 µM VX-770. (B) Quantification of C:B band ratio with chronic treatment of VX-770 at 5 µM. C:B band ratio was significantly decreased in NL cells chronically treated for 48 hrs with 5 µM VX-770, (*P = 0.008) (n = 3, cultures from 3 different NL individuals). The reduction of C:B band ratio in G551D/ΔF508 cells chronically treated for 48 hrs with 5 µM VX-770 was not statistically significant (n = 3, cultures from 3 different CF patients (G551D/ΔF508)). (C) Structural model showing positions of G551D and F508 in the CFTR molecule. (D) Illustration representing the proposed relationship between function and stability of CFTR variants. Wild-type CFTR has an intermediate stability that allows for optimal function. G551D CFTR is a more rigid protein that exhibits increased stability compared to wild-type CFTR but lacks sufficient function, presumably due to decreased flexibility. VX-770 decreases G551D CFTR stability and renders it a more flexible protein, resembling the stability and function of wild-type CFTR. However, VX-770 causes destabilization of wild-type CFTR and VX-809-corrected ΔF508 CFTR, diminishing their function. VX-809 increases the stability of ΔF508, bringing it closer to resembling wild-type CFTR, but this increased stability is diminished when VX-770 is present.