Pretreatment with arginase inhibitor CB-1158 increases NO pathway–driven CFTR channel activity in CFBE cells transduced with lv (lentivirus). SLC6A14. (A) Traces normalized to 5 minutes (timepoint at the end of baseline) represent change in FLIPR fluorescence from baseline (depolarization due to ACC: ΔF/F₀) relative to buffer control, as a measure of CFTR channel function in CFBE stably expressing F508del-CFTR with lv. SLC6A14. Cells were pretreated 1 hour with 10 µM CB-1158, 50 µM 1400 W, combination of CB-1158 and 1400 W, or buffer in the FLIPR dye solution. At the 5-minute point, 1 mM L-arginine was added into the apical bath, and, at 15 minutes, cAMP stimulation occurred due to 10 µM FSK addition. At 25 minutes, CFTR-specific inhibitor, CFTR_inh-172 (Inh-172), was added to confirm CFTR-specific function. (B) Bar graph represents maximum change in ACC fluorescence after Fsk addition to the peak of Fsk response (ΔFsk/F₀) following 1 mM L-arginine in the CFBE cell line from (A) (mean ± S.D.). One-way ANOVA with Tukey’s multiple comparison test was performed (ns = not statistically significant, *P < 0.05; **P < 0.01, N = 3 biologic replicates, n = 4 technical replicates).

Inhibition of arginase activity enhances rescue of F508del-CFTR channel activity at the surface of primary respiratory epithelial tissues by VX-809 plus VX-770. (A) Bar graph represents maximum change in ACC fluorescence from baseline (ΔF/F₀) normalized to mock activation (with DMSO as vehicle control for arginine or FSK plus VX-770) for F508del-CFTR primary bronchial cells with 48-hour chronic treatment with either DMSO or 3 µM VX-809 (mean S.D.). Cells were pretreated acutely for 1 hour with 1 mM L-arginine, 10 µM CB-1158, 10 µM ABH, or buffer in the FLIPR dye solution. WT baseline is based on the mean FSK-stimulated values of eight individual WT-CFTR primary bronchial cells. One-way ANOVA with Tukey’s multiple comparison test relative to VX-809 plus VX-770 was performed (**P < 0.007; ****P < 0.0001, N > 4 biologic replicates, n = 2 technical replicates). (B) Representative line graphs represent change in ACC fluorescence from baseline (ΔF/F₀) relative to DMSO control, as a measure of CFTR channel function in F508del-CFTR bronchial cells grown on inserts at ALI with 48-hour chronic treatment of 3 µM VX-809 or DMSO. Cells were pretreated with 10 µM CB-1158, or buffer in the FLIPR dye solution. At the 10-minute point, 1 mM L-arginine (Arg) was added into the apical FLIPR membrane dye solution, and, at 25 minutes, cAMP stimulation (Stim) occurred due to 10 µM FSK and 1 µM VX-770 addition. At 45 minutes, CFTR-specific inhibitor, CFTR_inh-172 (Inh172), was added as an indicator of CFTR-specific function. (C) Bar graph represents the maximum change in ACC fluorescence from baseline (ΔF/F₀) normalized to first DMSO control on F508del-CFTR primary nasal cells with 48-hour chronic treatment of either DMSO or 3 µM VX-809 (mean S.D.). Cells are pretreated for 1 hour with 10 µM CB-1158, or buffer in the FLIPR dye solution. WT baseline is based on the mean FSK-stimulated values of 16 individual WT-CFTR primary nasal cells. One-way ANOVA with Tukey’s multiple comparison test was performed (ns = not statistically significant, *P < 0.04; ***P = 0.005, N = 3 biologic replicates, n = 1 to 2 technical replicates). (D) Representative trace shows change in ACC fluorescence from baseline (ΔF/F₀) relative to DMSO control, as a measure of CFTR channel function in F508del-CFTR nasal cells grown on inserts at ALI for 14 days with 48-hour chronic treatment of 3 µM VX-809 or DMSO. Cells were pretreated with 10 µM CB-1158 or buffer in the FLIPR dye solution. At the 10-minute point, 1 mM L-arginine (Arg) was added into the apical buffer, and, at 25 minutes, cAMP stimulation (Stim) occurred due to 10 µM FSK and 1 µM VX-770 addition. At 45 minutes, CFTR-specific inhibitor, CFTR_inh-172 (Inh172), was added to confirm CFTR-specific function. (E) Representative trace shows Ussing chamber measurements of CFTR function in nasal cell culture from CF patients bearing F508del/F508del pretreated with VX-809 for 48 hours in absence (left) or presence of CB-1158 plus arginine (1 mM) for 1 hour (right). CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with 30 µM amiloride and cAMP activation with 10 µM FSK. The subsequent addition included 10 µM CFTRinh-172 to determine CFTR-specific activity. (F) Bar graph showing mean (±S.D.) FSK and VX-770–activated short-circuit transepithelial current (I_SC) for nasal cultures from three patients bearing F508del/F508del mutation, after pretreatment of 48 hours with VX-809 (3 µM) CB-1158 (1 µM) (mean S.D.). Unpaired t test was performed (**P = 0.0059, N = 3 biologic replicates, n = 1 technical replicate). For VX-809 or VX-809 plus CB1158 pretreatment, the confidence limits were 2.4–4.4 and 4.6–8.9 µA/cm², respectively, using a confidential level of 95%.

Inhibition of arginase activity enhances rescue of CBF at the surface of primary respiratory epithelial tissues by VX-809 plus VX-770. Top panel: Bar graph represents CBF (Hertz) on F508del-CFTR primary bronchial cells with 48-hour chronic treatment of either DMSO or 3 µM VX-809 (mean S.D.). Cells were pretreated with 1 mM L-arginine, 10 µM CB-1158, 10 µM ABH, or buffer in the HBSS solution following 1 hour of equilibration and 30 minutes of either 10 µM FSK (first bar) or 10 µM FSK with 1 µM VX-770 (remaining bars) apical addition to the HBSS. WT baseline is based on the mean FSK-stimulated values of eight individual WT-CFTR primary bronchial cells. One-way ANOVA with Tukey’s multiple comparison test was performed when comparing the first bar (VX-809 + VX-770) with the rest of the conditions (*P < 0.05; ****P < 0.0001, N > 3 biologic replicates, n = 2 technical replicates). Bottom panel: Bar graph represents FSK-stimulated CBF (Hertz) of the fully differentiated (day 30 postseeding) F508del-CFTR primary nasal cells with 48-hour chronic treatment of either DMSO or 3 µM VX-809 (mean S.D.). Cells were pretreated with 1 mM L-arginine, 10 µM CB-1158, 10 µM ABH, or buffer in the HBSS solution following 1 hour of equilibration and 30 minutes of either 10 µM FSK (first bar) or 10 µM FSK with 1 µM VX-770 (remaining bars) apical addition to the HBSS. One-way ANOVA with Tukey’s multiple comparison test was performed (ns = not statistically significant, ***P = 0.001; ****P < 0.0001, N = 3 biologic replicates, n = 2 technical replicates).

Velocity and pattern of bead movement are defective in F508del respiratory cultures. (A) Left: Representative MCCV of a WT bronchial epithelial insert. Each color represents an individual bead movement over the course of 10 seconds following 1-hour equilibration of WT-CFTR bronchial epithelial inserts in HBSS solution containing 0.02% polystyrene microspheres. MCCV appears as a mostly homogenous circular motion. Right: Representative MCCV of a F508del-CFTR bronchial epithelial insert shows less movement of each individual bead compared with the WT bronchial epithelial insert. (B) Bar graph represents the raw MCCV (micrometers per second) of either WT-CFTR or F508del-CFTR primary bronchial cells with 48-hour chronic treatment of DMSO (mean ± S.D.). Cells were pretreated with buffer in the HBSS solution containing 1 µm green or red polystyrene microspheres (0.02%) following 1 hour of equilibration. Unpaired t test was performed (****P < 0.0001, N > 8 biologic replicates, n = 1 technical replicate).

Inhibition of arginase activity enhances rescue of MCCV on the surface of primary respiratory epithelial tissues by VX-809 plus VX-770. (A) Bar graph represents MCCV (micrometers per second) of F508del-CFTR primary bronchial cells with 48-hour chronic treatment of either DMSO or 3 µM VX-809 (mean S.D.). Cells were pretreated with 1 mM L-arginine, 10 µM CB-1158, 10 µM ABH, or buffer in the HBSS solution containing 1 µm green or red polystyrene microspheres (0.02%) following 1 hour of equilibration and 30 minutes of either 10 µM FSK or 10 µM FSK with 1 µM VX-770 apical addition to the HBSS. WT baseline is based on the mean FSK-stimulated values of eight individual WT-CFTR primary bronchial cells. The MCCV values were normalized to each biologic replicate’s respective VX-809 plus VX-770 treatment. One-way ANOVA with Tukey’s multiple comparison test was performed (**P < 0.007; ****P < 0.0001, N > 4 biologic replicates, n = 3 technical replicates). (B) Connected line plot represents the FSK-stimulated MCCV (micrometers per second) of the fully differentiated (day 30 postseeding) F508del-CFTR primary nasal cells treated with 3 µM VX-809 following equilibration in HBSS solution containing 1 µm polystyrene microspheres (0.02%) for 1 hour. Each dot represents an individual biologic replicate (n = 2 to 3 technical replicates). Paired t test was performed (*P < 0.05, N = 3 biologic replicates, n = 2 technical replicates). (C) Bar graph represents MCCV of the fully differentiated (day 30 postseeding) F508del-CFTR primary nasal cells with 48-hour chronic treatment of either DMSO or 3 µM VX-809 (mean S.D.). Cells were pretreated with 1 mM L-arginine, 10 µM CB-1158, 10 µM ABH, or buffer in the HBSS solution containing 1 µM green or red polystyrene microspheres following 1 hour of equilibration and 30 minutes of either 10 µM FSK or 10 µM FSK with 1 µM VX-770 apical addition to the HBSS. The MCCV values were normalized to each biologic replicate’s respective VX-809 plus VX-770 treatment. One-way ANOVA with Tukey’s multiple comparison test was performed (ns = not statistically significant, *P < 0.05, N = 3 biologic replicates, n = 2 to 3 technical replicates).

Restoration of normal channel function translates to complete rescue of CBF but only partial rescue of MCCV in VX-809 plus VX-770–treated bronchial epithelia from patients homozygous for F508del. CFTR channel activity, CBF, and MCCV outcome measurements of three conditions treated with 10 µM FSK: 48-hour DMSO (red), 48-hour VX-809 plus VX-770 (blue), and 48-hour VX-809 plus VX-770 with CB-1158 and arginine (green), which were normalized to experimental WT controls and plotted against channel activity. Left: CFTR channel activity was plotted against CBF, and the equation of the line graph was generated by calculating linear regression using Spearman’s correlation test (****P < 0.0001, r = 0.867). Right: CFTR channel activity was plotted against MCCV, and the equation of the line graph was generated by calculating linear regression using Spearman’s correlation test (r = 0.8854).