Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product

Abstract

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR₁₂₈₁, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR₁₂₈₁ activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR₁₂₈₁, functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR₁₂₈₁-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR₁₂₈₁, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR₁₂₈₁ function ∼8-fold over that of VX-770 alone, normalizing CFTR₁₂₈₁ channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.

Keywords: cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); drug discovery; drug screening; small molecule.

FIGURE 1.

Biochemical rescue of CFTR₁₂₈₁ by correctors in CFBE cells. A, schematic of CFTR showing site of W1282X premature termination codon in nucleotide binding domain 2 (top). Wild type, mutated (W1282X-CFTR), and truncated (CFTR₁₂₈₁) expression constructs were used in this study (bottom). B, immunoblot of wild type CFTR (WT, one-fifth the amount of protein loaded), W1282X-CFTR, and CFTR₁₂₈₁ in CFBE cells. Arrowheads represent core-glycosylated (black arrowheads) and complex-glycosylated (open arrowheads) CFTR. C, immunoblot of wild type CFTR, W1282X-CFTR, and CFTR₁₂₈₁ in CFBE cells in response to ΔF508-CFTR correctors (3 μm VX-809 and 10 μm C4) and putative modulators of read-through (200 μg/ml G418, 200 μg/ml gentamycin, and 10 μm PTC124). Arrowheads represent core-glycosylated (black) and complex-glycosylated (open) CFTR. D, surface presentation of W1282X-CFTR and CFTR₁₂₈₁ in response to CFTR modulators and PTC124 measured by live cell ELISA (mean ± S.E., ANOVA with Dunnett's post hoc test compared with control (DMSO-treated) cells, *, p < 0.01). E, metabolic pulse-chase analysis of CFTR₁₂₈₁ maturation in response to VX-809. Right, quantification of VX-809 effect on CFTR₁₂₈₁ maturation (mean ± S.E., t test, *, p < 0.01). Data shown in B–E are representative of at least triplicate experiments. p, pulse; ch, chase.

FIGURE 2.

Functional rescue of CFTR₁₂₈₁ by known correctors and potentiators. A, design of assays to identify correctors (left) and potentiators (right). B, original YFP fluorescence quenching data from W1282X-CFTR-expressing FRT cells treated with the indicated CFTR modulators. Concentrations used are as follows: sodium butyrate (3 mm), VX-809 (3 μm), fsk (10 μm), genistein (gen, 50 μm) and VX-770 (5 μm). C, summary (mean ± S.E., n = 3–6) for experiments as in B in response to a panel of known CFTR correctors (at 3 μm) and potentiators (at 5 μm). Cells were treated with forskolin plus VX-770 for corrector assays, and cells were corrected with VX-809 and treated with forskolin for potentiator assays. Statistical analysis by ANOVA with Dunnett's post hoc test: for correctors, data were compared with cells treated with fsk/VX-770; for potentiators, data were compared with cells treated with fsk/VX-809 (*, p < 0.05; **, p < 0.001). D, original data (left) and summary (right; mean ± S.E., n = 3–6) for CFTR₁₂₈₁-mediated YFP quenching in FRT cells that are uncorrected or treated with correctors and/or potentiators (as in C). Statistical analysis as in C. Dashed lines in C and D represent unstimulated and maximal responses, respectively.

FIGURE 3.

Novel CFTR₁₂₈₁ correctors with greater efficacy than VX-809. A, chemical structures of novel correctors identified by screening and that of VX-809. B, concentration-dependent CFTR correction in FRT cells expressing CFTR₁₂₈₁ by W1282X_corr-A23 and W1282X_corr-B09 (mean ± S.E., n = 3–4). Dashed line represents response to VX-809 (3 μm). C, top, CFTR cell-surface presentation in FRT cells expressing W1282X-CFTR-3HA measured by chemiluminescence using a horseradish peroxidase-coupled antibody labeling assay (mean ± S.E., n = 10–24). Bottom, W1282X read-through in response to correctors (3 μm) and G418 (1 mg/ml) measured using a luciferase-based reporter in FRT cells (mean ± S.E., n = 6–30, ANOVA with Dunnett's post hoc test compared with control data, *, p < 0.05; **, p < 0.001). D, structure-activity analysis of class A correctors. E, short-circuit current measurement of corrector action in CFTR₁₂₈₁-expressing FRT cells. Data are shown for W1282X_corr-A23 (black traces) and VX-809 (gray trace; 10 μm). Data are representative of triplicate experiments. F, efficacy of correctors identified in the W1282X screen on ΔF508-CFTR. YFP fluorescence quenching data for ΔF508-CFTR-expressing FRT cells treated with W1282X_corr-A23 or VX-809 (3 μm). G, relative efficacy of correctors from classes A, B, and C (at 3 μm, 24 h) in CFTR₁₂₈₁ and ΔF508-CFTR-expressing FRT cells (mean ± S.E., n = 4–12). Data were normalized to VX-809 efficacy. Compound structure provided in Table 1. In B, E, F, and G cells were stimulated with forskolin (10 μm) and VX-770 (5 μm). CFTR_inh-172 was used at 10 μm in all studies.

FIGURE 4.

Novel CFTR₁₂₈₁ potentiators. A, left, concentration dependence of VX-770 in VX-809-corrected (3 μm, 24 h) CFTR₁₂₈₁ (left) and ΔF508-CFTR (right)-expressing FRT cells. B, chemical structures of potentiators identified in the screen, compared with VX-770. C, concentration-dependent CFTR₁₂₈₁ activity in FRT cells for indicated potentiators (mean ± S.E., n = 3–4). Dashed line indicates CFTR₁₂₈₁ activation produced by VX-770 (5 μm). Cells were corrected with VX-809 (3 μm, 24 h). D, short-circuit current in CFTR₁₂₈₁-expressing FRT cells in response W1282X_pot-A15 (top) or W1282X_pot-C01 (bottom). In all studies forskolin was used at 10 μm, and cells were corrected with VX-809 (3 μm, 24 h). Data in A and D are representative of 3–4 experiments.

FIGURE 5.

Marked synergy of CFTR₁₂₈₁ potentiators with VX-770. A, potentiator (25 μm) activity in the absence and presence of VX-770 (5 μm) in CFTR₁₂₈₁ (top) and ΔF508-CFTR (bottom)-expressing FRT cells measured by YFP quenching (mean ± S.E., n = 6–8, ANOVA with Dunnett's post hoc test compared with cells treated with fsk/VX-770. *, p < 0.01; **, p < 0.0001). B, left, short-circuit current of CFTR₁₂₈₁ responses in FRT cells to VX-770 and W1282X_pot-A15; right, synergy of VX-770 (5 μm) and W1282X_pot-A15 (10 μm) is not dependent on order of addition. C, per-channel activity of CFTR₁₂₈₁ relative to wild type CFTR. CFTR current was measured in FRT cells expressing wild type CFTR-3HA (top left) or W1282X-CFTR-3HA (bottom left). W1282X-CFTR-3HA cells were corrected with W1282X_corr-A23 (3 μm) and stimulated with VX-770 (5 μm) and W1282X_pot-A15 (30 μm). Right, normalized per-channel activity of CFTR₁₂₈₁ in response to VX-770 without or with W1282X_pot-A15 relative to wild type CFTR (assigned a value of 1). D, structure-activity analysis of class A potentiators. E, I_sc of CFTR₁₂₈₁ responses to potentiator combination in CFBE cells. Representative I_sc recording (left) and average data (right; mean ± S.E., ANOVA with Dunnett's post hoc test, **, p < 0.0001). F, short-circuit current of CFTR₁₂₈₁ in FRT cells in response to W1282X_pot-B01 (left) or W1282X_pot-E01 (right) in the absence (top) and presence (bottom) of VX-770 (5 μm). In A, B, E, and F cells were corrected with VX-809 (3 μm, 24 h). Forskolin was used at 10 μm in A–C, and F and 20 μm in E. Data in B, C, and E are representative of 3–4 experiments. Data in F are representative of 2–3 experiments.

FIGURE 6.

Synergy with VX-770 of bioactive small molecules identified in a synergy screen. A, schematic of screen used to identify bioactive molecules that act in synergy with VX-770. CFTR₁₂₈₁-expressing FRT cells were corrected with VX-809, and test compounds were added with forskolin and VX-770. B, structures of bioactive compounds that act in synergy with VX-770. C, left, YFP fluorescence quenching in CFTR₁₂₈₁-expressing FRT cells in response to VX-770 alone (top trace) and with indicated compounds. Right, concentration dependence of CFTR₁₂₈₁ activity in response to VX-770 and indicated compounds (mean ± S.E., n = 3–6). gen, genistein; api, apigenin; kam, kaempferol; iso, isoliquiritigenin. D, compounds do not activate CFTR₁₂₈₁ in FRT cells alone and produce limited activity in the presence of forskolin (mean ± S.E., n = 3–6). Statistical analysis was by ANOVA with Dunnett's post hoc test. For treatment of cells with compounds alone, or with fsk and compounds, data are compared with fsk-alone treatment; for cells treated with fsk/VX-770 and compound, data are compared with cells treated with fsk/VX-770 (*, p < 0.05; **, p < 0.0001). E, short-circuit current in FRT cells expressing CFTR₁₂₈₁ in response to VX-770 (5 μm) and apigenin. F, dose-response of CFTR₁₂₈₁ activation in FRT cells by VX-770 and apigenin. G, left, CFTR₁₂₈₁ activation in CFBE cells by apigenin and VX-770 is independent of addition order and (right) is more efficacious than genistein. Statistical analysis was by ANOVA with Dunnett's post hoc test (*, p < 0.05; **, p < 0.001). In C–G cells were corrected with VX-809 (3 μm, 24 h) and stimulated with forskolin (10 μm). Data in E–G are representative of 3–5 experiments.

FIGURE 7.

Testing of correctors and potentiators in human nasal epithelial cells from a single homozygous W1282X CF subject. A, short-circuit current in primary cultures (left) and CRCs (right) from a non-CF subject. amil, amiloride. B, short-circuit current in primary cultures (left) and CRCs (right) from a homozygous ΔF508-CFTR subject with (black traces) or without (gray traces) VX-809 correction. gen, genistein. C, short-circuit current in primary cultures from a homozygous W1282X-CFTR subject without (gray trace) or with (black trace) VX-809 correction. D, short-circuit current in W1282X-CFTR CRCs without (gray trace) or with (black traces) VX-809 correction. In some experiments, cells were treated for 24 h with PTC124 (ataluren; 25 μm), amlexanox (25 μm), or geneticin (0.5 mg/ml). Concentrations used were amiloride (20 μm), forskolin (20 μm), genistein (50 μm), ATP (100 μm), W1282X_pot-A15 (10 μm), and VX-809 (3 μm, 24 h). E, CFTR transcript levels (mean ± S.E., n = 3) in nasal brushings (top left), and P0 cultures (top right), or CRCs (bottom left) from a non-CF subject (open bars) and a homozygous W1282X-CFTR subject (black bars) (bottom right). Comparison of CFTR transcript levels in P0 cultures and CRCs. Statistical analysis was by t test (*, p < 0.05). Data in A–D are representative of 3–4 experiments.