Use of kinase inhibitors to correct ΔF508-CFTR function

Abstract

The most common mutation in cystic fibrosis (CF) is a deletion of Phe at position 508 (ΔF508-CFTR). ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma membrane. To identify compounds and drugs that rescue this trafficking defect, we screened a kinase inhibitor library enriched for small molecules already in the clinic or in clinical trials for the treatment of cancer and inflammation, using our recently developed high-content screen technology (Trzcinska-Daneluti et al. Mol. Cell. Proteomics 8:780, 2009). The top hits of the screen were further validated by (1) biochemical analysis to demonstrate the presence of mature (Band C) ΔF508-CFTR, (2) flow cytometry to reveal the presence of ΔF508-CFTR at the cell surface, (3) short-circuit current (Isc) analysis in Ussing chambers to show restoration of function of the rescued ΔF508-CFTR in epithelial MDCK cells stably expressing this mutant (including EC(50) determinations), and importantly (4) Isc analysis of Human Bronchial Epithelial (HBE) cells harvested from homozygote ΔF508-CFTR transplant patients. Interestingly, several inhibitors of receptor Tyr kinases (RTKs), such as SU5402 and SU6668 (which target FGFRs, VEGFR, and PDGFR) exhibited strong rescue of ΔF508-CFTR, as did several inhibitors of the Ras/Raf/MEK/ERK or p38 pathways (e.g. (5Z)-7-oxozeaenol). Prominent rescue was also observed by inhibitors of GSK-3β (e.g. GSK-3β Inhibitor II and Kenpaullone). These results identify several kinase inhibitors that can rescue ΔF508-CFTR to various degrees, and suggest that use of compounds or drugs already in the clinic or in clinical trials for other diseases can expedite delivery of treatment for CF patients.

Fig. 1.

Representative hits of the high-content screen. Average normalized fluorescence intensity values of ΔF508-CFTR cells (which co-express eYFP(H148Q/I152L) that were (A) transfected with shRNA for FGFR1, or (B) treated with 10 μm SB431542 (noncorrector), (C) (5Z)-7-Oxozeaenol, (D) SU5402, (E) GSK-3β Inhibitor II, (F), RDEA119, (G) Kenpaullone, (H), Ki8751, and grown at 37 °C. After 48 h (5 days in case of shRNA knockdown of FGFR1) cells were stimulated with FIG (25 μm Forskolin, 45 μm IBMX and 50 μm Genistein), and quenching of fluorescence during Cl⁻/I⁻ exchange of 70–300 cells was quantified simultaneously and recorded. FGFR1 knockdown was 90% (as determined by RT-qPCR). One representative shRNA clone out of 8 is shown.

Fig. 2.

Effect of select kinase inhibitors on ΔF508-CFTR maturation analyzed by immunoblotting. 293MSR-GT cells stably expressing ΔF508-CFTR were treated with 15 μm kinase inhibitors or 0.3% DMSO (vehicle control), as indicated, grown at 37 °C for 48 h, and the appearance of the mature protein, band C, monitored by immunoblotting with anti-CFTR antibodies. Band B represents the immature protein. DMSO represents vehicle-alone control, 27 °C represents temperature rescue of ΔF508-CFTR at 27 °C, 37 °C represents untreated ΔF508-CFTR control, and WT represents WT-CFTR. Top panels depict the anti-CFTR immunoblot and bottom panels depict actin (loading) control. ** represents cellular toxicity.

Fig. 3.

Effect of kinase inhibitors on cell surface expression of ΔF508-CFTR analyzed by flow cytometry. BHK cells stably expressing ΔF508-CFTR-3HA were placed at (A) 27 °C (positive control) for 48 h, or (B) treated with 10 μm (5Z)-7-oxozeaenol, (C) SU5402, (D) SU6668, or (E) RDEA-119/AR-119/BAY869766, at 37 °C. (F) BHK cells stably expressing WT-CFTR. Flow cytometry was then performed on nonpermeabilized cells following immunostaining for the HA epitope located at the ectodomain of ΔF508-CFTR or WT-CFTR, to quantify the amount of cell-surface CFTR in the analyzed cells. (G) Summary of increase in cell surface expression of ΔF508-CFTR (% change in fluorescence intensity) of the hits analyzed by flow cytometry (two independent experiments, 10,000 live cells per treatment per experiment).

Fig. 4.

Effect of compounds treatment on the ΔF508-CFTR channel activity in the MDCK cells stably expressing ΔF508-CFTR. Representative short-circuit current (Isc) traces of MDCK ΔF508-CFTR monolayers treated with vehicle (DMSO) alone, or 10 μm (A) (5Z)-7-oxozeaenol, (B) SU5402, (C) SU6668, (D) GSK-3β Inhibitor II, (E) 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine (C8863), for 48 h prior to analysis in Ussing chambers. ENaC sodium channels were inhibited with 10 μm amiloride; non-CFTR chloride channels were blocked with 250 μm DNDS. CFTR currents were stimulated with FIG (25 μm Forskolin, 25 μm IBMX and 50 μm Genistein) at time 0 and after the indicated times (arrows) inhibited using 15 μm GlyH-101 (Gly). F, Representative short-circuit currents mediated by MDCK cells that stably express WT-CFTR. G, Summary of the increase in short-circuit currents (ΔIsc) in MDCK cells stably expressing ΔF508-CFTR that were treated by the analyzed compounds (relative to DMSO vehicle control alone). Data are mean ± S.E. (n): number of experiments.

Fig. 5.

Effect of compounds treatment on ΔF508-CFTR activity in primary Human Bronchial Epithelial (HBE) cells harvested from lungs of ΔF508/ΔF508 homozygote patients undergoing lung transplant. Representative short-circuit currents (Isc) mediated by ΔF508-CFTR human bronchial epithelial (HBE) monolayers treated with vehicle (DMSO) alone, or 10 μm (A) (5Z)-7-oxozeaenol, (B) SU5402, (C) SU6668, (D) GSK-3β Inhibitor II, (E) 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine (C8863), (F) Kenpaullone, for 48 h prior to analysis in Ussing chambers. ENaC sodium channels were inhibited with 10 μm amiloride; non-CFTR chloride channels were blocked with 250 μm DNDS. CFTR currents were stimulated with FIG (25 μm Forskolin, 25 μm IBMX and 50 μm Genistein) as indicated, and after the indicated times (black arrows) inhibited using 15 μm (panels A, B, E, F) or 50 μm (panels C, D) GlyH-101 (Gly). In panels C and D, half of the Gly solution (25 μm) was added twice sequentially, as indicated. G, Representative short-circuit currents mediated by HBE cells from non-CF controls (WT-CFTR). H, Summary of increase in short-circuit currents (ΔIsc) in HBE cells stably expressing ΔF508-CFTR that were treated by analyzed compounds. Data from individual patients are shown. Where several replica were tested from the same patient (see Table I), the average value is shown. Bars represent median values. The baseline currents (before amiloride addition) ranged between 6–20 μAmp/cm² for WT-HBE and 19–40 μAmp/cm² for ΔF508-CFTR HBE. After adding amiloride, the currents for both WT and ΔF508-CFTR HBE were ∼0–3 μAmp/cm².

Fig. 6.

Dose response curves of select kinase inhibitors for rescue of ΔF508-CFTR expressed in the MDCK cells. Average increase in short-circuit currents (ΔIsc) of MDCK cell monolayers stably expressing ΔF508-CFTR (relative to DMSO vehicle control alone) treated for 48 h with 1, 10, 20, 100, 200, 1000, and 10,000 nm of the top inhibitor compounds, indicated in panels (A–K). Data are mean ± S.E. of (n) samples.