Use of Kinase Inhibitors to Correct ΔF508-CFTR Function*

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 EC50 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.

Cystic fibrosis (CF) 1 is a disease characterized by defective epithelial ion transport. In the lung airways, reduced Cl Ϫ transport caused by defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR), coupled with increased Na ϩ absorption caused by elevated activity of the Epithelial Na ϩ Channel (ENaC), result in dehydration and thickening of the mucosal fluid (1)(2)(3)(4). This predisposes patients to bacterial colonization, repeated pulmonary infections, and ultimately death. CF is associated with a wide-spread defect in the secretory processes of all secretory epithelia, including abnormalities in airways, gastrointestinal and genitourinary tracts, and elevated sweat electrolyte concentrations.
CF is caused by mutations in the cystic fibrosis gene (CFTR). CFTR encodes a 1480 amino acid polypeptide, called CFTR, which functions as a chloride channel in epithelial membranes (4 -6). Besides its function as a chloride channel, CFTR regulates other apical membrane conductance pathways, such as the Epithelial Na ϩ Channel, ENaC (1), and bicarbonate secretion (7). The CFTR protein in healthy individuals is found in the apical membrane of epithelial cells, which lines the airways, gastrointestinal tract, and other exocrine ducts in the body.
Although many (ϳ1900) mutations in CFTR have been identified to date (www.genet.sickkids.on.ca/cftr), the most common mutation found in Ͼ70% of patients of European ancestry is a deletion of phenylalanine at position 508 (⌬F508-CFTR) (8,9). The F508 deletion, located in the nucleotide binding domain 1 (NBD1) of CFTR, alters the folding and prevents the full maturation of the ⌬F508-CFTR protein, which is subsequently degraded in the proteasome very early during biosynthesis. This abnormal folding of the ⌬F508-CFTR mutant is thought to be responsible for its improper cellular localization. As ⌬F508-CFTR is a trafficking-impaired mutant that is retained in the ER, its level at the apical membrane is reduced dramatically, precluding proper Cl Ϫ secretion, which leads to CF (10 -13). Efforts to enhance exit of ⌬F508-CFTR from the ER and its trafficking to the plasma membrane are therefore of utmost importance for the development of treatment for this disease. Indeed, over the past few years several groups have identified a few small molecules that can correct the trafficking and functional defects of the ⌬F508 mutant, including corrector (corr)-3a and corr-4a, carboplatin, sildenafil, or its analogs glafenine, VX-325, VX-640, and in particular, the promising compound VX-809 (14 -20). However, although VX-809 was recently tested in a phase II clinical trial, its effectiveness in alleviating the lung disease of CF patients was rather limited, underscoring the urgent need to identify new correctors (21). We had previously developed a highcontent screen aimed at identifying proteins and small molecules that correct the trafficking defect of ⌬F508-CFTR using human HEK293 MSR GripTite cells that stably express ⌬F508-CFTR (22). Using this approach we recently performed a kinase inhibitor screen to identify kinases that, when inhibited, rescue ⌬F508-CFTR. Here we describe a screen of a kinase inhibitor library biased toward compounds that are already in the clinic or in clinical trials for the treatment of other diseases, such as cancer and inflammation. Our screen identified several small molecule kinase inhibitors (and their signaling cascades) that rescue ⌬F508-CFTR function, with some of these compounds already in clinical trials, thus potentially accelerating their use for the treatment of CF.

EXPERIMENTAL PROCEDURES
Media and Reagents-Dulbecco's Modified Eagle's Medium (DMEM), F12 nutrient mixture, Dulbecco's Phosphate Buffered Saline (D-PBS) with and without calcium or magnesium, fetal bovine serum (FBS), trypsin, G418, Blasticidin, and Zeocin were obtained from Invitrogen (Carlsbad, CA). SuperSignal West Femto Maximum Sensitivity kit was from Pierce (Rockford, IL), and Affinipure goat antimouse antibody (Cat.#115005062) was from Jackson Immuno-Research (West Grove, PA). The small molecules kinome library was obtained from the Ontario Institute for Cancer Research (OICR-see below). The mouse M3A7 anti-CFTR monoclonal antibody was obtained from Millipore (Billerica, MA), and the anti-␤-actin monoclonal antibody was from Sigma (A5441). Mouse anti-HA.11 monoclonal antibody was from Covance (MMS-101R), and Alexa Fluor 647-labeled goat anti-mouse antibody was from Invitrogen (A21236). The small molecules kinase inhibitors used for validation of the compound kinome screen were from Tocris (Bristol, UK), Selleck Chemicals and EMD Chemicals (San Diego, CA). shRNA TRC clones for FGFR1 knockdown were a kind gift from Dr. Jason Moffat (University of Toronto).
Small Molecules Kinase Inhibitor Library-The OICR Kinase Inhibitor Cassette that was screened contains 231 compounds that are reported to inhibit at least 68 kinases (supplemental Table S1). These inhibitors were purchased from a panel of more than 20 different vendors, or synthesized when not commercially available. The library was designed to cover as many targets and drug-like compounds as possible. In cases where there are multiple compounds targeting the same primary kinase, it was anticipated that having multiple chemotypes with different properties and selectivity profiles would enrich the screening set. Approximately 25% of the library consists of inhibitors that have made it into the clinic, an additional 25% being compounds in different phases of discovery (lead generation or optimization), and the remaining 50% are tool compounds that have not been advanced to the clinic but are known to be active inhibitors against various kinase targets.
Cellomics YFP Halide Exchange Screen-Cellomics halide exchange assay was performed as described previously (22). Briefly, 50,000 293MSR-GT cells (stably expressing eYFP(H148Q/I152L) (14) and ⌬F508-CFTR) per well were seeded in the 96-well plates. The next day ⌬F508-CFTR cells were treated (in triplicate) with 10 M small molecule kinome library, 0.2% DMSO (vehicle control), or corr-4a (positive control) at 37°C, or incubated at 27°C (positive control). A 10 M dose was chosen based on a preliminary screen data (not shown) as a dose that covers a wide range of inhibiting concentrations but is not toxic to ⌬F508-CFTR cells. After  , and a modified target activation algorithm, objects (individual cells or sometimes clusters of cells) were defined by eYFP(H148Q/I152L) fluorescence intensity, and the decrease in fluorescence intensity over 24-s time course, at 30°C, 5% CO 2 was recorded. The number of primary objects was used as an indicator of cell toxicity (cell death). Valid wells contained between 70 and 300 objects per field. After collecting and analyzing data, a second run of the screen was performed with compounds preselected based on the first run (ϳ100 compounds, each in triplicate).
Data Analysis-Compounds with a difference in fluorescence intensity between unstimulated (ϪFIG) and stimulated (ϩFIG) samples lower than 0.08 were rejected after the first run of the screen. The rest of the compounds were subjected to the secondary screen. Only the compounds that exhibited a difference in average fluorescence intensity between unstimulated and stimulated cells of at least 0.10 were further analyzed (supplemental Table S2). Compounds that displayed a difference in average fluorescence intensity of at least 0.17 were considered Tier I hits. Compounds that showed a difference in average fluorescence intensity lower than 0.17 were considered Tier II hits. Representative compounds of both groups were selected for further validation of the ⌬F508-CFTR rescue.
Knockdowns were validated by two-step RT-qPCR. Total RNA was isolated using the RNeasy 96 kit (Qiagen, Dorking, Surrey, UK), and cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real time PCR reactions were performed using Platinum® SYBR® Green qPCR-SuperMix-UDG (Invitrogen) and CFX96 Real-Time System (BioRad). Primers were obtained from Integrated DNA Technologies. For standard curves, real time PCR was performed on a fivefold dilution series DNA.
Flow Cytometry-The rescue of ⌬F508-CFTR was also validated by flow cytometry as described previously (22). Briefly, at 48 h after adding 10 M kinase inhibitor or 0.2% DMSO (vehicle control), BHK cells were trypsinized, washed, and resuspended in ice-cold FACS buffer (PBS supplemented with 2% FBS). To stain CFTR at the cell surface, cells were incubated with anti-HA.11 monoclonal antibody (1:25) or AF647-labeled goat anti-mouse antibody (1:200) as a control, for 1 h at 4°C. Subsequently, the cells were washed with the cold FACS buffer and incubated with AF647-conjugated goat anti-mouse antibody (1:200) at 4°C for 1 h. They were then washed as above and resuspended in FACS buffer with 1 g/ml propidium iodide (PI). The flow-cytometric analysis was performed using LSRII System (BD Biosciences). The data from 10,000 live (propidium iodide negative) cells were collected and analyzed with FlowJo v.7.6.4 software. Cell toxicity, as defined as Ͼ10% of cells staining positive for PI, was only observed for Ki8751 treatment. Alsterpaullone treatment resulted in altered cellular morphology (increased cell granularity and size) but not toxicity.
Short-circuit Current (Isc) Measurements in Ussing Chambers-Cell inserts or Snapwells, seeded with polarized MDCK or HBE cells (expressing ⌬F508 or WT -CFTR), were mounted on an Ussing chamber apparatus (Physiological Instruments, San Diego, CA) and studied under voltage clamp conditions as previously described (23)(24)(25). Briefly, ENaC channels were inhibited with 10 M amiloride (Sigma), and non-CFTR chloride channels were blocked with 250 M DNDS (4,4Ј-dinitrostilbene-2,2Ј-disulfonate, Sigma). CFTR currents were then stimulated using 25 M Forskolin, 25 M IBMX, and 50 M Genistein (FIG), and after the indicated time (min) inhibited using 15-50 M GlyH-101 (Gly). Data were recorded and analyzed using Analyzer 2.1.3. Dose-response analyses (EC 50 ) for the top kinase inhibitor hits were carried out with increasing inhibitor doses between 1 nM to 10 M, applied to MDCK cells stably expressing ⌬F508-CFTR. A few of the tested compounds (PKC412, GDC0941, PD184352, Go6976, Alsterpaullone, Kenpaullone) were toxic to MDCK cells, resulting in loss of cell monolayer integrity and loss of resistance, detected in the Ussing chambers. These were thus excluded from the data analysis.

Identification of Kinase Inhibitors that Correct ⌬F508-CFTR
Function Using the YFP High-content Assay-To systematically identify proteins and pathways that are responsible for correction of the trafficking defect of ⌬F508-CFTR we previously developed a high-content functional assay or screen that allows for the identification of proteins and small molecules that correct ⌬F508-CFTR function in multiple individual cells simultaneously, using Cellomics VTI Array Scan technology (22). For this, we generated cell lines in which halidesensitive eYFP(H148Q/I152L) mutant (14) was stably transfected into HEK293 MSR GripTite (293MSR-GT) cells stably expressing wild type CFTR (WT-CFTR) or ⌬F508-CFTR (22). Expression of WT-CFTR and ⌬F508-CFTR in these cells was verified by immunoblotting with antibodies to CFTR (supplemental Fig. S1A), and expression of eYFP(H148Q/I152L) was verified by fluorescence microscopy (supplemental Fig. S1B).
Validation of the Hits Maturation of ⌬F508-CFTR-To further analyze and validate the hits, we employed alternative methods to demon-  (Table I and supplemental Table S2). First, we tested for the appearance of a mature ⌬F508-CFTR protein represented by band C in an immunoblot. Fig. 2 shows that ⌬F508-CFTR migrates primarily as a 140 -150 kDa protein (band B) when analyzed by SDS-PAGE, whereas the mature wild type CFTR protein migrates primarily as a 170 -180 kDa protein (band C). Treatment of ⌬F508-CFTR cells with the indicated compounds (at 15 M) led to the appearance of the mature band C in some of them, similar to that observed following low temperature (27°C) treatment (albeit not as robustly). As 293MSR-GT cells showed increased sensitivity toward some of the analyzed compounds (see supplemental Table  S2) because of drug toxicity, we were unable to successfully test these compounds by immunoblotting. Moreover, we wanted to directly demonstrate the presence of ⌬F508-CFTR at the plasma membrane. Therefore, we tested the appearance of ⌬F508-CFTR protein at the plasma membrane of nonpermeabilized BHK cells (which are not as sensitive to those compounds as the 293MSR-GT cells) using flow cytometry. BHK cells stably expressing ⌬F508-CFTR-3HA were treated with 10 M kinase inhibitors or 0.2% DMSO (vehicle control), or grown at 27°C (positive control) for 48 h. Flow cytometry was then performed on nonpermeabilized cells following immunostaining for the HA epitope located at the ectodomain of ⌬F508-CFTR, to quantify the amount of cellsurface ⌬F508-CFTR. Fig. 3 depicts different degrees of ⌬F508-CFTR cell surface expression obtained in cells treated with (5Z)-7-oxozeaenol, SU5402, SU6668, RDEA-119/AR-119/BAY869766 and other compounds.

Functional Analysis of Correction of ⌬F508-CFTR by the
Kinase Inhibitors-To determine whether treatment with the above kinase inhibitors can lead to correction of ⌬F508-CFTR function, we performed short-circuit current (Isc) analysis in Ussing chambers on epithelial MDCK cells that stably express ⌬F508-CFTR (supplemental Fig. S1C) to assess CFTR chloride channel activity. MDCK cells were treated with 10 M kinase inhibitor or 0.2% DMSO (vehicle control) and grown at 37°C for 48 h. The effect of treatment with representative compounds (e.g. (5Z)-7-oxozeaenol, SU5402, SU6668, GSK-3␤ Inhibitor II, 7-Cyclopentyl-5-(4-phenoxyphenyl)-7Hpyrrolo[2,3-d]pyrimidin-4-ylamine/C8863), and others on ⌬F508-CFTR trafficking and function (i.e. chloride channel activity) is shown in Fig. 4 (A-E), and summarized in Fig. 4G, demonstrating various degrees of functional rescue of ⌬F508-CFTR with several of these hit compounds. We noted small channel activity ("leakage") of untreated ⌬F508-CFTR at 37°C (Fig. 4F), suggesting some escape from the ER of this mutant. DMSO alone did not add further to this "leakage," indicating that at the concentration used in our experiments (0.2%), it does not contribute to rescue of ⌬F508-CFTR (This is different from the observed rescue by 2% DMSO reported earlier (26)).

Effect of Kinase Inhibitors on ⌬F508-CFTR Chloride Channel Activity in Primary Human Bronchial Epithelial (HBE) Cells
Harvested from a CF Patient-Although the above compounds appear to rescue ⌬F508-CFTR in tissue culture cells, it is essential to determine if they can also rescue function in HBE cells harvested from CF patients. To this end, we investigated the effect of treatment with select validated kinase inhibitors in primary cultures of HBE cells obtained from transplant patients homozygous for the ⌬F508-CFTR mutation. The effect of compound treatment was compared with control (vehicle alone) on monolayers obtained from the same patient,

FIG. 2. Effect of select kinase inhibitors on ⌬F508-CFTR maturation analyzed by immunoblotting.
293MSR-GT cells stably expressing ⌬F508-CFTR were treated with 15 M 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 ⌬F508-CFTR at 27°C, 37°C represents untreated ⌬F508-CFTR control, and WT represents WT-CFTR. Top panels depict the anti-CFTR immunoblot and bottom panels depict actin (loading) control. ** represents cellular toxicity. which allowed us to eliminate the influence of patient-topatient variability. Fig. 5 (A-F) shows examples from several ⌬F508/⌬F508 patients (as well as two control "normal" individuals expressing WT-CFTR, panel G), demonstrating enhanced activity of the mutant CFTR after treatment of cells with (5Z)-7-oxozeaenol, SU5402, SU6668, GSK-3␤ Inhibitor II, 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo [2,3-d]pyrimidin-4-ylamine (C8863), and Kenpaullone. Other com-pounds exhibited very little or variable rescue (Fig. 5H). These findings suggest that cell surface expression of ⌬F508-CFTR is enhanced in HBE cells by delivering small molecule kinase inhibitors designed to correct the trafficking or maturation defect of this mutant protein, although from these latter results we cannot preclude the possibility that these compounds also potentiate ⌬F508-CFTR activity once at the plasma membrane. All validation data are summarized in Table I and supplemental Table S2.
Dose Response Curves of Rescue of ⌬F508-CFTR in MDCK Cells Treated with Select Kinase Inhibitors-To assess the effect of increasing doses of kinase inhibitors on correcting ⌬F508-CFTR function, we treated MDCK cells (stably expressing ⌬F508-CFTR) with increasing concentrations (1 nM to 10 M range) of the top hit compounds prior to Isc analysis in Ussing chambers ( Fig. 6 and Table I). The estimated half maximal effective concentrations (EC 50 ) of the analyzed compounds were in the nanomolar range (with an exception of SU6668). PD17304 and FPA124 showed rescue of ⌬F508-CFTR function only at a concentration of 10 M. Rescue of ⌬F508-CFTR by kinase inhibitors was also seen in other independently derived MDCK clones stably expressing ⌬F508-CFTR (e.g. supplemental Fig. S2 for (5Z)-7-oxozeaenol). Because of limited supply of cells from patients, we could not perform dose-response analyses of these compounds on HBE cells.

DISCUSSION
In this paper we report novel correctors (kinase inhibitors) of the ⌬F508-CFTR defect. In addition to performing the functional kinase inhibitor screen and functional validation using Isc in Ussing chambers, we carried out biochemical and flow cytometry analyses to demonstrate maturation and trafficking of ⌬F508-CFTR to the plasma membrane. Nevertheless, we cannot currently preclude the possibility that at least some of the kinase inhibitors we identified may also potentiate activity of rescued ⌬F508-CFTR.
The inhibitors present in the kinase inhibitor library chosen for our screen comprised a substantial number of compounds that are already used in the clinic or are in clinical trials for the treatment of other diseases, such as cancer and inflammation. Thus, potential use of any "hits" from the screen, once validated, can be moved to clinical trials for CF more quickly. Indeed, some of the identified kinase inhibitors that rescue ⌬F508-CFTR are already in clinical trials for the treatment of other diseases. For example, E6201, a (5Z)-7-Oxozeaenol derivative, is now in clinical trials for the treatment of cancer (phase I (ClinicalTrials.gov identifier: NCT00794781)) and Psoriasis (phase II (ClinicalTrials.gov identifiers: NCT01268527, NCT00539929)) (27,28). SU6668 (Orantinib) is currently in clinical trials for advanced solid tumors (phase I completed (ClinicalTrials.gov identifier: NCT00024206)) and AZD0530 (Saracatinib) is in phase II clinical trials for prostate, pancreatic, breast, colorectal, bone, and ovarian cancers (ClinicalTrials.gov identifiers: NCT01267266, NCT00735917, NCT00558272, NCT00397878, NCT00610714). RDEA119/ BAY869766 (in combination with Sorafenib (ClinicalTrials.gov identifiers: NCT01204177, NCT00785226) or Gemcitabine (ClinicalTrials.gov identifier: NCT01251640)) and PD0325901 (in combination with PF-04691502 (ClinicalTrials.gov identifier: NCT01347866)) are in clinical trials in late-stage cancer patients (phase II and phase I, respectively). Therefore, our identification of the kinase inhibitors that rescue ⌬F508-CFTR, which are already in clinical trials for other diseases, can accelerate use of these compounds for the treatment of CF patients, most of whom carry the ⌬F508 mutation.
Our results reveal that in HBE cells from CF patients, CFTR activity of ⌬F508-CFTR treated with several of the hit compounds (relative to the vehicle-treated controls) was ϳ10 -30% of untreated WT-CFTR (although in some cases ϳ50% rescue was observed). It should be kept in mind that even a partial correction of the ⌬F508-CFTR defect can be very beneficial, because several reports proposed that as low as 10 -25% rescue of CFTR activity may be sufficient to restore airway epithelial function (29,30), and CF patients and mice with residual CFTR activity exhibit milder disease than those lacking it altogether (31)(32)(33). Also, recent studies showed that a more complex approach is required to fully restore normal G, Representative short-circuit currents mediated by HBE cells from non-CF controls (WT-CFTR). H, Summary of increase in short-circuit currents (⌬Isc) in HBE cells stably expressing ⌬F508-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 Amp/ cm 2 for WT-HBE and 19 -40 Amp/ cm 2 for ⌬F508-CFTR HBE. After adding amiloride, the currents for both WT and ⌬F508-CFTR HBE were ϳ0 -3 Amp/cm 2 .
⌬F508-CFTR biogenesis (34). The proposed correction strategy employs a two-step folding model in which the correction of both ⌬F508-CFTR NBD1 stability and the NBD1-MSD2 domain interaction are necessary. These findings may explain the limited efficacy of ⌬F508-CFTR correctors currently undergoing clinical trials, and suggest that a combination drug therapy for a complete correction of the ⌬F508-CFTR defect may be required.
The vast majority of the top hit compounds reported in our paper rescue ⌬F508-CFTR at nanomolar concentrations. The estimated half maximal effective concentration (EC 50 ) for the analyzed molecules were calculated from the data of functional assays (Isc) using Ussing chambers, and they should not be directly compared with IC 50 values obtained from biochemical experiments. To better illustrate this point, SU6668 (Orantinib) corrects ⌬F508-CFTR function with EC 50 of ϳ1 M (Table I). In in vitro assays, SU6668 inhibits PDGFR␤, VEGFR2, and FGFR1 autophosphorylation at IC 50 values of 8 nM, 21 nM, and 1.2 M, respectively (35). For comparison, in cellular assays SU6668 inhibits VEGF-and PDGF-dependent signaling with IC 50 values of 0.5 and 1 M, respectively. The SU6668 pharmacokinetic analysis performed on animal plasma samples revealed an inhibition of VEGFR2 phosphorylation at concentrations of Ն 1 g/ml, whereas the pharmacokinetic profile of patients with advanced solid tumors showed the maximal plasma concentrations (C max ) of 3.0 g/ml (day 1) and 2.0 g/ml (day 22) at a fed dose of 200 mg/m 2 /day (36,37).
Our identification of several kinase inhibitors that promote rescue of ⌬F508-CFTR suggests that these kinases normally inhibit maturation or trafficking of this CFTR mutant, probably by affecting the function of specific chaperones. Although a large body of literature describes destabilization of numerous activated kinases and oncogenes by Hsp90 inhibitors (38), the effect of kinases and their downstream signaling proteins on chaperones has not been extensively studied.
ERK1/2 were shown to be potent inhibitors of Heat Shock Factor 1 (HSF-1) activity and thus have an inhibitory effect on production of heat shock proteins (Hsps) such as Hsp70 (50 -52). Inactive, monomeric HSF-1 exists in a complex with either Hsp70 (53) or Hsp90 (54). This repressed state of HSF-1 is maintained through inhibitory phosphorylation of the specific serine residues by ERK1/2, GSK-3, PKC␣, and PKC kinases (51,52). ERK1/2 role in the negative regulation of HSF-1 activity is mediated by its phosphorylation of HSF-1 on Ser307. This initial phosphorylation marks HSF-1 for a secondary phosphorylation on Ser303, which represses HSF-1 function. Ser303 is phosphorylated by glycogen synthase kinase 3 (GSK-3), which inactivates this transcription factor and inhibits subsequent expression of Hsps (51). Thus, HSF1 function is antagonized by ERK1/2 in concert with GSK-3 kinase activity.
As noted in Table I, three of our hits (FPA-124, PI-103, and 10-DEBC) target PI3K/Akt/mTOR signaling. So far, it is not known whether or how this pathway may regulate ⌬F508-CFTR maturation. Although Akt was shown to promote cAMP-mediated trafficking of WT-CFTR to the plasma membrane (72), its possible effect on trafficking of ⌬F508-CFTR that escaped the ER is unknown. Moreover, in some cases (e.g. AQP2, Norepinephrine transporter) Akt signaling actually decreases cell surface expression of plasma membrane proteins (73,74). Thus, how PI3K/Akt/mTOR inhibitors may promote rescue of ⌬F508-CFTR awaits future investigation.
In summary, our work here has demonstrated the rescue of ⌬F508-CFTR by several kinase inhibitors in 293MSR-GT cells, BHK cells, epithelial MDCK cells and, importantly, in primary HBE cells from CF patients. Because some of our identified kinase inhibitors that rescued ⌬F508-CFTR are already used in the clinic or are in clinical trials for the treatment of cancer or inflammatory disease, their potential testing or use for treatment of CF patient carrying the ⌬F508 mutation (i.e. the majority of CF patients) can be greatly expedited. Moreover, these kinase inhibitors may be useful for the treatment of other "trafficking diseases," in which proteins are stuck in the ER much like ⌬F508-CFTR.