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Rescue of F508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Vertex Pharmaceuticals, San Diego, California

Submitted 15 April 2005 ; accepted in final form 8 January 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl^– channel. The most common mutation results in a deletion of phenylalanine at position 508 (F508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of F508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores F508-CFTR-mediated Cl^– transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of F508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

high-throughput screening; human bronchial epithelia; Cl^– channels; corrector; potentiator

Pharmacological agents that increase PKA-regulated Cl^– channel gating of CFTR are called potentiators and include the isoflavone genistein (24). The efficacy of genistein in vivo, however, may ultimately be limited because of its low potency, rapid in vivo metabolism, and lack of selectivity (3, 24, 31). With the use of high-throughput screening (HTS), several different structural classes of potentiators have been identified (23, 29, 32, 62), including phenylglycine (38), sulfonamides (38), and tetrahydrobenzothiophenes (62). In contrast to potentiators, there are relatively few correctors, small molecules that rescue the folding and/or trafficking of F508-CFTR to increase the cell surface density of F508-CFTR. The most studied corrector to date is 4-phenylbutyrate (4-PBA), which produces a small increase in F508-CFTR cell surface density in vitro (44) but has shown poor efficacy in clinical trials (45). HTS efforts identified 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid diamides as potent (10 nM) modulators of CFTR activity in expression systems (22); however, these compounds have not been shown to be active in primary epithelia. Recently, Egan et al. (14) proposed that curcumin was a corrector of F508-CFTR; however, the activities of curcumin in vitro and in vivo are inconsistent (53). HTS approaches by Verkman and colleagues (37) have recently identified other structural classes of small-molecule correctors of F508-CFTR trafficking with potency in the micromolar range. Indeed, there is a continued need for potent and efficacious correction agents that act either alone or in combination with potentiators of channel gating to restore ion channel function of F508-CFTR. Here, we describe efficacious small molecules that rescue either the defective trafficking or gating of F508-CFTR in both recombinant cells and human bronchial epithelia (HBE) isolated from F508-homozygous patients.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Cell culture. NIH/3T3 mouse fibroblasts stably expressing wt-CFTR (wt-NIH/3T3) or F508-CFTR (F508-NIH/3T3) were cultured as previously described (11). HEK-293 cells expressing F508-CFTR or wt-CFTR were grown as previously described (51). Assay medium used for NIH/3T3 and HEK-293 experiments was HyQ CCM5 (HyClone, Logan, UT) with 1% heat-inactivated FBS. Non-CF and CF airway epithelia were isolated from bronchial tissue, cultured as previously described (19), and plated onto Costar Snapwell filters that were precoated with NIH/3T3-conditioned medium. After 4 days, the apical medium was removed, and the cells were grown at an air-liquid interface for >14 days before use. This resulted in a monolayer of fully differentiated columnar cells that were ciliated, features that are characteristic of airway epithelia. Non-CF HBE were isolated from nonsmokers who had no known lung disease. CF-HBE were isolated from patients homozygous for F508-CFTR.

HTS assay. NIH/3T3 cells expressing F508-CFTR were used to develop a HTS assay to identify modulators of F508-CFTR. To monitor compound-induced changes in anion flux through CFTR, we used voltage-sensitive assays based on the change in fluorescence resonance energy transfer between a membrane-soluble voltage-sensitive dye, bis-(1,2-dibutylbarbituic acid)trimethine oxonol [DiSBAC2(3)] and a plasma membrane-localized fluorescent, coumarin-linked phospholipid (CC2-DMPE; Invitrogen, Madison, WI) (20). We excited the coumarin-lipid donor at 405 nm using a 300-W xenon arc lamp directed toward the cells with a 425-nm dichroic; the emitted light was passed through 460-nm and 580-nm filters, which we collected at 1 Hz using photomultiplier tubes. The data were normalized to a starting ratio of 1 and reported as R_F/R_I (where R_F is final response and R_I is initial response).

For the corrector HTS assay, F508-CFTR-NIH/3T3 cells were preincubated with compound to allow for de novo synthesis and processing and trafficking to the membrane. After the preincubation, the compound was removed, and membrane potential changes in response to F508-CFTR activation by genistein and forskolin were monitored with a cell-based fluorescence assay of membrane potential (Fig. 1A). F508-CFTR-NIH/3T3 cells were seeded at a density of 10,000/well into poly-L-lysine-coated 384-well microtiter plates and incubated in assay medium (HyQ CCM5 medium with 1% FBS) with test compound (10 µM in 0.1% DMSO) or 0.1% DMSO (control) for 16 h at 37°C and 5% CO₂. Because low-temperature incubation is known to correct the trafficking of F508-CFTR (11), we used temperature correction to validate the assay and then as a positive control for correction. Microtiter plates containing F508-CFTR-NIH/3T3 cells were incubated for 16 h at 37°C with or without compound or at 27°C and 5% CO₂. Before activity was monitored, the compound was washed out with three changes of bath medium containing (in mM) 160 NaCl, 4.5 KCl, 2.0 CaCl₂, 1 MgCl₂, 10 D-glucose, and 10 HEPES (ph 7.4 with NaOH). The cells were then incubated with 20 µM CC2-DMPE for 30 min, rinsed three times, and then incubated with 3 µM DiSBAC2 (3) for 20 min with bath medium in the dark at room temperature. After the fluorescence resonance energy transfer-based membrane potential probes were loaded, the microtiter plates were placed into an integrated liquid handler and fluorescent detector (VIPR-II) to monitor changes in fluorescent intensity in response to membrane potential changes. After baseline readings were obtained, Cl^–-free medium and genistein were added to the bath to promote Cl^– efflux and potentiate channel gating, respectively. Forskolin (10 µM) was then added to activate F508-CFTR; the maximum increase in absolute ratio of the R_F and R_I was monitored and expressed as R_F/R_I. As expected, the response in temperature-corrected cells was significantly higher than that in uncorrected cells (Fig. 1B). No response was observed in temperature-corrected NIH/3T3 cells not expressing CFTR. The statistical robustness of the assay was tested by running six plates on 3 days, with one-half of the plates incubated for 16 h at 27°C and the other one-half at 37°C. From data shown in Fig. 1C, the 1-z' screening window parameter (63) was 0.64, indicating that this assay is appropriate for HTS.

View larger version (16K): [in this window] [in a new window]   Fig. 1. High-throughput screening (HTS) assay validation and protocol in NIH/3T3 cells expressing F508-CFTR. A: schematic of the cell-based correction (top) and potentiator (bottom) HTS assay formats based on voltage-sensitive fluorescent probes. For the corrector HTS assay, cells were incubated in the presence of compound (CMPD) for 16 h before monitoring of forskolin (Fsk)-stimulated activity in the presence of Cl–-free medium and genistein (Genist). For the potentiator HTS assay, cells were incubated at 27°C for 16 h, and compounds from the screening library were added in place of genistein. B: membrane potential response [ratio of final response to initial response (RF/RI)] to Cl–-free medium + genistein addition (first arrow) and forskolin (FSK) addition (second arrow) in temperature-corrected () and uncorrected () F508-NIH/3T3 cells. C: assay window for the HTS was determined by monitoring the forskolin- and genistein-induced response in cells incubated at 37°C () and 27°C () for 16 h over 3 consecutive screening days.

(1)

where X is R_F/R_I in the presence of compound and where 27°C and DMSO is R_F/R_I for the positive and negative controls, respectively.

For the potentiator HTS assay, F508-CFTR-NIH/3T3 cells in assay medium were seeded at 30,000 cells/well into 384-well microtiter plates and incubated at 27°C for 16 h. The cells were subsequently washed, loaded with the fluorescent membrane potential probes, and placed into the VIPR-II as described above. After baseline readings were obtained, Cl^–-free medium and compound were added 15 s before 10 µM forskolin addition. The maximum increase in R_F/R_I was measured, and the data were normalized to the positive (genistein) and negative (DMSO-treated) controls using Eq. 2

(2)

where X is R_F/R_I in the presence of compound and where genistein and DMSO is R_F/R_I for the positive and negative controls, respectively. In this assay, genistein potentiated the forskolin-induced membrane depolarization by 73 ± 7% above the untreated control with an EC₅₀ of 19.2 ± 1.9 µM (n = 35).

cAMP measurements. The level of cAMP in NIH/3T3 cells after forskolin or test compound application was determined with an adenylyl cyclase activation FlashPlate assay, according to the manufacturer’s directions (Perkin-Elmer Life Sciences, Boston, MA). Briefly, NIH/3T3 cells were incubated for 30 min with forskolin or test compounds in the presence or absence of IBMX. The cells were then lysed and transferred along with the medium to a 96-well FlashPlate precoated with anti-cAMP antibody. A fixed concentration of radiolabeled cAMP was added to the plates and incubated overnight at 22°C. The FlashPlates were read in a microplate scintillation counter, and the cAMP concentrations were determined with a cAMP standard curve that was present in each plate.

Ussing chamber recordings. Fischer rat thyroid (FRT) epithelia expressing F508-CFTR or G551D-CFTR were grown on Costar Snapwell cell culture inserts for 4 days. The epithelia were then mounted in an Ussing chamber (Physiologic Instruments, San Diego, CA), and the monolayers were continuously short circuited with a voltage-clamp system (Department of Bioengineering, University of Iowa). Transepithelial resistance was measured by applying a 2-mV pulse. For measurement of short-circuit current (I_sc), the basolateral bath solution contained (in mM) 135 NaCl, 1.2 CaCl₂, 1.2 MgCl₂, 2.4 K₂HPO₄, 0.6 KHPO₄, 10 HEPES, and 10 dextrose (titrated to pH 7.4 with NaOH). To set up a basolateral-to-apical Cl^– concentration gradient, the basolateral membrane was permeabilized with nystatin (360 µg/ml). All experiments were performed 30 min after nystatin permeabilization. The apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large Cl^– concentration gradient across the epithelium. The solutions were maintained at 37°C and bubbled with air. The electrode offset potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the flow of Cl^– through CFTR expressed in the apical membrane. The I_sc was digitally acquired using an MP100A-CE interface and AcqKnowledge software (version 3.2.6; BIOPAC Systems, Santa Barbara, CA). Forskolin (10 µM) and all test compounds were added to both sides of the cell culture inserts.

HBE grown on Costar Snapwell cell culture inserts were mounted in an Ussing chamber (Physiologic Instruments), and the transepithelial resistance and I_sc in the presence of a basolateral-to-apical Cl^– gradient were measured with a voltage-clamp system (Department of Bioengineering, University of Iowa). Briefly, HBE were examined under voltage-clamp recording conditions (holding voltage = 0 mV) at 37°C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, and 10 HEPES (pH adjusted to 7.35 with NaOH), and the apical solution contained (in mM) 145 sodium gluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, and 10 HEPES (pH adjusted to 7.35 with NaOH).

Patch-clamp recordings. Total Cl^– current in F508-NIH/3T3 cells was monitored with the perforated-patch recording configuration as previously described (42). Voltage-clamp recordings were performed at 22°C using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). The pipette solution contained (in mM) 150 N-methyl-D-glucamine (NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 µg/ml amphotericin B (pH adjusted to 7.35 with HCl). The extracellular medium contained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, and 10 HEPES (pH adjusted to 7.35 with HCl). Pulse generation, data acquisition, and analysis were performed with a personal computer equipped with a Digidata 1320 A/D interface in conjunction with Clampex 8 (Axon Instruments). To activate F508-CFTR, 10 µM forskolin and 20 µM genistein were added to the bath, and the current-voltage relation was monitored every 30 s.

Gating activity of wt-CFTR and temperature-corrected F508-CFTR expressed in NIH/3T3 cells was observed with excised inside-out membrane patch recordings as previously described (8) using an Axopatch 200B patch-clamp amplifier (Axon Instruments). The pipette contained (in mM) 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2 MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bath contained (in mM) 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Tris base (pH adjusted to 7.35 with HCl). After excision, both wt-CFTR and F508-CFTR were activated by adding 1 mM MgATP, 75 nM of the catalytic subunit of PKA (Promega, Madison, WI), and 10 mM NaF to inhibit protein phosphatases, which prevented current rundown. The pipette potential was maintained at 80 mV.

Mutant hERG trafficking assay. The cell surface expression of a trafficking deficient mutant human ether-a-go-go (hERG) channel (G601S-hERG) was monitored with a hERG-Lite assay developed by ChanTest (Cleveland, OH) as described by Wible et al. (61). Briefly, HEK-293 cells expressing G601S-hERG with an HA epitope in the extracellular loop spanning transmembrane domains S1 and S2 were incubated for 16 h at 37°C with or without test compound. After preincubation, the cells were first incubated with rat anti-HA (1:500; Roche) for 2 h, after which they were rinsed three times and then incubated for 1 h with a secondary antibody cocktail containing; horseradish peroxidase-conjugated goat anti-rat IgG (Jackson Laboratories; 1:1,000) in blocking buffer plus SYBR green (Molecular Probes; 1:10,000). After cells were rinsed, SYBR green fluorescence was measured in a ThermoElectron Fluoroskan Ascent microplate reader (wavelengths: excitation, 485 nm; emission, 537 nm). To capture chemiluminescent signals, SuperSignal ELISA Femto maximum sensitivity substrate (Pierce, Rockford, IL; 100 ml/well) was added to each well. For each compound, the chemiluminescence signal in each well was normalized to the control (DMSO) to give a relative surface expression level. A significant increase in cell surface expression was observed when the test compound mean was above the vehicle control, mean ± 3 control SD.

Immunoblot analysis. HEK-293 cells expressing F508-CFTR or HBE natively expressing wt-CFTR or F508-HBE were incubated for 16 h at 37°C with or without test compound in assay medium. After incubation, cells were harvested in ice-cold PBS (without calcium and magnesium) plus 1 mM EDTA and pelleted at 1,000 g at 4°C. Cell pellets were lysed in 1% NP-40, 0.5% sodium deoxycholate, 200 mM NaCl, 10 mM Tris, pH 7.8, and 1 mM EDTA plus protease inhibitor cocktail (Sigma) used 1:250 for 30 min on ice. Lysates were spun clear for 10 min at 10,000 g at 4°C to pellet nuclei and insoluble material. Approximately 10 µg (HEK-293 cells) or 150 µg (HBE cells) total protein were heated in sample buffer without bromophenol blue at 37°C for 5 min and loaded onto a 3–8% Tris-acetate gel (Invitrogen). The gel was transferred to nitrocellulose and processed for Western blotting with monoclonal CFTR antibody, M3A7 (Upstate). Blots were developed by enhanced chemiluminescence. We quantified the relative amounts of bands B and C using NIH Image analysis of scanned autoradiograms.

Metabolic pulse-chase analysis. HEK-293 wt-CFTR and F508-CFTR cells were incubated for 16 h in assay medium with DMSO or compound. Metabolic labeling was performed as described (55) with some modifications. Cells were starved 30 min in DMEM without cysteine and methionine with 1% dialyzed FBS in the presence of compound. Cells were then pulsed with [³⁵S]methionine and cysteine EXPRES³⁵S³⁵ label (Perkin-Elmer) for 15 or 60 min (labeling times for individual experiments are noted in the figure legends). Cells were washed and chased in assay medium with compound for 0–23 h. At each time point, cells were harvested and lysed in RIPA buffer, and CFTR was immunoprecipitated with M3A7. Samples were separated by SDS-PAGE and analyzed by autoradiography. Radioactivity was quantified by Phosphorimager analysis (GE Healthcare, Piscataway, NJ).

Cell surface biotinylation. HEK-293 wt-CFTR and F508-CFTR cells were incubated for 16 h in assay medium with DMSO or compound. Cell surface glycoproteins were labeled with biotin-LC-hydrazide (Pierce) as described by Prince et al. (40). Cells were immediately harvested and lysed after labeling. Postnuclear supernatants were immunoprecipitated for CFTR by M3A7. Samples were separated by SDS-PAGE, and proteins were transferred to nitrocellulose. Biotinylated proteins were visualized by horseradish peroxidase-streptavidin (GE Healthcare) via enhanced chemiluminescence.

Real-time quantitative PCR. Cells were pretreated for 16 h with DMSO or compound in assay medium and harvested, and total RNA was extracted with RNeasy (Qiagen, Valencia, CA). cDNA was synthesized by iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR was carried out for CFTR and 18S transcripts with iQ SYBR Green Supermix (Bio-Rad) using the iCyler (Bio-Rad). CFTR primers used were forward (5'- GCAGCCTTACTTTGAAACTC-3') and reverse (5'AACAGCAATGAAGAAGATGAC-3'). Cycle thresholds were determined for CFTR and normalized to total mRNA.

Analysis of ubiquitin proteasome activity in vitro. The activity of the ubiquitin proteasome was monitored with a model substrate as previously described (54). Briefly, Jurkat cells expressing 2XUb--lactamase reporter were plated in 96-well plates at a density of 1.5 x 10⁶ cells/ml and incubated with the various concentrations of compounds. The cells were treated for 1 h with 100 µg/ml cycloheximide and then loaded with 1 µM CCF2-acetoxymethylester (AM) (Invitrogen). -Lactamase activity was measured by quantifying the fluorescence emission from the cells with a CytoFluor 4000 plate fluorimeter (PerSeptive Biosystems, Foster City, CA). Background-subtracted emission values at 460 and 530 nm were expressed as a ratio of 460 to 530 nm, where a high ratio represents high -lactamase activity.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
HTS assays for potentiators and correctors of F508-CFTR. To identify small-molecule modulators of F508-CFTR density and/or gating in the plasma membrane, we developed cell-based assays of membrane potential for either potentiators or correctors using NIH/3T3 cells expressing F508-CFTR (Fig. 1). The assays report F508-CFTR activity as a forskolin-stimulated depolarization in the presence of a Cl^– gradient. Two separate HTS assays were validated to detect potentiators or correctors using genistein and temperature correction, respectively (see MATERIALS AND METHODS for details).

Identification of F508-CFTR correctors. To identify novel chemical scaffolds that correct the trafficking of F508-CFTR to the cell surface, we screened 164,000 synthetic compounds at a final concentration of 10 µM. The library consisted of chemically diverse druglike and leadlike compounds obtained from several commercial vendors and internal medicinal chemistry programs. Individual compounds were selected for further testing if their activity was >2.5 SD from the mean, which corresponded to 32% of the 27°C control. This resulted in the identification of 1,028 compounds, 185 of which confirmed in retests at 10 µM, giving a positive hit rate for the assay of 0.11%. Of these, 67 compounds were removed because of impurities (<85% purity by LC/MS) and poor chemical attractiveness, including 1) high molecular weight and/or cLogP, 2) known toxic or reactive functionalities, 3) chemotypes with limited protein interaction sites, and 4) structures not amenable to relatively rapid analog synthesis. The remaining compounds were selected for dose-response analysis with the HTS assay and were counterscreened in NIH/3T3 cells that did not express F508-CFTR. A total of 108 compounds, comprising 13 structurally distinct scaffolds, were selected for further study. As independent confirmation of corrector activity, these compounds were then tested in the FRT epithelial cell line expressing F508-CFTR using Ussing chamber measurement of I_sc. Six of the 13 structurally distinct scaffolds identified in the HTS were confirmed in FRT-F508 epithelia (data not shown). None of the compounds was active in untransfected FRT cells that did not express F508-CFTR (data not shown). The quinazolinone shown in Fig. 2A (VRT-422) was among the most potent (EC₅₀ = 3.7 ± 0.9 µM) and efficacious (47 ± 4%, 27°C) compounds identified in the HTS (Fig. 2B; Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Identification of small-molecule correctors of F508-CFTR expressed in NIH/3T3 cells by HTS. A: structure of the primary hit (VRT-422) and the medicinal chemistry optimized hit (VRT-325) (experimental procedure for the preparation of VRT-325 is described in supplemental materials; see http://ajplung.physiology.org/cgi/content/full/00169.2005/DC1). B: membrane potential response (RF/RI) to FSK in 0, 0.75, 2.2, and 6.7 µM VRT-422-pretreated (a) and VRT-325-pretreated (b) (16 h) F508-NIH3T3 cells. The axis bars show the relative distance on the plots corresponding to 0.5 RF/RI and 60 s.

We next investigated whether the increase in F508-CFTR activity in response to either VRT-422 or VRT-325 corresponded to changes in F508-CFTR processing and transport to the cell surface. We conducted many of the biochemical studies in HEK-293 cells expressing F508-CFTR. This system yields a high expression of F508 CFTR, making it well suited for antibody-labeling studies (59). To evaluate effects on processing, we compared the glycosylation pattern of F508-CFTR using immunoblots of cell lysates from HEK-293 cells expressing F508-CFTR in the presence and absence of compound. Incubation of HEK-293 cells expressing F508-CFTR for 16 h in the presence of either VRT-422 or VRT-325 resulted in a dose-dependent increase in the steady-state levels of extensively glycosylated (150–170 kDa), mature F508-CFTR (so-called band C; Fig. 3A), which is indicative of passage through the Golgi complex (27). In addition, there was an increase in the steady-state levels of the core glycosylated form (so-called band B; Fig. 3A). Quantification of the amounts of F508-CFTR in the core-glycosylated (band B) and mature band C forms yielded an approximate EC₅₀ of 4.0 µM for VRT-422 and 0.8 µM for VRT-325 (Fig. 3B). Similar results were obtained in NIH/3T3 cells expressing F508-CFTR (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of VRT-422 and VRT-325 on the steady-state levels of core-glycosylated and mature F508-CFTR expressed in HEK-293 cells. A: glycosylation pattern of F508-CFTR in HEK-293 cells treated for 16 h with 0–20 µM VRT-422 (top) and VRT-325 (bottom) or incubated at 27°C for 16 h. B: dose response to VRT-422 and VRT-325. The ratio of band C to band B determined from quantification of scanned autoradiograms is plotted against the compound concentration, and each point represents the data from 3 separate replicates (means ± SE).

F508

F508

F508

View larger version (11K): [in this window] [in a new window]   Fig. 4. Quinazolinone correctors increase the cell surface density of F508-CFTR in the plasma membrane. A: cell surface biotinylation of wild-type (wt)-CFTR and F508-CFTR in HEK-293 cells after 16-h preincubation with DMSO or 6.7 µM VRT-325. B: representative current-voltage relationship for the FSK- and genistein-stimulated F508-CFTR current (IF508) in NIH/3T3 cells preincubated with DMSO (thin line) or 6.7 µM VRT-325 (thick line) for 16-h. Vm, membrane voltage. C: quantitative data of the IF508 in F508-CFTR-NIH/3T3 cells pretreated for 16 h with DMSO, 6.7 µM VRT-422, or 6.7 µM VRT-325 or incubated at 27°C for 16 h. The number of replicates is shown in the parentheses.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Quinazolinone correctors increase the persistence of core-glycosylated F508-CFTR in the endoplasmic reticulum. A: effect of corrector compounds on CFTR transcription. NIH/3T3 F508 cells were treated for 16 h in DMSO, 6.7 µM VRT-325, 6.7 µM VRT-422, or the known HDAC inhibitor sodium butyrate (NaButyrate). CFTR transcript levels were determined by RT-PCR and are shown normalized to total mRNA. B: effects of the correctors on cellular proteasome activity. A ubiquitin--lactamase fusion protein (2XUb--lactamase) was used to assess proteasome activity in Jurkat cells. Cellular -lactamase activity remaining after 1 h cycloheximide treatment of cells is used as a reporter for proteasome activity. The known protease inhibitors (ALLN and MG-132) are shown as positive controls. Background-subtracted emission values at 460 and 530 nm were expressed as a ratio of 460/530, where a high ratio represents high -lactamase activity. Each time point represents the mean of 4 replicates. C: pulse-chase analysis of F508-CFTR in the absence (top) or in the presence of VRT-422 (middle) or VRT-325 (bottom). F508-CFTR-HEK-293 cells were incubated for 16 h with DMSO or 6.7 µM VRT-422 or VRT-325, pulse labeled for 15 min with [35S]methionine/cysteine, and subsequently chased for the indicated times. F508-CFTR was immunoprecipitated using M3A7-antibody and analyzed by SDS-PAGE. D: quantification of core-glycosylated F508-CFTR in DMSO-treated, 6.7 µM VRT-422-treated, or 6.7 µM VRT-325-treated cells using PhosphorImager analysis of radioactivity in immunoprecipitated CFTR separated by SDS-PAGE.

In addition to rapid degradation in the ER, F508-CFTR also exhibits increased cell surface turnover relative to wt-CFTR (60). To further investigate the effects of the quinazolinone correctors on the maturation efficiency and stability of F508-CFTR, we used pulse-chase experiments to examine F508-CFTR maturation in the presence and absence of 6.7 µM VRT-325 (Fig. 6). In HEK-293 cells expressing F508-CFTR, 16-h incubation with VRT-325 increased the amount of mature F508-CFTR, with a maximum conversion of core-glycosylated F508-CFTR to the mature form of 6.8 ± 2% in 6.7 µM VRT-325-treated cells vs. 3.4 ± 0.8% for DMSO treatment (Fig. 6A). The conversion of core-glycosylated F508-CFTR to the mature form in compound-treated cells represents 15% of the maturation efficiency observed for wt-CFTR (Fig. 6A). In addition, 6.7 µM VRT-325 (n = 3) increased t_1/2 of band C from 4.9 ± 0.1 to 7.7 ± 1.1 h (n = 3) compared with the DMSO-treated controls (Fig. 6, B and C), suggesting that the compound-corrected form of F508-CFTR is more stable at the cell surface.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effect of VRT-325 on maturation and stability of F508-CFTR in HEK-293 cells. A: maturation efficiencies of band B-to-C conversion determined from 15 min [35S]methionine/cysteine pulse-chase experiment were monitored for wt-CFTR and F508-CFTR after 16-h treatment with DMSO and 6.7 µM VRT-325. Efficiencies are expressed as amount of band C present at each time point divided by band B at time zero (%total). B: representative gels from 60-min [35S]methionine/cysteine pulse-chase experiments. C: Maturation efficiencies are shown over the long chase times for F508-CFTR after pretreatment with DMSO and 6.7 µM VRT-325, as well as for wild-type CFTR. Each time point represents the data collected from 3 replicate experiments.

Selectivity of the quinazolinone correctors for F508-CFTR.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Quinazolinone correctors increase trafficking of endoplasmic reticulum-arrested mutant G601S- human ether-a-go-go (hERG). Cell surface expression of G601S-hERG expressed in HEK-293 cells treated for 16 h with 1, 10, and 30 µM VRT-422 or VRT-325. Average surface expression in the presence of the test article was normalized to the average for vehicle control and indicated as the relative surface expression.

Identification of novel F508-CFTR potentiators using HTS.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Identification of small-molecule potentiators of temperature-corrected F508-CFTR expressed in NIH/3T3 cells. A: structure of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-y)phenol]. Experimental procedure for the preparation of VRT-532 is described in the supplemental materials (see http://ajplung.physiology.org/cgi/content/full/00169.2005/DC1). B: representative tracing of the response to 0, 0.75, 2.2, 6.7, and 20 µM VRT-532. C: representative single-channel currents before, during, and after application of 20 µM VRT-532 in excised inside-out membrane patches (pipette voltage = 80 mV). The bath contained 1 mM ATP with 75 nM PKA to activate F508-CFTR. C, closed; O, open.

Potentiator activity was confirmed by inside-out patch-clamp recording in F508-NIH/3T3 cells. Treatment with 20 µM VRT-532 in the presence of ATP and PKA increased the open probability (P_o) of F508-CFTR from 0.09 ± 0.04 to 0.39 ± 0.1 (n = 3; P < 0.05, paired t-test), primarily by increasing the open burst duration, and had no effect on single-channel conductance (Fig. 8C). The P_o of F508-CFTR in the presence of VRT-532 (0.39 ± 0.1) was similar to that of wt-CFTR (0.36 ± 0.04; n = 3) under identical recording conditions. VRT-532 did not increase the total cAMP concentration in NIH/3T3 cells in the presence or absence of the phosphodiesterase inhibitor IBMX, nor did it directly inhibit phosphodiesterases in in vitro enzyme assays (data not shown). Together, these results suggest that VRT-532 acts on F508-CFTR to potentiate its gating activity.

Rescue of F508-CFTR activity in primary CF airway cultures by correctors and potentiators. To test the activity of the corrector VRT-325 and the potentiator VRT-532 in a physiologically relevant system, we used monolayers of HBE obtained from the airways of F508-homozygous CF patient donors (F508-HBE). HBE cultures represent highly differentiated, polarized surface airway cells and allow assessment of CFTR-mediated Cl^– secretion with I_sc under voltage-clamp control conditions (19). To monitor CFTR-dependent I_sc in F508-HBE cultures, amiloride was added to the apical side to block epithelial Na⁺ channels, and a basolateral-to-apical Cl^– gradient was established. Figure 9, A and B, shows validation of the F508-HBE model with low-temperature correction, potentiation with genistein, and inhibition by the known CFTR blockers, CFTR_inh-172 (29) and glibenclamide (47). A small forskolin-stimulated current corresponding to 5% of wt-CFTR activity was observed in uncorrected cells and appears to be mediated by F508-CFTR on the basis of its forskolin sensitivity, potentiation by genistein, insensitivity to Ca²⁺-activated Cl^– channel inhibitor DIDS (data not shown), and inhibition by CFTR_inh-172 or glibenclamide (Fig. 9).

View larger version (35K): [in this window] [in a new window]   Fig. 9. Rescue of F508-CFTR in human bronchial epithelia (HBE) isolated from F508-homozygous cystic fibrosis (CF) patients. A: representative tracing of the short-circuit current (Isc) response to 10 µM amiloride, 10 µM FSK, 30 µM genistein, and 10 µM CFTRinh-172 in temperature-corrected () and uncorrected () F508-HBE. B: cumulative data (n = 6) of the response to FSK ± genistein in 27°C-corrected (open bars) and uncorrected (solid bars) F508-HBE. C: representative Isc after 48-h preincubation of F508-HBE with DMSO or 6.7 µM VRT-325. D: cumulative data (n = 17) of the response in DMSO-treated (solid bars) and 6.7 µM VRT-325-treated (open bars) F508-HBE. E: dose response to VRT-325 in FSK-stimulated F508-HBE (n = 5). F: glycosylation pattern of wt-CFTR in non-CF HBE and F508-CFTR in DMSO- and 6.7 µM VRT-325-treated F508-HBE (48-h incubation in compound). C, band C; B, band B. *Significant difference (P < 0.05) from 37°C (B) or DMSO-treated (D) groups.

View larger version (12K): [in this window] [in a new window]   Fig. 10. Kinetics of compound correction in F508-HBE. A: time course of VRT-325-induced correction in F508-HBE (n = 3). F508-HBE were preincubated with 6.7 µM VRT-325 for 4–96 h and the FSK-stimulated Isc in compound-treated cells was normalized to that in DMSO-treated cells (%correction). B: membrane stability of F508-CFTR was determined by first incubating HBE cultures for 96 h with 6.7 µM VRT-325 or at 27°C. Response to 10 µM FSK and VRT-532 was then monitored from 10 min to 72 h after compound removal or the temperature was returned to 37°C (%uncorrected). Representative Isc results in uncorrected (DMSO) and 0, 2, 24, and 36 h after removal of 6.7 µM VRT-325 are shown. C: cumulative data for VRT-325-treated (n = 3) and 27°C-treated (n = 6) F508-HBE.

Estimating potential clinical efficacy of correctors and potentiators. Estimates of the amount of F508-CFTR rescue believed to ameliorate the decline of lung function in CF patients range from 5% to 30% of wt-CFTR, based on correlations of CFTR genotype and Cl^– secretion in nasal potential difference and sweat Cl^– measurements with the clinical phenotype of CF patients with mild, moderate, and severe CF disease (9, 34–36). To evaluate the potential for the corrector VRT-325 to ameliorate lung disease in CF patients alone or in combination with the potentiator VRT-532, we compared the amount of Cl^– secretion in compound-treated F508-HBE with that in non-CF HBE endogenously expressing native wt-CFTR (Fig. 11). This comparison of in vitro activities is important because of the lack of clinically predictive animal models of CF lung disease (21). In non-CF HBE, forskolin stimulated a large, biphasic increase in Cl^– secretion with net changes in I_sc for the peak and steady-state levels of 53.0 ± 6.3 and 34.1 ± 4.5 µA/cm², respectively (n = 30). A 48-h incubation with VRT-325 increased the forskolin response in CF-HBE from 2.0 ± 0.3 to 3.9 ± 0.5 µA/cm² (n = 17), which is >10% of the sustained CFTR-dependent Cl^– secretion in non-CF HBE (Fig. 11, A and B). In the absence of a basolateral-to-apical Cl^– gradient and in the presence of amiloride, the peak forskolin response in untreated and VRT-325-treated F508-HBE was 1.2 ± 0.2 and 2.0 ± 0.2 µA/cm² (n = 6; P < 0.05, paired t-test), respectively. For comparison, the peak forskolin response in non-CF HBE under identical recording conditions was 13.9 ± 1.5 µA/cm² (14 ± 1.5% of non-CF HBE; n = 4). These data indicate that the amount of correction is 14.1 ± 1.5% of the response observed in non-CF HBE, which is similar to the amount of correction observed in the presence of a basolateral-to-apical Cl^– gradient (Fig. 11, A and B). Subsequent addition of the potentiator VRT-532 further increased F508-CFTR-dependent Cl^– secretion to levels >20% of that observed in non-CF HBE (Fig. 11, A and B), indicating that the two mechanisms can be combined to achieve additional efficacy and possibly synergy. In addition, VRT-325-induced correction was significantly greater than that with 1.5 mM 4-PBA and with temperature correction in F508-HBE (Fig. 11, C and D). Other reported correctors (curcumin and compound 9) were not active in F508-HBE (Fig. 11D). These results indicate that VRT-325 is significantly more efficacious than other known correctors of F508-CFTR trafficking in HBE isolated from CF patients.

View larger version (20K): [in this window] [in a new window]   Fig. 11. Efficacy of VRT-325 compared with wt-CFTR, 4-phenylbutyrate (4-PBA), and 27°C correction in F508-HBE. A: representative Isc of the response to 10 µM FSK and glibenclamide (Glib) in non-CF HBE (dashed line) and uncorrected (thin line) and VRT-325-corrected (thick line) F508-HBE. VRT-532 was added subsequent to FSK only in F508-HBE. B: cumulative data for uncorrected and VRT-325-corrected F508-HBE with and without addition of VRT-532 normalized to the steady-state FSK response in non-CF HBE (%wild-type activity). C, top: representative Isc in uncorrected () and 6.7 µM VRT-325-treated (48 h; ) F508-HBE. C, bottom: representative Isc in uncorrected () and 1,500 µM 4-PBA-treated (48 h; ) F508-HBE. D: response to 10 µM FSK in 1,500 µM 4-PBA-, 10 µM curcumin-, 10 µM compound 9- (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid diamide), 6.7 µM VRT-422-, 6.7 µM VRT-325-, and 27°C-treated CF-HBE normalized to untreated controls (%uncorrected). *P < 0.05 compared with untreated controls. **P < 0.01 compared with untreated controls.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

The mechanistic data on the quinazolinone corrector compounds presented here suggest a model in which the compounds act primarily or initially at the level of the ER to facilitate the folding and export of F508-CFTR. Data supporting compound action in the ER include the increased persistence of core-glycosylated F508-CFTR and lack of effects on CFTR transcription and proteasome activity. The relatively slow increase in functional cell surface F508-CFTR observed in CF-HBE (Fig. 10) is likely due to several compounding actions that result in an accumulation of corrected cell surface F508-CFTR over the course of several days. First, the fact that <10% of newly synthesized F508-CFTR is converted to the mature form (Fig. 6A) suggests that corrector compound action is able to catalyze an export-competent conformation in a relatively small fraction of the ER pool of F508-CFTR. Second, the slow decrease in cell surface CFTR activity in F508-HBE after compound washout (Fig. 10) suggests that the F508-CFTR that escapes the ER due to compound treatment possesses a conformation similar to wt-CFTR. These two effects would result in a situation where a small amount of correctly folded F508-CFTR is produced on compound addition, and this stable form accumulates at the cell surface until ER exit is balanced by cell surface turnover such that steady-state is achieved several days later. The possibility that the corrector compounds result in properly folded F508-CFTR suggests additional parameters for future investigation, including cell surface recycling rate, channel gating efficiency (P_o), and CFTR folding in vitro. In addition, further studies in F508-HBE could be used to investigate the functional consequences of F508-CFTR upregulation, including effects on ion transport, airway surface fluid transport, and mucociliary clearance (2, 33). The HBE system also allows the direct comparison of Cl^– secretion and HBE function not only between CF and non-CF epithelia but also between CF patients with genotypes other than F508, which could lead to insights regarding the relationship between CFTR activity and clinical phenotype.

Our results showing that VRT-325 increased trafficking of G601S-hERG (Fig. 7), as well as those of Loo et al. (26) showing a similar effect on the G268V P-gp mutant and other CFTR mutations, indicate that VRT-325 is not a specific corrector of F508-CFTR, and it is possible that the molecular target of VRT-325 is a component of the ER folding/quality control machinery. Possible targets include molecular chaperones such as Hsc70 and Hsp90, which have been reported to act on G601S-hERG (16). Further work is needed to characterize the mechanism of action of VRT-325 and to identify which classes of proteins are affected by this compound. However, these data suggest the intriguing possibility that correction compounds may be useful for the pharmacological treatment of other diseases known to be caused by protein misfolding (1, 58).

Although several potent CFTR potentiators have been reported in the literature, CFTR potentiator VRT-532 appears to be distinguished by its ability to potentiate the gating of several forms of CFTR, including F508-, G551D-, and wt-CFTR. Like genistein (38), VRT-532 had an approximately fivefold lower affinity for G551D-CFTR than F508-CFTR. Other structural classes of F508-CFTR potentiators such as phenylglycines show greater potency shifts vs. G551D (70 nM and 1,100 nM against F508- and G551D-CFTR, respectively) or are inactive against G551D-CFTR (benzothiophenes and sulfonamides) (38, 62). Further characterization of the ability of VRT-532 to potentiate other CFTR mutations is warranted. Patch-clamp recording and cell signaling analysis suggest that VRT-532 acts directly on the channel to increase the channel P_o to levels observed for wt-CFTR. Further studies, however, are required to confirm that VRT-532 binds directly to CFTR to alter its activity.

Under our culture and recording conditions, a small forskolin-stimulated Cl^– current was observed in uncorrected HBE isolated from F508 homozygous patients, and this current was increased by either genistein or VRT-532. The total Cl^– current observed in F508-HBE was 5% of that observed in non-CF HBE, was blocked by CFTR inhibitors, as well as by the basolateral Cl^– transport inhibitor, bumetanide. In contrast, the Ca²⁺-activated Cl^– channel blocker DIDS had no effect. These data suggest the presence of residual F508-CFTR activity in F508-HBE. The presence of residual F508-CFTR activity in airway is controversial. For example, residual Cl^– current has been reported in primary cultures of nasal (48) and airway epithelia (12), and immunohistochemical studies have demonstrated the presence of apical localized F508-CFTR in primary cultures of respiratory tissue (13, 39). However, other studies have failed to demonstrate the presence of F508-CFTR on the apical membrane of cultured HBE (25). The extent to which the presence of residual F508-CFTR activity in vitro is related to the culture conditions is not clear. The ability to detect residual activity in our study could be due to the use of a basolateral-to-apical Cl^– gradient, which markedly increased the signal-to-noise ratio compared with that observed in the absence of a Cl^– gradient. However, there is growing evidence that residual activity is present in a fraction of the F508-homozygous patient population (15, 23, 56). These patients exhibit a milder CF phenotype, including improved lung function (forced expiratory volume in 1 s) and pancreatic sufficiency. This is an important issue to resolve from a clinical perspective because it is possible that such patients could benefit from a potentiator therapy.

A key question in the design of CFTR modulators to treat CF is this: how much CFTR-mediated Cl^– secretion is needed to achieve clinical benefit? Genotype/phenotype correlations of Cl^– channel function with disease severity show that mutations resulting in a only modest recovery toward wild-type levels (e.g., 5–30%) increase in CFTR expression or activity (e.g., A455E, 2,789 + 5GA, 5T, R334W, R347P, and R117H) and are typically associated with pancreatic sufficiency, a slower rate of pulmonary function decline, and a better severity index than that shown in patients with severe disease genotypes (5, 6, 10, 36, 49). We have used HBE cells in an attempt to benchmark the effects of small molecules for potential efficacy. VRT-325 alone or in combination with VRT-532 increased Cl^– secretion to >10–20% of non-CF HBE, levels expected to provide therapeutic benefit in CF patients (9, 34–36). These findings support the prospect for small-molecule therapies to restore clinically significant function of CFTR in a majority of CF patients.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Vertex Pharmaceuticals has a financial interest in the discovery and development of small-molecule therapies for CF.

	ACKNOWLEDGMENTS

 
We thank Cystic Fibrosis Foundation Therapeutics for generous financial support, as well as Melissa Ashlock, Robert Beall, Preston Campbell, John Chabala, Kevin Foskett, Ray Frizzell, Eric Gordon, Diana Wetmore, William Guggino, Christopher M. Penland, Bruce Stanton, and Jeffrey Wine for guidance and support. We also thank Dr. Michael Welsh for providing wt-CFTR and F508-expressing NIH/3T3 and FRT cells, Neil Bradbury for providing F508-expressing HEK-293 cells, and National Disease Research Interchange for providing lung samples for HBE cells.