Cystic fibrosis (CF), a relatively common hereditary disease in white populations, can produce chronic lung infection and deterioration of lung function, pancreatic insufficiency, male infertility, and meconium ileus (Pilewski and Frizzell, 1999). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated Cl^– channel expressed in airway, pancreatic, intestinal, testicular, and other epithelia (Sheppard and Welsh, 1999). ΔF508 is by far the most common CFTR mutation causing CF, being present in ∼60% of CF genes and in ∼90% of CF subjects as at least one allele (Bobadilla et al., 2002). The ΔF508 mutation is believed to produce Cl^–-impermeable epithelial cells by aberrant protein folding and consequent defects in cellular processing and channel gating (Dalemans et al., 1991; Denning et al., 1992; Haws et al., 1996; Kopito, 1999). Most ΔF508-CFTR protein is retained at the endoplasmic reticulum and is degraded rapidly (Jensen et al., 1995; Ward et al., 1995). Many other CFTR mutations causing CF are targeted to the cell plasma membrane but produce chloride-impermeable cells by a primary defect in channel gating. The most common of the CFTR gating mutants is G551D, with a worldwide frequency of 3.1% among CF chromosomes (Hamosh et al., 1992), although people of Celtic descent have frequencies as high as 8% (Cashman et al., 1995).

Small-molecule activators/correctors of mutant CFTRs may provide a strategy for treatment of CF that corrects the underlying defect. Activation of mutant CFTRs avoids potential concerns about treating the wrong cells and/or losing physiological CFTR regulation as might occur with gene therapy or activation of alternative chloride channels. Restoration of cAMP-regulated chloride permeability in epithelial cells expressing ΔF508-CFTR would probably require compound(s) that correct the underlying defects in cellular processing and channel gating, although there may exist a subset of subjects with enough plasma membrane ΔF508-CFTR expression (Penque et al., 2000; Sermet-Gaudelus et al., 2002) to be benefited by a corrector of defective channel gating (“potentiator”). Potentiators may also be useful as monotherapy for CF caused by gating mutants of CFTR such as G551D.

Various small molecules have been found to have potentiator activity for correction of the ΔF508-CFTR gating defect. Relatively high concentrations of flavones such as genistein (>50 μM) and xanthines such as isobutylmethylxanthine (>1 mM) can restore normal or near-normal ΔF508-CFTR channel gating when given with cAMP agonists (Drumm et al., 1991; Haws et al., 1996; Hwang et al., 1997). Flavones at high concentrations also are able to correct defective gating in G551D-CFTR (Illek et al., 1999; Zegarra-Moran et al., 2002). We identified previously a benzothiophene class of ΔF508-CFTR potentiators by high-throughput screening of 100,000 small molecules (Yang et al., 2003). After compound optimization by structure-activity studies, benzothiophenes were identified that rapidly restored near-normal ΔF508-CFTR channel gating with K_a ∼ 0.5 μM, as measured by short-circuit current analysis. However, activation required high concentrations of cAMP agonists, and the benzothiophenes did not activate CFTR gating mutants such as G551D.

In this study, we carried out high-throughput screening to identify novel classes of correctors of defective ΔF508-CFTR channel gating, focusing on compounds with very high potency, potentiator activity in human airway epithelial cells from CF subjects, and activity against other CFTR gating mutants. Two novel classes of potentiators emerged from primary screening and secondary evaluation: phenylglycines and sulfonamides. These compounds were potent in ΔF508-CFTR–transfected and natively expressing human cells, active in the presence of relatively low concentrations of cAMP agonists, and active against multiple CFTR gating mutants, including G551D. To evaluate their potential usefulness for drug development, the phenylglycines and sulfonamides were subject to analysis of structure-activity relationships, single-channel electrophysiology, and metabolic stability/in vivo pharmacology.

Materials and Methods

Cell Lines. Fischer rat thyroid (FRT) epithelial cells stably coexpressing human ΔF508-CFTR and the high-sensitivity halide-sensing green fluorescent analog YFP-H148Q/I152L (Galietta et al., 2001a) were generated as described previously (Yang et al., 2003). FRT cells were cultured on plastic in Coon's modified F-12 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. For primary screening, cells were plated using a multidrop dispenser (Thermo Electron Corp., Woburn, MA) into black 96-well microplates (Corning-Costar, Acton, MA) at 50,000 cells/well. Screening was done 18 to 24 h after plating. For short-circuit current measurements, cells were cultured on Snapwell permeable supports (Corning-Costar) at 500,000 cells/insert. Human nasal epithelial cells from subjects with CF were cultured on Snapwell inserts and were allowed to differentiate in a hormone-supplemented medium as described previously (Galietta et al., 1998). Some measurements were done using stably transfected FRT cells expressing YFP-H148Q and wild-type or G551D-CFTR (Galietta et al., 2001b). Patch-clamp experiments were done on ΔF508-CFTR expressing FRT cells plated on 35-mm Petri dishes.

Compounds. A collection of 50,000 diverse drug-like compounds (>90% with molecular size of 250–500 Da; ChemDiv, San Diego, CA) was used for initial screening. The compounds were cherry-picked for favorable drug-like properties, maximal chemical diversity, and minimal overlap with 100,000 compounds tested previously. For optimization, >1000 analogs of activators identified in the primary screen were purchased from ChemDiv. Compounds were prepared as 10 mM stock solutions in DMSO. Secondary plates containing one or four compounds per well were prepared for screening (1 mM in DMSO). Compounds for secondary analysis were resynthesized, purified, and confirmed by NMR and liquid chromatography/mass spectrometry.

Screening Procedures. Screening was carried out using a Beckman integrated system containing a 3-m robotic arm, a CO₂ incubator containing microplate carousel, plate washer, liquid-handling workstation, barcode reader, delidding station, plate sealer, and two FluoStar fluorescence plate readers (Optima; BMG LABTECH, Durham, NC), each equipped with dual syringe pumps and HQ500/20X (500 ± 10 nm) excitation and HQ535/30M (535 ± 15 nm) emission filters (Chroma Technology Corp., Brattleboro, VT). For assay of ΔF508-CFTR potentiator activity, FRT cells were incubated at 27°C (90% humidity, 5% CO₂) to allow the rescue of mutant CFTR. After 18- to 24-h incubation, plates (40–50 per day) were washed with PBS, and cells were incubated with 60 μl of PBS containing forskolin (20 μM) and test compounds (2.5 μM). After 15 min, the 96-well plate was transferred to a plate reader for fluorescence assay. Each well was assayed individually for I^– influx by recording fluorescence continuously (200 ms per point) for 2 s (baseline) and then for 12 s after rapid (<1 s) addition of 165 μl of PBS, in which 137 mM Cl^– was replaced by I^–. I^– influx rate was computed by fitting the final 11.5 s of the data to an exponential for extrapolation of initial slope and normalizing for total fluorescence (background-subtracted initial fluorescence). All compound plates contained negative controls (DMSO vehicle alone) and positive controls (genistein, 5 and 50 μM). Assay analysis indicated a Z′ factor (Zhang et al., 1999) of >0.7.

Synthetic Chemistry.¹H spectra were obtained in CDCl₃ or d₆-DMSO using a Mercury 400 MHz spectrometer. Flash-column chromatography was done using EM silica gel (230–400 mesh). Thin-layer chromatography was carried out on Merk silica gel 60 F254 plates and visualized under a UV lamp. Microwave reactions were carried out on a synthesizer (Emrys, Charlottesville, VA). Representative synthetic schemes for a phenylglycine and sulfonamide follow (Fig. 2A).

For synthesis of phenylglycine-01 (PG-01), to a solution of N-tert-butoxycarbonyl-N-methylphenylgycine (compound I) (1.26 g, 4.75 mmol) at room temperature was added p-isopropylaniline (705 mg, 5.22 mmol), 4-(N,N-dimethylamino)pyridine (DMAP) (116 mg, 0.92 mmol) in CH₂Cl₂ (25 ml) and 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDCI; 1.00 g, 5.22 mmol). The reaction mixture was stirred for 2 h and then quenched by pouring over saturated NH₄Cl. After extraction with CH₂Cl₂, the organic layer was washed successively with water and brine, dried (Na₂SO₄), and concentrated in vacuo. Column chromatography of the crude residue gave [(4-isopropylphenylcarbamoyl)-phenylmethyl]-methylcarbamic acid tert-butyl ester (compound IIA) as a white solid (1.67 g, 92%). Compound IIA (300 mg, 0.785 mmol) was dissolved in a minimal quantity of trifluoroacetic acid, maintained at room temperature for 15 min, poured over aqueous NaHCO₃, and extracted with CH₂Cl₂. Washing, drying, and evaporation of the organic layer gave compound II as a yellow oil (218 mg, 98%). To a mixture of compound II (177 mg, 0.620 mmol), indole-3-acetic acid (114 mg, 0.651 mmol) and DMAP (15 mg, 0.124 mmol) in CH₂Cl₂ (5 ml), EDCI (131 mg, 0.682 mmol) was added at room temperature. The reaction mixture was worked up as for compound IIA and recrystallized from CH₂Cl₂/MeOH (9:1) to give PG-01 as a white solid (1.67 g, 92%). Mass (ES+): m/z = 440 [M + 1]⁺; ¹H NMR δ 1.21 (d, 6H, J = 6.9 Hz), 2.85 (sep, 1H, J = 6.9 Hz), 2.95 (s, 3H), 3.91 (s, 2H), 6.55 (s, 1H), 7.08 to 7.40 (m, 13H), 7.59 (d, 1H, J = 7.8 Hz), 7.88 (bs, 1H), 8.13 (bs, 1H).

For synthesis of sulfonamide-03 (SF-03), compound III (Blus, 1999) (2.21 g, 8.0 mmol) and diethylethoxymethylenemalonate (1.81 g, 8.4 mmol) were dissolved in tetrahydrofuran (4 ml), and the solution was heated to 140°C for 30 min until the tetrahydrofuran and ethanol by-product evaporated. The residue was diluted with ethyl acetate, washed with brine, dried with Na₂SO₄, and evaporated to dryness. Flash chromatography gave light yellow solid compound IIIB (3.29 g, 90%). To a solution of phenyl ether (Ph₂O, 3 ml) and compound IIIB (130 mg, 0.30 mmol) in an Emrys microwave reaction vessel was added 4-chlorobenzoic acid (1 mg, 0.02 mmol). The solution was microwave-irradiated at 250°C for 75 min. The white precipitate was filtered and washed with hexane to yield compound IV (48 mg, 42%). To an Emrys microwave reaction vessel (0.2–0.5 ml) containing compound IV (65 mg, 0.083 mmol) was added o-methoxybenzyl amine (200 mg, 1.4 mmol) and microwave-irradiated at 180°C for 30 min. The resulting solution was diluted with dichloromethane and water and extracted with ethyl acetate three times. After washing, drying, and evaporation, the residue was purified by flash chromatography giving SF-03 as a white powder (27 mg, 35%). Mass (ES+): m/z = 492 [M + 1]⁺; ¹H NMR CDCl₃ δ 1.08 (t, 3H, J = 7.2 Hz), 3.65 (q, 2H, J = 7.2 Hz), 3.79 (s, 3H), 4.70 (d, 2H, J = 6.0 Hz), 6.81 (m, 2H), 7.02 (m, 2H), 7.16 (td, 1H, J = 8.0, 1.6 Hz), 7.23 (d, 1H, J = 7.2 Hz), 7.29 (m, 2H), 7.37 (d, 1H, J = 8.4 Hz), 7.53 (dd, 1H, J = 8.8, 2.0 Hz), 8.77 (d, 1H, J = 2.0 Hz), 8.83 (d, 1H, J = 6.4 Hz), 10.74 (t, 1H, J = 5.6 Hz), 12.30 (d, 1H, J = 4.4 Hz).

Assays of cAMP. cAMP activity was measured using the BIOTRAK enzymatic immunoassay (Amersham Biosciences Inc., Piscataway, NJ) on FRT cell lysates after incubation with activators for 10 min in the presence of 0.5 μM forskolin.

Short-Circuit Current Measurements. For Ussing chamber experiments, ΔF508-CFTR–expressing FRT cells were seeded on Snapwell inserts and cultured for 7 to 9 days. The basolateral solution contained 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1 mM CaCl₂, 0.5 mM MgCl₂, 10 mM glucose, and 10 mM sodium HEPES, pH 7.3. In the apical bathing solution, 65 mM NaCl was replaced by sodium gluconate, and CaCl₂ was increased to 2 mM. Solutions were bubbled with air and maintained at 37°C. The basolateral membrane was permeabilized with 250 μg/ml amphotericin B. For human bronchial epithelial cells, apical and basolateral chambers contained 126 mM NaCl, 0.38 mM KH₂PO₄, 2.1 mM K₂HPO₄, 1 mM MgSO₄, 1 mM CaCl₂, 24 mM NaHCO₃, and 10 mM glucose (basolateral membrane not permeabilized). Hemichambers were connected to a DVC-1000 voltage clamp (World Precision Instruments, Inc., Sarasota, FL) via Ag/AgCl electrodes and 1 M KCl agar bridges for recording short-circuit current.

Patch-Clamp Analysis. Experiments were performed in the cell-attached configuration of the patch-clamp technique on FRT cells expressing ΔF508-CFTR. Cells were plated at a density of 10⁴ cells/well, grown at 37°C for 24 to 48 h, and then incubated for 24 to 48 h at 27°C to allow trafficking of the ΔF508 protein to the plasma membrane.

Single channel recordings were obtained using an EPC-7 patch-clamp amplifier (List Medical Instruments, Darmstadt, Germany). Data were filtered at 250 Hz and digitized at 500 Hz using an ITC-16 data translation interface (InstruTECH Corporation, Port Washington, NY). The pipette solution contained 120 mM CsCl, 10 mM TEA chloride, 0.5 mM EGTA, 1 mM MgCl₂, 40 mM mannitol, and 10 mM cesium HEPES, pH 7.3. The bath solution contained 130 mM KCl, 2 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, 20 mM mannitol, and 10 mM sodium HEPES, pH 7.3. Channel activity in the patches was recorded before and after stimulation with forskolin (20 μM), with and without potentiators. Most experiments were done with a pipette voltage of –60 mV (referred to the bath). Analysis of open-channel probability (P_o), mean channel open time (T_o), and mean channel closed time (T_c) was done using recordings of at least 3 min as described previously (Taddei et al., 2004).

Pharmacokinetics. To increase compound solubility, potentiators were dissolved in a liposomal formulation containing 5 mg of potentiator in 21.3 mg of hydrogenated soy phosphatidylcholine, 5.2 mg of cholesterol, 8.4 mg of distearoylphosphatidylglycerol, and 90 mg of sucrose in 5 ml of PBS. A bolus of potentiator-containing solution (5 mg/kg) was administered intravenously in rats over 1 min (male Sprague-Dawley rats, 360–420 g) by a jugular vein catheter. Arterial blood samples (∼1 ml) were obtained at predetermined times for liquid chromatography/mass spectrometry (LCMS) analysis. Animal procedures were approved by the University of California, San Francisco, Committee on Animal Research.

LCMS. For analysis of blood samples, collected plasma was chilled on ice, and ice-cold acetonitrile (2:1, v/v) was added to precipitate proteins. Samples were centrifuged at 4°C at 20,000g for 10 min. Supernatants (supplemented with sulforhodamine 101 as internal standard) were analyzed for PG-01 and SF-03 by extraction with C-18 reversed-phase cartridges (1 ml; Alltech Associates, Inc., Deerfield, IL) by standard procedures. The eluate was evaporated, and the residue was reconstituted in 100 μl of mobile phase for HPLC analysis. Reversed-phase HPLC separations were carried out using a C18 column (2.1 × 100 mm, 3 μm particle size; Supelco, Bellefonte, PA) connected to a solvent delivery system (model 2690; Waters, Milford, MA). The solvent system consisted of a linear gradient from 20% CH₃CN/10 mM KH₂PO_4, pH 3, to 95% CH₃CN/10 mM KH₂PO₄, pH 3, over 10 min, followed by 6 min at 95% CH₃CN/20 mM NH₄OAc (0.2 ml/min flow rate). PG-01 and SF-03 were detected at 256 nm, after establishing a linear standard calibration curve in the range of 20 to 5000 nM. The detection limit was 10 nM, and recovery was >90%. Mass spectra were acquired on a mass spectrometer (Alliance HT 2790 + ZQ; Waters) using positive ion detection, scanning from 200 to 800 Da as described previously (Sonawane et al., 2004).

Stability in Hepatic Microsomes. PG-01 and SF-03 (10 μM each) were incubated separately with a phosphate-buffered (100 mM) solution of rat liver microsomes (2 mg of protein/ml; Sigma-Aldrich, St. Louis, MO) containing NADPH (0 or 1 mM) for 60 min at 37°C. After 60 min, the mixture was chilled on ice, and 0.5 ml of ice-cold acetonitrile was added to precipitate the proteins for LCMS analysis as described above.

Results

Compounds were screened at a concentration of 2.5 μM (in the presence of forskolin, 20 μM) in ΔF508-CFTR–expressing FRT cells after low-temperature rescue. CFTR-dependent I^– influx was determined from the time course of decreasing cellular YFP fluorescence. The screening revealed many compounds that at 2.5 μM increased I^– influx as much as the reference compound genistein at 50 μM and substantially greater than forskolin (20 μM) alone (Fig. 1A). Most of these active compounds had PG and SF scaffolds (Fig. 1B); in addition, some active compounds were related structurally to tetrahydrobenzothiophene potentiators identified previously (Yang et al., 2003). Dose-response analysis of more than 1000 analogs of each chemical class not included in the primary library established a structure-activity relationship database. An example of dose-response analysis of phenylglycine analogs is shown in Fig. 1C, with compounds having a wide range of activating potencies. Dose-response data from the fluorescence assay for the most active compound of each class is shown in Fig. 1D, with data for comparison shown for genistein and the tetrahydrobenzothiophene ΔF508_act-02. Activation of ΔF508-CFTR was confirmed for each of the compounds by showing no activity on nontransfected FRT cells and near-complete inhibition of the increased I^– influx by the thiazolidinone CFTR_inh-172 (Ma et al., 2002a) at 10 μM (data not shown).

Table 1 summarizes structure-activity relationship (SAR) data for the most potent PG and SF analogs; data for a larger series of less-active analogs is provided as Supplemental Table S1. Fig. 2B summarizes the principal conclusions from SAR analysis. Active PGs contained a disubstituted glycyl amine with amide of aromatic amines. Substitutions at R1 had relatively little effect on compound activity. Most active compounds had as R1 4-isopropylphenyl, with reduced activity for R1 as 2,3-diH-1,4-benzodioxin-6-yl in (PG-02 and -04) or 4-methoxyphenyl (PG-05). Evaluation of R2 substitutions indicated that replacement of hydrogen by methyl (PG-07) or methoxy (PG-10) strongly reduced potency. The R2 phenyl group seemed to be important for activity because its replacement by indol-3-methyl reduced activity. All potent compounds had as R3 a methyl, because its replacement by hydrogen (PG-06) or furfuryl-2-methyl reduced activity. Most active compounds had as R4 an indolyl-3-acetyl, because substitution by thiophene-2-acetyl or diphenyl acetyl resulted in loss of activity. Thus, the greatest ΔF508-CFTR–activating potency was produced by hydrophobic R1, R2, and R3, with R4 as indolyl-2 (or 3)-acetyl.

SAR analysis of sulfonamides supported the requirement of 3-carboxamide and 6-aminosulfo groups. All active quinolone compounds had as R1 hydrophobic groups such as alkoxy-, dialkyl-, alkyl-, and halo-substituted phenyl or cyclohexyl (SF-10) groups. The greatest activity was found for R2 as nonpolar alkyl chains (ethyl, methyl, 2-propenyl). The most potent compounds (SF-02 to -04) contained an ethyl group at R2 in combination with phenyl as R1 and an alkyl group as R3. Substitutions at R3 with nonpolar linear or branched alkyl or cycloalkyl groups improved activity. In general, the greatest potency was found with hydrophobicnonpolar substitutions on sulfonamide and carboxamide moieties.

Apical membrane current in FRT cells was measured to verify the activation of ΔF508-CFTR Cl^– conductance and to determine compound potency. Apical membrane current was measured after permeabilization of the basolateral membrane with amphotericin B in the presence of a Cl^– gradient (apical, 65 mM Cl^–; basolateral, 130 mM). After maximal forskolin (20 μM), test compounds were added at increasing concentrations as shown in Fig. 3A, followed by CFTR_inh-172. The small effect of forskolin alone demonstrated defective ΔF508-CFTR gating, because a 10-fold lower concentrations of forskolin fully activated wild-type CFTR in this assay (Galietta et al., 2001c). PG-01 and SF-01 gave ΔF508-CFTR Cl^– currents with potencies greater than 100 nM (Fig. 3B), and maximal currents comparable with or greater than that produced by 50 μM genistein. It is interesting that these compounds were substantially less effective for the activation of wild-type CFTR. When stimulated with submaximal forskolin, PG-01 and SF-01 produced only a fraction (40–60%) of the current elicited by genistein (data not shown).

Experiments were also done by adding the potentiator first, followed by increasing concentrations of forskolin. Forskolin alone at 0.5 and 2 μM gave little apical membrane current (Fig. 3C, top left). However, PG-01, which did not itself activate ΔF508-CFTR, produced substantial ΔF508-CFTR Cl^– current after the addition of 0.5 and 2 μM forskolin (Fig. 3C, bottom left). Data are summarized in Fig. 3C (right), showing significant synergy of these potentiators with forskolin. The correction of ΔF508-CFTR gating in the presence of relatively low concentrations of cAMP agonists is a desirable property of these compounds (see Discussion).

An initial analysis of compound specificity was done. Cells were incubated with potentiators in the presence of a low concentration of forskolin (0.5 μM), lysed, and assayed for cAMP. PG-01 and SF-01 did not increase cAMP above the level induced by forskolin 0.5 μM alone (Fig. 4A), whereas the compound CFTR_act-16, an indirect activator of CFTR (Ma et al., 2002b), strongly increased cAMP. MDR-1 activity was assayed by intracellular accumulation of the fluorescent probe rhodamine-123. Two cell lines were used, the parental human tracheal cell line 9HTEo-, and its multidrug-resistant subclone 9HTEo-/Dx that strongly expresses MDR-1 (Rasola et al., 1994). 9HTEo-/Dx cells accumulate much less rhodamine-123 than 9HTEo-cells as a consequence of MDR-1–mediated dye extrusion. Dye accumulation was increased significantly by the MDR-1 inhibitor verapamil but was not affected by PG-01 or SF-01 (Fig. 4B). Last, effects on the UTP/calcium-activated Cl^– channel were measured from short-circuit current measurements on human bronchial epithelial cells. There was no effect of PG-01 or SF-01 on the magnitude or kinetics of the calcium-activated Cl^– current (Fig. 4C).

The ΔF508-CFTR–activating mechanism was investigated by cell-attached patch-clamp measurements. The addition of 20 μM forskolin produced low channel activity, with a P_o of 0.04 (Fig. 5A). Channel openings were separated by long-duration closures, in agreement with previous observations on ΔF508-CFTR (Dalemans et al., 1991; Haws et al., 1996). Although all patches contained more than one channel, simultaneous channel openings were rarely seen because of the low P_o. PG-01 or SF-01 at 100 nM strongly stimulated channel activity with multiple channel openings observed. P_o after activation (0.3–0.4) was comparable with that of wild-type CFTR (Dalemans et al., 1991; Haws et al., 1996) (Fig. 5B). Analysis of gating kinetics indicated that the increase in P_o was caused by a reduction in mean channel closed time (T_c) rather than an increase in T_o (Fig. 5B).

The possibility was evaluated that the PG or SF ΔF508-CFTR potentiators might correct defective gating in other mutant CFTRs that cause CF in humans. Measurements were done in the “class III” mutants G551D and G1349D, which produce a severe gating defect without impairment in protein trafficking (Gregory et al., 1991). These mutations affect the glycine residues in NBD1 and NBD2 that are highly conserved in ATP-binding cassette proteins (Hyde et al., 1990; Logan et al., 1994). The G551D and G1349D mutant CFTRs produced little Cl^– current after the addition of maximal forskolin (Fig. 6, A and B). Genistein, a known activator of G551D- and G1349D-CFTR, increased Cl^– current substantially, albeit at high micromolar concentrations (Fig. 6, A and B, top curves). PG-01 produced large currents in both G551D- and G1349D-CFTR–expressing cells as shown in Fig. 6, A and B (bottom curves) and summarized in Fig. 6, C and D. The currents were sensitive to CFTR_inh-172 and were not seen in nontransfected cells. In contrast, PG-01, SF-01, and the benzothiophene ΔF508_act-02 did not increase Cl^– currents in G551D- and G1349D-CFTR–expressing cells (data not shown).

The ability of PG-01 and SF-01 to correct defective CFTR channel gating in CF human airway epithelial cells was tested (Fig. 7). Human nasal epithelial cells from ΔF508 homozygote subjects were cultured as polarized monolayers on permeable supports for transepithelial short-circuit current measurement. After blocking the epithelial Na⁺ channel with amiloride, forskolin (20 μM) was applied, followed by genistein, PG-01, or SF-01. CFTR_inh-172 was applied at the end of each study to determine total CFTR-dependent current. Cells maintained at 37°C had little CFTR current, in agreement with the expected intracellular retention of ΔF508-CFTR. Low-temperature rescue by incubation at 27°C for 20 to 24 h produced greater ΔF508-CFTR current, with significant activation by PG-01 and SF-01 at nanomolar concentrations (Fig. 7A). Stimulation by forskolin plus PG-01 or SF-01 was blocked by CFTR_inh-172. Genistein was comparably effective but at much higher concentrations. Primary cell cultures from subjects carrying CFTR mutations causing pure gating defects were also tested. For these studies, cells were cultured at 37°C. Nasal epithelial cells from a subject with the G551D mutation (Zegarra-Moran et al., 2002) showed a large response to PG-01 after forskolin stimulation (Fig. 7B). Cells were also tested from a subject having D1152H and ΔF508 CFTR mutations, with the former mutation affecting the second nucleotide-binding domain and causing a decrease in channel activity (Vankeerberghen et al., 1998). The D1152H/ΔF508 cells maintained at 37°C showed large CFTR currents in response to PG-01 (Fig. 7C).

To predict hepatic clearance of PG-01 and SF-03, in vitro incubations were done with rat hepatic microsomes for 1 h at 37°C in the absence (control) and presence of NADPH followed by LCMS analysis. SF-03 was chosen for these studies as the most potent of the SF compounds. Figure 8A (top, left and right) shows representative HPLC chromatograms, with PG-01 eluting at 7.85 min and its two major metabolites (M1 and M2) eluting at 7.16 and 6.88 min. Mass spectrometry identified the original compound, and M1 and M2 with m/z 456 (∼PG-01+OH; [M + 1]⁺) and 472 (∼PG-01 + 2OH; [M + 1]⁺), respectively (Fig. 8A, top, middle). A minor metabolite was also detected at 7.43 min with m/z 428. Approximately 90% of the PG-01 was metabolized after incubation with microsomes for 1 h in the presence of NADPH, and nonmetabolized PG-01 was not detectable after 2 h (data not shown). Figure 8A (bottom, left and right) shows the HPLC profile for SF-03 and its two major metabolites eluting at 7.44 min and 7.16/6.77 min, respectively, with corresponding molecular ion peaks (Fig. 8A, bottom, middle) at m/z 492 (SF-03, [M + 1]⁺), 508 (∼SF-03+OH, [M + 1]⁺) and 389. SF-03 was ∼35% degraded after a 1-h incubation with liver microsomes in the presence of NADPH.

Pharmacokinetic analysis of PG-01 and SF-03 in rats was done by serial measurements of plasma concentrations after single bolus infusions (5 mg/kg). Figure 8B (left) shows HPLC chromatograms for PG-01 and SF-03 (each at 50 nM added to control plasma and supplemented with sulforhodamine 101 as internal standard), demonstrating the sensitivity of the assay. PG-01 pharmacokinetics fitted a two-compartment model with half-times of <5 min and 130 min with volume of distribution ∼4 L, whereas SF-03 clearance had elimination half-times of ∼7 and 110 min with volume of distribution ∼2 L (Fig. 8B, right).

Discussion

The purpose of this study was to identify new classes of drug-like compounds that strongly activate CF-causing mutant CFTRs. Our strategy was to carry out high-throughput screening for ΔF508-CFTR potentiators using a collection of 50,000 diverse, drug-like small molecules. The screening yielded two novel classes of ΔF508-CFTR potentiators having phenylglycine and sulfonamide scaffolds. Several rounds of optimization involving testing of analogs of each compound class produced ΔF508-CFTR potentiators that fully activated ΔF508-CFTR with potencies greater than 100 nM. Many active phenylglycine and sulfonamide analogs of widely differing activities were identified, which is an important prerequisite for the development of these compounds as drugs to treat CF. Analysis of phenylglycine properties revealed a number of favorable properties, including the ability to correct defective channel gating in several different CFTR mutants and synergy with cAMP agonists. The phenylglycine PG-01 was metabolized rapidly in hepatic microsomes, suggesting the possibility of aerosol delivery for CF therapy in which any absorbed compound would be inactivated rapidly by hepatic metabolism. The sulfonamides were relatively stable metabolically and did not correct defective gating in non-ΔF508 CFTR mutants, although they did show synergy with cAMP agonists.

Measurement of transepithelial chloride current in FRT cells confirmed the correction of defective ΔF508-CFTR gating by the phenylglycine and sulfonamide compounds. In one protocol, cells were stimulated with maximal forskolin, followed by increasing concentrations of test compounds. Total current activated by forskolin plus potentiators was blocked by CFTR_inh-172. In a different protocol, a dose-response to forskolin was done with versus without prior potentiator addition. Little response to forskolin was seen in the absence of potentiator, and only at high forskolin concentrations. The addition of the potentiator first, which did not itself activate ΔF508-CFTR, restored substantial sensitivity to forskolin. Measurements of cellular cAMP concentrations indicated that the apparent synergy of the potentiators with forskolin is not caused by cAMP elevation. We propose a direct interaction between the phenylglycine and sulfonamide potentiators with ΔF508-CFTR. The lack of effect of these compounds in the absence of cAMP-elevating agents and the apparent synergy with cAMP-elevating agents are favorable properties in that near-native CFTR regulation is recapitulated.

Cell-attached patch-clamp experiments were carried out to investigate the mechanism of channel activation. In the presence of forskolin alone, ΔF508-CFTR produced bursts of channel openings separated by long closures lasting for several seconds, resulting in reduced open-channel probability. The potentiators strongly increased channel activity, remarkably reducing the time spent in the closed state. The resulting open-channel probability was comparable with that of wild-type CFTR.

The phenyglycines corrected defective gating in a number of CF-causing CFTR mutants including ΔF508, G551D, G1349D, and D1152H. G551D and G1349D affect critical glycine residues in nucleotide binding domains 1 and 2 of CFTR, respectively (Hyde et al., 1990), producing a severe gating defect (Gregory et al., 1991; Logan et al., 1994; Derand et al., 2002; Zegarra-Moran et al., 2002). Forskolin alone produced little activation of these mutant CFTRs even at high concentrations, whereas PG-01 after forskolin produced a >10-fold elevation in current. The apparent K_d for PG-01 for G551D-CFTR activation was ∼1 μM, approximately 100-fold better than that of genistein. The potency for activation G1349D-CFTR by PG-01 was even better at ∼40 nM. In contrast to ΔF508, other cystic fibrosis mutations, of which more than 1000 have been identified, have a relatively very low frequency. The fraction of CF mutations that cause a pure gating defect (class III mutants) is unknown but is likely to be substantial. The phenylglycines may be useful in monodrug therapy for many of these mutations. Further studies are warranted to establish the molecular mechanism by which a small molecule is able to correct defective channel gating in quite different CFTR mutants.

Transepithelial current measurements on primary cultures of human airway epithelia indicated that the phenylglycine and sulfonamide potentiators identified here are also effective in a native epithelium. This finding is not unexpected because these compounds probably bind to mutant CFTRs directly, and so their activity should be cell-context–independent. The best phenylglycine was also effective on cells cultured from subjects with CF having G551D and D1152H CFTR mutations, supporting the possible use of this class of compounds for monotherapy of CF caused by some mutations. For treatment of CF caused by the ΔF508-CFTR mutation, the potentiators would probably need to be combined with compounds that correct defective ΔF508-CFTR cellular processing.