Phenylglycine and sulfonamide correctors of defective ΔF508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating

Article abstract of DOI:10.1124/mol.105.010959

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The ΔF508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defec

Full text of DOI:10.1124/mol.105.010959

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2005/02/23/mol.105.010959.DC1.html
0026-895X/05/6705-1797–1807$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 67:1797–1807, 2005
Vol. 67, No. 5
10959/1201294
Printed in U.S.A.
Phenylglycine and Sulfonamide Correctors of Defective ⌬F508
and G551D Cystic Fibrosis Transmembrane Conductance
□
S
Regulator Chloride-Channel Gating
Nicoletta Pedemonte, N. D. Sonawane, Alessandro Taddei, Jie Hu, Olga Zegarra-Moran,
Yat Fan Suen, Lori I. Robins, Christopher W. Dicus, Dan Willenbring, Michael H. Nantz,
Mark J. Kurth, Luis J. V. Galietta, and A. S. Verkman
Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California (N.P., N.D.S., J.H.,
A.S.V.); Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genova, Italy (N.P., A.T., O.Z.-M., L.J.V.G.); and
Department of Chemistry, University of California, Davis, Davis, California (Y.F.S., L.I.R., C.W.D., D.W., M.H.N., M.J.K.)
Received January 4, 2005; accepted February 18, 2005
ABSTRACT
Mutations in the cystic fibrosis transmembrane conductance CFTR in the presence of forskolin with Ka ϳ 70 nM and also
regulator (CFTR) chloride channel cause cystic fibrosis. The activated the CFTR gating mutants G551D and G1349D with Ka
⌬F508 mutation produces defects in channel gating and cellu- values of ϳ1100 and 40 nM, respectively. The most potent
lar processing, whereas the G551D mutation produces primar- sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydro-
ily a gating defect. To identify correctors of gating, 50,000 quinoline-3-carboxylic acid cycloheptylamide, had Ka ϳ 20 nM
diverse small molecules were screened at 2.5 ␮M (with forsko- for activation of ⌬F508-CFTR. In cell-attached patch-clamp
lin, 20 ␮M) by an iodide uptake assay in epithelial cells coex- experiments, phenylglycine-01 (PG-01) and sulfonamide-01
pressing ⌬F508-CFTR and a fluorescent halide indicator (yel- (SF-01) increased channel open probability Ͼ5-fold by the re-
low fluorescent protein-H148Q/I152L) after ⌬F508-CFTR duction of interburst closed time. An interesting property of
rescue by 24-h culture at 27°C. Secondary analysis and testing these compounds was their ability to act in synergy with cAMP
of Ͼ1000 structural analogs yielded two novel classes of cor- agonists. Microsome metabolism studies and rat pharmacoki-
rectors of defective ⌬F508-CFTR gating (“potentiators”) with netic analysis suggested significantly more rapid metabolism of
nanomolar potency that were active in human ⌬F508 and PG-01 than SF-03. Phenylglycine and sulfonamide compounds
G551D cells. The most potent compound of the phenylglycine may be useful for monotherapy of cystic fibrosis caused by
class, 2-[(2–1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopro- gating mutants and possibly for a subset of ⌬F508 subjects
pylphenyl)-2-phenylacetamide, reversibly activated ⌬F508- with significant ⌬F508-CFTR plasma-membrane expression.
Cystic fibrosis (CF), a relatively common hereditary dis- membrane conductance regulator (CFTR) protein, a cAMP-
activated ClϪ channel expressed in airway, pancreatic, intes-
ease 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 trans-
tinal, testicular, and other epithelia (Sheppard and Welsh,
1999). ⌬F508 is by far the most common CFTR mutation
causing CF, being present in ϳ60% of CF genes and in ϳ90%
of CF subjects as at least one allele (Bobadilla et al., 2002).
The ⌬F508 mutation is believed to produce ClϪ-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 ⌬F508-CFTR protein is retained at the endoplas-
mic reticulum and is degraded rapidly (Jensen et al., 1995;
This work was supported by the Cystic Fibrosis Foundation and by grants
HL73856, HL59198, EB00415, EY13574, and DK35124 from the National
Institutes of Health. Dr. Galietta acknowledges grant GP0296Y01 from Tele-
thon-Italy.
□S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.105.010959.
ABBREVIATIONS: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; YFP, yellow fluorescent protein; PG,
phenylglycine; SF, sulfonamide; MDR-1, multidrug resistance protein 1; FRT, Fischer rat thyroid; DMSO, dimethyl sulfoxide; PBS, phosphate-
buffered saline; DMAP, 4-(N,N-dimethylamino)pyridine; EDCI, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide; LCMS, liquid chromatography/
mass spectrometry; HPLC, high-performance liquid chromatography; SAR, structure-activity relationship; Po, open-channel probability; To, mean
channel open time; Tc, mean channel closed time.
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Pedemonte et al.
Ward et al., 1995). Many other CFTR mutations causing CF ing green fluorescent analog YFP-H148Q/I152L (Galietta et al.,
2001a) were generated as described previously (Yang et al., 2003).
are targeted to the cell plasma membrane but produce chlo-
FRT cells were cultured on plastic in Coon’s modified F-12 medium
ride-impermeable cells by a primary defect in channel gating.
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100
U/ml penicillin, and 100 ␮g/ml streptomycin. For primary screening,
cells were plated using a multidrop dispenser (Thermo Electron
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).
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
⌬F508-CFTR expressing FRT cells plated on 35-mm Petri dishes.
Compounds. A collection of 50,000 diverse drug-like compounds
(Ͼ90% 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 min-
imal overlap with 100,000 compounds tested previously. For optimi-
zation, Ͼ1000 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, pu-
rified, and confirmed by NMR and liquid chromatography/mass spec-
trometry.
Screening Procedures. Screening was carried out using a Beck-
man integrated system containing a 3-m robotic arm, a CO2 incuba-
tor 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 Ϯ 10 nm) excitation and HQ535/30M (535 Ϯ 15 nm) emis-
sion filters (Chroma Technology Corp., Brattleboro, VT). For assay of
⌬F508-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 ␮l of PBS containing forskolin
(20 ␮M) and test compounds (2.5 ␮M). After 15 min, the 96-well plate
was transferred to a plate reader for fluorescence assay. Each well
was assayed individually for IϪ influx by recording fluorescence
continuously (200 ms per point) for 2 s (baseline) and then for 12 s
after rapid (Ͻ1 s) addition of 165 ␮l of PBS, in which 137 mM ClϪ
was replaced by IϪ. IϪ 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 ini-
tial fluorescence). All compound plates contained negative controls
(DMSO vehicle alone) and positive controls (genistein, 5 and 50 ␮M).
Assay analysis indicated a ZЈ factor (Zhang et al., 1999) of Ͼ0.7.
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). Represen-
tative synthetic schemes for a phenylglycine and sulfonamide follow
(Fig. 2A).
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 poten-
tial concerns about treating the wrong cells and/or losing
physiological CFTR regulation as might occur with gene
therapy or activation of alternative chloride channels. Resto-
ration of cAMP-regulated chloride permeability in epithelial
cells expressing ⌬F508-CFTR would probably require com-
pound(s) that correct the underlying defects in cellular pro-
cessing and channel gating, although there may exist a sub-
set of subjects with enough plasma membrane ⌬F508-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 ⌬F508-CFTR gating defect. Rela-
tively high concentrations of flavones such as genistein (Ͼ50
␮M) and xanthines such as isobutylmethylxanthine (Ͼ1 mM)
can restore normal or near-normal ⌬F508-CFTR channel gat-
ing when given with cAMP agonists (Drumm et al., 1991; Haws
et al., 1996; Hwang et al., 1997). Flavones at high concentra-
tions 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 ⌬F508-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 ⌬F508-CFTR channel gating with Ka ϳ 0.5 ␮M, as
measured by short-circuit current analysis. However, activation
required high concentrations of cAMP agonists, and the benzo-
thiophenes 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 ⌬F508-CFTR
channel gating, focusing on compounds with very high po-
tency, 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 ⌬F508-
CFTR–transfected and natively expressing human cells, ac-
tive 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.
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
Materials and Methods
Cell Lines. Fischer rat thyroid (FRT) epithelial cells stably coex- stirred for 2 h and then quenched by pouring over saturated NH4Cl.
pressing human ⌬F508-CFTR and the high-sensitivity halide-sens- After extraction with CH2Cl2, the organic layer was washed succes-

Potentiators of ⌬F508-CFTR Gating
1799
sively with water and brine, dried (Na2SO4), and concentrated in and water and extracted with ethyl acetate three times. After wash-
vacuo. Column chromatography of the crude residue gave [(4-isopro- ing, drying, and evaporation, the residue was purified by flash chro-
pylphenylcarbamoyl)-phenylmethyl]-methylcarbamic acid tert-butyl matography giving SF-03 as a white powder (27 mg, 35%). Mass
ester (compound IIA) as a white solid (1.67 g, 92%). Compound IIA
(ESϩ): m/z ϭ 492 [M ϩ 1]ϩ; 1H NMR CDCl3 ␦ 1.08 (t, 3H, J ϭ 7.2 Hz),
(300 mg, 0.785 mmol) was dissolved in a minimal quantity of triflu- 3.65 (q, 2H, J ϭ 7.2 Hz), 3.79 (s, 3H), 4.70 (d, 2H, J ϭ 6.0 Hz), 6.81
oroacetic acid, maintained at room temperature for 15 min, poured (m, 2H), 7.02 (m, 2H), 7.16 (td, 1H, J ϭ 8.0, 1.6 Hz), 7.23 (d, 1H, J ϭ
over aqueous NaHCO3, and extracted with CH2Cl2. Washing, drying, 7.2 Hz), 7.29 (m, 2H), 7.37 (d, 1H, J ϭ 8.4 Hz), 7.53 (dd, 1H, J ϭ 8.8,
and evaporation of the organic layer gave compound II as a yellow oil 2.0 Hz), 8.77 (d, 1H, J ϭ 2.0 Hz), 8.83 (d, 1H, J ϭ 6.4 Hz), 10.74 (t,
(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
1H, J ϭ 5.6 Hz), 12.30 (d, 1H, J ϭ 4.4 Hz).
Assays of cAMP. cAMP activity was measured using the BIO-
mmol) in CH2Cl2 (5 ml), EDCI (131 mg, 0.682 mmol) was added at TRAK enzymatic immunoassay (Amersham Biosciences Inc., Pisca-
room temperature. The reaction mixture was worked up as for com- taway, NJ) on FRT cell lysates after incubation with activators for 10
pound IIA and recrystallized from CH2Cl2/MeOH (9:1) to give PG-01 min in the presence of 0.5 ␮M forskolin.
as a white solid (1.67 g, 92%). Mass (ESϩ): m/z ϭ 440 [M ϩ 1]ϩ; 1H
Short-Circuit Current Measurements. For Ussing chamber
NMR ␦ 1.21 (d, 6H, J ϭ 6.9 Hz), 2.85 (sep, 1H, J ϭ 6.9 Hz), 2.95 (s, experiments, ⌬F508-CFTR–expressing FRT cells were seeded on
3H), 3.91 (s, 2H), 6.55 (s, 1H), 7.08 to 7.40 (m, 13H), 7.59 (d, 1H, J ϭ Snapwell inserts and cultured for 7 to 9 days. The basolateral solu-
7.8 Hz), 7.88 (bs, 1H), 8.13 (bs, 1H).
tion contained 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1 mM
For synthesis of sulfonamide-03 (SF-03), compound III (Blus, CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM sodium HEPES,
1999) (2.21 g, 8.0 mmol) and diethylethoxymethylenemalonate (1.81 pH 7.3. In the apical bathing solution, 65 mM NaCl was replaced by
g, 8.4 mmol) were dissolved in tetrahydrofuran (4 ml), and the sodium gluconate, and CaCl2 was increased to 2 mM. Solutions were
solution was heated to 140°C for 30 min until the tetrahydrofuran bubbled with air and maintained at 37°C. The basolateral membrane
and ethanol by-product evaporated. The residue was diluted with was permeabilized with 250 ␮g/ml amphotericin B. For human bron-
ethyl acetate, washed with brine, dried with Na2SO4, and evaporated chial epithelial cells, apical and basolateral chambers contained 126
to dryness. Flash chromatography gave light yellow solid compound mM NaCl, 0.38 mM KH2PO4, 2.1 mM K2HPO4, 1 mM MgSO4, 1 mM
IIIB (3.29 g, 90%). To a solution of phenyl ether (Ph2O, 3 ml) and CaCl2, 24 mM NaHCO3, and 10 mM glucose (basolateral membrane
compound IIIB (130 mg, 0.30 mmol) in an Emrys microwave reaction not permeabilized). Hemichambers were connected to a DVC-1000
vessel was added 4-chlorobenzoic acid (1 mg, 0.02 mmol). The solu- voltage clamp (World Precision Instruments, Inc., Sarasota, FL) via
tion was microwave-irradiated at 250°C for 75 min. The white pre- Ag/AgCl electrodes and 1 M KCl agar bridges for recording short-
cipitate was filtered and washed with hexane to yield compound IV circuit current.
(48 mg, 42%). To an Emrys microwave reaction vessel (0.2–0.5 ml)
Patch-Clamp Analysis. Experiments were performed in the cell-
containing compound IV (65 mg, 0.083 mmol) was added o-methoxy- attached configuration of the patch-clamp technique on FRT cells
benzyl amine (200 mg, 1.4 mmol) and microwave-irradiated at 180°C expressing ⌬F508-CFTR. Cells were plated at a density of 104 cells/
for 30 min. The resulting solution was diluted with dichloromethane well, grown at 37°C for 24 to 48 h, and then incubated for 24 to 48 h
Fig. 1. Identification of ⌬F508-CFTR potentiators
by high-throughput screening. A, original traces
showing quenching of cellular YFP fluorescence by
IϪ addition with saline alone and after additions of
forskolin (20 ␮M) alone or forskolin plus genistein
(50 ␮M), SF-01 (2.5 ␮M), or PG-01 (2.5 ␮M). B,
chemical structures of potent PG-01 and SF-01 com-
pounds. C and D, dose-response analysis of indi-
cated compounds (mean Ϯ S.E., n ϭ 4), including
the tetrahydrobenzothiophene ⌬F508act-02 (Yang et
al., 2003).

1800
Pedemonte et al.
at 27°C to allow trafficking of the ⌬F508 protein to the plasma sis. Animal procedures were approved by the University of Califor-
membrane.
nia, San Francisco, Committee on Animal Research.
Single channel recordings were obtained using an EPC-7 patch-
LCMS. For analysis of blood samples, collected plasma was chilled
clamp amplifier (List Medical Instruments, Darmstadt, Germany). on ice, and ice-cold acetonitrile (2:1, v/v) was added to precipitate
Data were filtered at 250 Hz and digitized at 500 Hz using an ITC-16 proteins. Samples were centrifuged at 4°C at 20,000g for 10 min.
data translation interface (InstruTECH Corporation, Port Washing- Supernatants (supplemented with sulforhodamine 101 as internal
ton, NY). The pipette solution contained 120 mM CsCl, 10 mM TEA
standard) were analyzed for PG-01 and SF-03 by extraction with
chloride, 0.5 mM EGTA, 1 mM MgCl2, 40 mM mannitol, and 10 mM C-18 reversed-phase cartridges (1 ml; Alltech Associates, Inc., Deer-
cesium HEPES, pH 7.3. The bath solution contained 130 mM KCl, 2 field, IL) by standard procedures. The eluate was evaporated, and
mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, 20 mM the residue was reconstituted in 100 ␮l of mobile phase for HPLC
mannitol, and 10 mM sodium HEPES, pH 7.3. Channel activity in analysis. Reversed-phase HPLC separations were carried out using a
the patches was recorded before and after stimulation with forskolin C18 column (2.1 ϫ 100 mm, 3 ␮m particle size; Supelco, Bellefonte,
(20 ␮M), with and without potentiators. Most experiments were done PA) connected to a solvent delivery system (model 2690; Waters,
with a pipette voltage of Ϫ60 mV (referred to the bath). Analysis of Milford, MA). The solvent system consisted of a linear gradient from
open-channel probability (Po), mean channel open time (To), and 20% CH3CN/10 mM KH2PO4, pH 3, to 95% CH3CN/10 mM KH2PO4,
mean channel closed time (Tc) was done using recordings of at least pH 3, over 10 min, followed by 6 min at 95% CH3CN/20 mM NH4OAc
3 min as described previously (Taddei et al., 2004).
(0.2 ml/min flow rate). PG-01 and SF-03 were detected at 256 nm,
Pharmacokinetics. To increase compound solubility, potentia- after establishing a linear standard calibration curve in the range of
tors were dissolved in a liposomal formulation containing 5 mg of 20 to 5000 nM. The detection limit was 10 nM, and recovery was
potentiator in 21.3 mg of hydrogenated soy phosphatidylcholine, 5.2 Ͼ90%. Mass spectra were acquired on a mass spectrometer (Alliance
mg of cholesterol, 8.4 mg of distearoylphosphatidylglycerol, and 90 HT 2790 ϩ ZQ; Waters) using positive ion detection, scanning from
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
200 to 800 Da as described previously (Sonawane et al., 2004).
Stability in Hepatic Microsomes. PG-01 and SF-03 (10 ␮M
(male Sprague-Dawley rats, 360–420 g) by a jugular vein catheter. each) were incubated separately with a phosphate-buffered (100
Arterial blood samples (ϳ1 ml) were obtained at predetermined mM) solution of rat liver microsomes (2 mg of protein/ml; Sigma-
times for liquid chromatography/mass spectrometry (LCMS) analy- Aldrich, St. Louis, MO) containing NADPH (0 or 1 mM) for 60 min at
TABLE 1
Structure-activity relationship analysis of phenylglycine and sulfonamide ⌬F508-CFTR potentiators
Phenylglycines
Sulfonamides
Compound
R1
R2
R3
R4
Ka
␮M
PG-01
PG-02
PG-03
PG-04
PG-05
PG-06
PG-07
PG-08
PG-09
PG-10
SF-01
SF-02
SF-03
SF-04
SF-05
SF-06
SF-07
SF-08
SF-09
SF-10
SF-11
SF-12
SF-13
SF-14
SF-15
SF-16
SF-17
SF-18
SF-19
SF-20
4-Isopropyl-Ph
H
H
4-OMe
H
H
H
Me
Indol-3-actyl
Ac-NHCH2CO-
Indol-3-actyl
Indol-3-acetyl
Indol-3-acetyl
Indol-3-acetyl
Indol-3-acetyl
Indol-3-acetyl
Indol-2-acetyl
Indol-3-acetyl
0.30
0.30
0.34
0.40
0.70
0.88
1.33
2.13
2.33
2.71
0.30
0.02
0.03
0.03
0.06
0.11
0.12
0.13
0.14
0.14
0.16
0.19
0.20
0.20
0.22
0.24
0.24
0.27
0.29
0.33
2,3-diH-1,4-benzodioxin-6-yl
Me
4-Isopropyl-Ph
2,3-diH-1,4-benzodioxin-6-yl
Me
Me
4-OMe-Ph
4-Isopropyl-Ph
1,3-benzodioxol-5-yl
4-OMe-Ph
Me
H
4-Me
4-OMe
4-Me
4-OMe
Me
Me
Me
2,3-diH-1,4-benzodioxin-6-yl
H
2,3-diH-1,4-benzodioxin-6-yl
Me
2-OEt-Ph
2-Propenyl
Cycloheptyl
Ph
Et
Ph
Et
2-OMe-Ph-methyl
Cyclohexyl
n-Pentyl
n-Butyl
Cycloheptyl
2-Pyridinylmethyl
(3-OMe)-propyl
3(N-(n-butyl)phenylamino)propyl
2-Pyridinylmethyl
n-Hexyl
Ph
Et
OMe-Ph
Me
Ph
Ph
2-Propenyl
2-Propenyl
Me
2,5-di-Me-Ph
Ph
Et
CH2-CH2-CH(Me)-CH2-CH2-
Ph
Ph
2-propenyl
2-Propenyl
2-Me-Ph
2-EtO-Ph
3-Me-Ph
Ph
Me
Me
Me
Et
n-Butyl
(Tetrahydro-2-furanyl)methyl
n-Pentyl
2-(1-Cyclohexen-1-yl)ethyl
(Tetrahydro-2-furanyl)methyl
2-Pyridinylmethyl
3-OMe-propyl
Ph
Et
2-Et-Ph
2,5-di-Me-Ph
2,6-di-Me-Ph
Me
Me
Me
n-Butyl

Potentiators of ⌬F508-CFTR Gating
1801
Fig. 2. Synthesis and structure-activity analysis of
⌬F508-CFTR potentiators. A, top, synthesis of phe-
nylglycine PG-01. Conditions: a, p-isopropylaniline,
EDCI, catalytic amount DMAP, CH2Cl2, 22°C, 2 h,
yield 92%; b, trifluoroacetic acid, 22°C, 15 min, 98%;
c, indole-3-acetic acid, EDCI, catalytic amount
DMAP, CH2Cl2, 22°C, 2 h, 92%. Bottom, synthesis of
sulfonamide SF-03. Conditions: d, diethyl ethoxy-
methylene-malonate, 140°C, 1 h, 95%; e, catalytic
amount p-chlorobenzoic acid, Ph2O, 250°C, 45%; f,
o-methoxybenzyl-amine, neat, 180°C, 35%. B, con-
clusions from SAR analysis of PG and SF analogs.
See Results for explanations.
Fig. 3. Activation of cell-membrane ClϪ current by
⌬F508-CFTR potentiators. Apical membrane ClϪ
current measured in FRT cells expressing ⌬F508-
CFTR after low-temperature rescue. A, representa-
tive traces showing currents activated by forskolin
(fsk, 20 ␮M) and ⌬F508-CFTR potentiators PG-01
and SF-01 and inhibited by CFTRinh-172. B, average
dose-responses for potentiators, with genistein data
shown for comparison (S.E., n ϭ 4). C, representa-
tive curves (left) and averaged data (S.E., n ϭ 4,
right) from experiments showing forskolin dose-
response with versus without the prior addition of
potentiators (2 ␮M).

1802
Pedemonte et al.
37°C. After 60 min, the mixture was chilled on ice, and 0.5 ml of tetrahydrobenzothiophene potentiators identified previously
ice-cold acetonitrile was added to precipitate the proteins for LCMS
analysis as described above.
(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 data-
base. 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 ⌬F508act-02.
Activation of ⌬F508-CFTR was confirmed for each of the
compounds by showing no activity on nontransfected FRT
cells and near-complete inhibition of the increased IϪ influx
by the thiazolidinone CFTRinh-172 (Ma et al., 2002a) at 10
Results
Compounds were screened at a concentration of 2.5 ␮M (in
the presence of forskolin, 20 ␮M) in ⌬F508-CFTR–expressing
FRT cells after low-temperature rescue. CFTR-dependent IϪ
influx was determined from the time course of decreasing
cellular YFP fluorescence. The screening revealed many com-
pounds that at 2.5 ␮M increased IϪ influx as much as the
reference compound genistein at 50 ␮M and substantially
greater than forskolin (20 ␮M) alone (Fig. 1A). Most of these
active compounds had PG and SF scaffolds (Fig. 1B); in
addition, some active compounds were related structurally to ␮M (data not shown).
Fig. 4. Specificity of ⌬F508-CFTR potentiators. A,
intracellular cAMP concentration after forskolin ad-
dition without and with potentiators (2 ␮M). Effects
of PG-01 and SF-01 not significant. B, MDR-1 activ-
ity shown as rhodamine-123 accumulation in mul-
tidrug-sensitive (9HTEo-) and multidrug-resistant
(9HTEo-/Dx) cells. Significant accumulation was
found in 9HTEo-/Dx cells for verapamil (100 ␮M)
but not for the potentiators (5 ␮M). C, activation of
ClϪ current by apical UTP in polarized human bron-
chial epithelia. Pretreatment with ⌬F508-CFTR ac-
tivators (2 ␮M) did not affect the maximum current
or time course of the UTP response.
Fig. 5. Single-channel patch-clamp analysis of
⌬F508-CFTR channel stimulation by potentiators.
A, representative recordings showing activity in
multichannel patches. Broken line indicates current
with channels closed. Pipette voltage was Ϫ60 mV.
Downward deflections represent channel openings
(ClϪ ions moving from pipette to cell). B, mean Po,
Tc, and To in the presence of forskolin alone or in
combination with indicated potentiators.

Potentiators of ⌬F508-CFTR Gating
1803
Table 1 summarizes structure-activity relationship (SAR) alkoxy-, dialkyl-, alkyl-, and halo-substituted phenyl or cy-
data for the most potent PG and SF analogs; data for a larger clohexyl (SF-10) groups. The greatest activity was found for
series of less-active analogs is provided as Supplemental R2 as nonpolar alkyl chains (ethyl, methyl, 2-propenyl). The
Table S1. Fig. 2B summarizes the principal conclusions from most potent compounds (SF-02 to -04) contained an ethyl
SAR analysis. Active PGs contained a disubstituted glycyl group at R2 in combination with phenyl as R1 and an alkyl
amine with amide of aromatic amines. Substitutions at R1 group as R3. Substitutions at R3 with nonpolar linear or
had relatively little effect on compound activity. Most active branched alkyl or cycloalkyl groups improved activity. In
compounds had as R1 4-isopropylphenyl, with reduced activ- general, the greatest potency was found with hydrophobic-
ity for R1 as 2,3-diH-1,4-benzodioxin-6-yl in (PG-02 and -04) nonpolar substitutions on sulfonamide and carboxamide
or 4-methoxyphenyl (PG-05). Evaluation of R2 substitutions moieties.
indicated that replacement of hydrogen by methyl (PG-07) or
Apical membrane current in FRT cells was measured to
methoxy (PG-10) strongly reduced potency. The R2 phenyl verify the activation of ⌬F508-CFTR ClϪ conductance and to
group seemed to be important for activity because its replace- determine compound potency. Apical membrane current was
ment by indol-3-methyl reduced activity. All potent com- measured after permeabilization of the basolateral mem-
pounds had as R3 a methyl, because its replacement by brane with amphotericin B in the presence of a ClϪ gradient
hydrogen (PG-06) or furfuryl-2-methyl reduced activity. Most (apical, 65 mM ClϪ; basolateral, 130 mM). After maximal
active compounds had as R4 an indolyl-3-acetyl, because forskolin (20 ␮M), test compounds were added at increasing
substitution by thiophene-2-acetyl or diphenyl acetyl re- concentrations as shown in Fig. 3A, followed by CFTRinh-172.
sulted in loss of activity. Thus, the greatest ⌬F508-CFTR– The small effect of forskolin alone demonstrated defective
activating potency was produced by hydrophobic R1, R2, and ⌬F508-CFTR gating, because a 10-fold lower concentrations
R3, with R4 as indolyl-2 (or 3)-acetyl.
of forskolin fully activated wild-type CFTR in this assay
SAR analysis of sulfonamides supported the requirement (Galietta et al., 2001c). PG-01 and SF-01 gave ⌬F508-CFTR
of 3-carboxamide and 6-aminosulfo groups. All active quino- ClϪ currents with potencies greater than 100 nM (Fig. 3B),
lone compounds had as R1 hydrophobic groups such as and maximal currents comparable with or greater than that
Fig. 6. Activation of G551D- and G1349D-CFTR
mutants. A and B, stimulation of apical membrane
ClϪ current by genistein (top) and PG-01 (bottom) in
G551D- and G1349D-CFTR–expressing FRT cells.
Cells were pretreated with forskolin (fsk, 20 ␮M). C
and D, dose-responses for the PG-01 and genistein
for activation of G551D- and G1349D-CFTR (S.E.,
n ϭ 4).

1804
Pedemonte et al.
produced by 50 ␮M genistein. It is interesting that these mediated dye extrusion. Dye accumulation was increased
compounds were substantially less effective for the activation significantly by the MDR-1 inhibitor verapamil but was not
of wild-type CFTR. When stimulated with submaximal fors- affected by PG-01 or SF-01 (Fig. 4B). Last, effects on the
kolin, PG-01 and SF-01 produced only a fraction (40–60%) of UTP/calcium-activated ClϪ channel were measured from
the current elicited by genistein (data not shown).
short-circuit current measurements on human bronchial ep-
Experiments were also done by adding the potentiator ithelial cells. There was no effect of PG-01 or SF-01 on the
first, followed by increasing concentrations of forskolin. For- magnitude or kinetics of the calcium-activated ClϪ current
skolin alone at 0.5 and 2 ␮M gave little apical membrane (Fig. 4C).
current (Fig. 3C, top left). However, PG-01, which did not
The ⌬F508-CFTR–activating mechanism was investigated
itself activate ⌬F508-CFTR, produced substantial ⌬F508- by cell-attached patch-clamp measurements. The addition of
CFTR ClϪ current after the addition of 0.5 and 2 ␮M forsko- 20 ␮M forskolin produced low channel activity, with a Po of
lin (Fig. 3C, bottom left). Data are summarized in Fig. 3C 0.04 (Fig. 5A). Channel openings were separated by long-
(right), showing significant synergy of these potentiators duration closures, in agreement with previous observations
with forskolin. The correction of ⌬F508-CFTR gating in the on ⌬F508-CFTR (Dalemans et al., 1991; Haws et al., 1996).
presence of relatively low concentrations of cAMP agonists is Although all patches contained more than one channel, si-
a desirable property of these compounds (see Discussion).
multaneous channel openings were rarely seen because of
An initial analysis of compound specificity was done. Cells the low Po. PG-01 or SF-01 at 100 nM strongly stimulated
were incubated with potentiators in the presence of a low channel activity with multiple channel openings observed. Po
concentration of forskolin (0.5 ␮M), lysed, and assayed for after activation (0.3–0.4) was comparable with that of wild-
cAMP. PG-01 and SF-01 did not increase cAMP above the type CFTR (Dalemans et al., 1991; Haws et al., 1996) (Fig.
level induced by forskolin 0.5 ␮M alone (Fig. 4A), whereas 5B). Analysis of gating kinetics indicated that the increase in
the compound CFTRact-16, an indirect activator of CFTR (Ma Po was caused by a reduction in mean channel closed time
et al., 2002b), strongly increased cAMP. MDR-1 activity was (Tc) rather than an increase in To (Fig. 5B).
assayed by intracellular accumulation of the fluorescent
The possibility was evaluated that the PG or SF ⌬F508-
probe rhodamine-123. Two cell lines were used, the parental CFTR potentiators might correct defective gating in other
human tracheal cell line 9HTEo-, and its multidrug-resistant mutant CFTRs that cause CF in humans. Measurements
subclone 9HTEo-/Dx that strongly expresses MDR-1 (Rasola were done in the “class III” mutants G551D and G1349D,
et al., 1994). 9HTEo-/Dx cells accumulate much less rhoda- which produce a severe gating defect without impairment in
mine-123 than 9HTEo- cells as a consequence of MDR-1– protein trafficking (Gregory et al., 1991). These mutations
Fig. 7. Stimulation of ClϪ secretion in CF human
airway epithelial cells. Transepithelial short-circuit
ClϪ current measured in response to genistein and
indicated ⌬F508-CFTR potentiators. A, nasal epi-
thelial cells from a ⌬F508 homozygous patient. Cells
were incubated at 27°C for 24 h where indicated. B,
G551D-CFTR cells. C, D1152H-CFTR cells.

Potentiators of ⌬F508-CFTR Gating
1805
affect the glycine residues in NBD1 and NBD2 that are CFTR current, with significant activation by PG-01 and
highly conserved in ATP-binding cassette proteins (Hyde et SF-01 at nanomolar concentrations (Fig. 7A). Stimulation
al., 1990; Logan et al., 1994). The G551D and G1349D mu- by forskolin plus PG-01 or SF-01 was blocked by CFTRinh
-
tant CFTRs produced little ClϪ current after the addition of 172. Genistein was comparably effective but at much
maximal forskolin (Fig. 6, A and B). Genistein, a known higher concentrations. Primary cell cultures from subjects
activator of G551D- and G1349D-CFTR, increased ClϪ cur- carrying CFTR mutations causing pure gating defects
rent substantially, albeit at high micromolar concentrations were also tested. For these studies, cells were cultured at
(Fig. 6, A and B, top curves). PG-01 produced large currents 37°C. Nasal epithelial cells from a subject with the G551D
in both G551D- and G1349D-CFTR–expressing cells as mutation (Zegarra-Moran et al., 2002) showed a large re-
shown in Fig. 6, A and B (bottom curves) and summarized in sponse to PG-01 after forskolin stimulation (Fig. 7B). Cells
Fig. 6, C and D. The currents were sensitive to CFTRinh-172 were also tested from a subject having D1152H and ⌬F508
and were not seen in nontransfected cells. In contrast, PG-01, CFTR mutations, with the former mutation affecting the
SF-01, and the benzothiophene ⌬F508act-02 did not increase second nucleotide-binding domain and causing a decrease
ClϪ currents in G551D- and G1349D-CFTR–expressing cells in channel activity (Vankeerberghen et al., 1998). The
(data not shown).
The ability of PG-01 and SF-01 to correct defective CFTR CFTR currents in response to PG-01 (Fig. 7C).
channel gating in CF human airway epithelial cells was To predict hepatic clearance of PG-01 and SF-03, in vitro
D1152H/⌬F508 cells maintained at 37°C showed large
tested (Fig. 7). Human nasal epithelial cells from ⌬F508 incubations were done with rat hepatic microsomes for 1 h at
homozygote subjects were cultured as polarized monolay- 37°C in the absence (control) and presence of NADPH fol-
ers on permeable supports for transepithelial short-circuit lowed by LCMS analysis. SF-03 was chosen for these studies
current measurement. After blocking the epithelial Naϩ as the most potent of the SF compounds. Figure 8A (top, left
channel with amiloride, forskolin (20 ␮M) was applied, and right) shows representative HPLC chromatograms, with
followed by genistein, PG-01, or SF-01. CFTRinh-172 was PG-01 eluting at 7.85 min and its two major metabolites (M1
applied at the end of each study to determine total CFTR- and M2) eluting at 7.16 and 6.88 min. Mass spectrometry
dependent current. Cells maintained at 37°C had little identified the original compound, and M1 and M2 with m/z
CFTR current, in agreement with the expected intracellu- 456 (ϳPG-01ϩOH; [M ϩ 1]ϩ) and 472 (ϳPG-01 ϩ 2OH; [M ϩ
lar retention of ⌬F508-CFTR. Low-temperature rescue by 1]ϩ), respectively (Fig. 8A, top, middle). A minor metabolite
incubation at 27°C for 20 to 24 h produced greater ⌬F508- was also detected at 7.43 min with m/z 428. Approximately
Fig. 8. Liquid chromatography/mass spectrometry
analysis of microsomal metabolites of PG-01 and
SF-03, and rat pharmacokinetics. A, microsomes
were incubated with PG-01 or SF-03 (each 10 ␮M) in
the absence (control) or presence of NADPH for 1 h
at 37°C, and processed as described under Materials
and Methods. HPLC chromatograms at 256 nm for
control (left) and NADPH (right) samples, and cor-
responding ion current chromatograms for positive
ion electrospray mass spectrometry for indicated
m/z (middle). M1, metabolite 1; M2, metabolite 2. B,
pharmacokinetic analysis. Left, HPLC chromato-
gram of PG-01 and SF-03 demonstrating assay sen-
sitivity to greater than 50 nM. Right, pharmacoki-
netics of PG-01 (E) and SF-03 (F) after 5 mg/kg
intravenous bolus injection (mean Ϯ S.E., n ϭ 3–4
rats).

1806
Pedemonte et al.
90% of the PG-01 was metabolized after incubation with is not caused by cAMP elevation. We propose a direct inter-
microsomes for 1 h in the presence of NADPH, and nonme- action between the phenylglycine and sulfonamide potentia-
tabolized PG-01 was not detectable after 2 h (data not tors with ⌬F508-CFTR. The lack of effect of these compounds
shown). Figure 8A (bottom, left and right) shows the HPLC in the absence of cAMP-elevating agents and the apparent
profile for SF-03 and its two major metabolites eluting at 7.44 synergy with cAMP-elevating agents are favorable properties
min and 7.16/6.77 min, respectively, with corresponding mo- in that near-native CFTR regulation is recapitulated.
lecular ion peaks (Fig. 8A, bottom, middle) at m/z 492 (SF-03,
Cell-attached patch-clamp experiments were carried out to
[M ϩ 1]ϩ), 508 (ϳSF-03ϩOH, [M ϩ 1]ϩ) and 389. SF-03 was investigate the mechanism of channel activation. In the pres-
ϳ35% degraded after a 1-h incubation with liver microsomes ence of forskolin alone, ⌬F508-CFTR produced bursts of
in the presence of NADPH.
channel openings separated by long closures lasting for sev-
Pharmacokinetic analysis of PG-01 and SF-03 in rats was eral seconds, resulting in reduced open-channel probability.
done by serial measurements of plasma concentrations after The potentiators strongly increased channel activity, re-
single bolus infusions (5 mg/kg). Figure 8B (left) shows HPLC markably reducing the time spent in the closed state. The
chromatograms for PG-01 and SF-03 (each at 50 nM added to resulting open-channel probability was comparable with that
control plasma and supplemented with sulforhodamine 101 of wild-type CFTR.
as internal standard), demonstrating the sensitivity of the
The phenyglycines corrected defective gating in a number
assay. PG-01 pharmacokinetics fitted a two-compartment of CF-causing CFTR mutants including ⌬F508, G551D,
model with half-times of Ͻ5 min and 130 min with volume of G1349D, and D1152H. G551D and G1349D affect critical
distribution ϳ4 L, whereas SF-03 clearance had elimination glycine residues in nucleotide binding domains 1 and 2 of
half-times of ϳ7 and 110 min with volume of distribution ϳ2 CFTR, respectively (Hyde et al., 1990), producing a severe
L (Fig. 8B, right).
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
Discussion
The purpose of this study was to identify new classes of a Ͼ10-fold elevation in current. The apparent Kd for PG-01
drug-like compounds that strongly activate CF-causing mu- for G551D-CFTR activation was ϳ1 ␮M, approximately 100-
tant CFTRs. Our strategy was to carry out high-throughput fold better than that of genistein. The potency for activation
screening for ⌬F508-CFTR potentiators using a collection of G1349D-CFTR by PG-01 was even better at ϳ40 nM. In
50,000 diverse, drug-like small molecules. The screening contrast to ⌬F508, other cystic fibrosis mutations, of which
yielded two novel classes of ⌬F508-CFTR potentiators having more than 1000 have been identified, have a relatively very
phenylglycine and sulfonamide scaffolds. Several rounds of low frequency. The fraction of CF mutations that cause a
optimization involving testing of analogs of each compound pure gating defect (class III mutants) is unknown but is
class produced ⌬F508-CFTR potentiators that fully activated likely to be substantial. The phenylglycines may be useful in
⌬F508-CFTR with potencies greater than 100 nM. Many monodrug therapy for many of these mutations. Further
active phenylglycine and sulfonamide analogs of widely dif- studies are warranted to establish the molecular mechanism
fering activities were identified, which is an important pre- by which a small molecule is able to correct defective channel
requisite for the development of these compounds as drugs to gating in quite different CFTR mutants.
treat CF. Analysis of phenylglycine properties revealed a
Transepithelial current measurements on primary cul-
number of favorable properties, including the ability to cor- tures of human airway epithelia indicated that the phenyl-
rect defective channel gating in several different CFTR mu- glycine and sulfonamide potentiators identified here are also
tants and synergy with cAMP agonists. The phenylglycine effective in a native epithelium. This finding is not unex-
PG-01 was metabolized rapidly in hepatic microsomes, sug- pected because these compounds probably bind to mutant
gesting the possibility of aerosol delivery for CF therapy in CFTRs directly, and so their activity should be cell-context–
which any absorbed compound would be inactivated rapidly independent. The best phenylglycine was also effective on
by hepatic metabolism. The sulfonamides were relatively cells cultured from subjects with CF having G551D and
stable metabolically and did not correct defective gating in D1152H CFTR mutations, supporting the possible use of this
non-⌬F508 CFTR mutants, although they did show synergy class of compounds for monotherapy of CF caused by some
with cAMP agonists.
mutations. For treatment of CF caused by the ⌬F508-CFTR
Measurement of transepithelial chloride current in FRT mutation, the potentiators would probably need to be com-
cells confirmed the correction of defective ⌬F508-CFTR gat- bined with compounds that correct defective ⌬F508-CFTR
ing by the phenylglycine and sulfonamide compounds. In one cellular processing.
protocol, cells were stimulated with maximal forskolin, fol-
lowed by increasing concentrations of test compounds. Total
current activated by forskolin plus potentiators was blocked
by CFTRinh-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
⌬F508-CFTR, restored substantial sensitivity to forskolin.
Measurements of cellular cAMP concentrations indicated
that the apparent synergy of the potentiators with forskolin
Acknowledgments
We thank Drs. R. Kip Guy and A. Shelat for computational anal-
ysis for compound selection and for advice on SAR analysis.
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