Cyanoquinolines with independent corrector and potentiator activities restore ΔPhe508-cystic fibrosis transmembrane conductance regulator chloride channel function in cystic fibrosis

Article abstract of DOI:10.1124/mol.111.073056

The ΔPhe508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein impairs its folding, stability, and chloride channel gating. Although small molecules that separately correct defective ΔPhe508-CFTR folding/cellular processing

Full text of DOI:10.1124/mol.111.073056

0026-895X/11/8004-683–693$25.00
MOLECULAR PHARMACOLOGY
Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 80:683–693, 2011
Vol. 80, No. 4
73056/3715523
Printed in U.S.A.
Cyanoquinolines with Independent Corrector and Potentiator
Activities Restore ⌬Phe508-Cystic Fibrosis Transmembrane
Conductance Regulator Chloride Channel Function in Cystic
Fibrosis
Puay-Wah Phuan, Baoxue Yang, John M. Knapp, Alex B. Wood,
Gergely L. Lukacs, Mark J. Kurth, and A. S. Verkman
Departments of Medicine and Physiology, University of California, San Francisco, California (P.-W.P., B.Y., A.S.V.); Department
of Chemistry, University of California, Davis, California (J.M.K., A.B.W., M.J.K.); and Department of Physiology and Groupe de
Recherche Axe´ sur la Structure des Prote´ ine (GRASP), McGill University, Montreal, Quebec, Canada (G.L.L.)
Received April 16, 2011; accepted July 5, 2011
ABSTRACT
The ⌬Phe508 mutation in the cystic fibrosis transmembrane con- only potentiator activity. N-(2-((3-Cyano-5,7-dimethylquinolin-2-
ductance regulator (CFTR) protein impairs its folding, stability, and yl)amino)ethyl)-3-methoxybenzamide (CoPo-22), which was syn-
chloride channel gating. Although small molecules that separately thesized in six steps in 52% overall yield, had low micromolar
correct defective ⌬Phe508-CFTR folding/cellular processing EC50 for ⌬Phe508-CFTR corrector and potentiator activities by
(“correctors”) or chloride channel gating (“potentiators”) have short-circuit current assay. Maximal corrector and potentiator ac-
been discovered and are in clinical trials, single compounds with tivities were comparable with those conferred by the bithiazole
bona fide dual corrector and potentiator activities have not been Corr-4a and the flavone genistein, respectively. CoPo-22 also
identified. Here, screening of ϳ110,000 small molecules not activated wild-type and G551D CFTR chloride conductance
tested previously revealed a cyanoquinoline class of compounds within minutes in a forskolin-dependent manner. Compounds with
with independent corrector and potentiator activities (termed dual corrector and potentiator activities may be useful for single-
CoPo). Analysis of 180 CoPo analogs revealed 6 compounds with drug treatment of cystic fibrosis caused by ⌬Phe508 mutation.
dual corrector and potentiator activities and 13 compounds with
the most common CF-causing CFTR mutation, is present in at
Introduction
least one allele in ϳ90% of patients with CF. Whereas the
Cystic fibrosis (CF) is caused by mutations in the gene encod-
wild-type CFTR protein functions as a chloride channel at the
ing the cystic fibrosis transmembrane conductance regulator
(CFTR), a cAMP-regulated chloride channel expressed in air-
apical plasma membrane of target cells, the ⌬Phe508-CFTR
protein is misfolded and largely retained at the endoplasmic
way, intestinal, pancreatic, and other epithelia. Deletion of phe-
reticulum (ER) for degradation by the ubiquitin proteasome
nylalanine at residue 508 (⌬Phe508) in CFTR, which is by far
system. A small fraction of newly synthesized ⌬Phe508-CFTR,
however, can reach the cell surface in a tissue-specific manner
but exhibits impaired channel gating and metabolic stability
This work was supported by the National Institutes of Health National
Institute of Diabetes and Digestive and Kidney Diseases [Grants DK72517,
(Cheng et al., 1990; Dalemans et al., 1991; Lukacs et al., 1994).
Multiple structural defects have been identified in ⌬Phe508-
CFTR, affecting four of its five domains to various extents,
including the first nucleotide binding domain, in which the
⌬Phe508 mutation is located, the nucleotide binding domain 2,
and membrane-spanning domains 1 and 2 (Du and Lukacs,
2009; Thibodeau et al., 2010).
DK075302, DK86125, DK35124]; the National Institutes of Health National
Heart, Lung, and Blood Institute [Grant HL73856]; the National Institute of
Health National Institute of Biomedical Imaging and Bioengineering [Grant
EB00415]; the National Institute of Health National Eye Institute [Grant
EY135740]; the Cystic Fibrosis Foundation; the Canadian Cystic Fibrosis
Foundation; and a Canada Research Chair (to G.L.L.).
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.073056.
ABBREVIATIONS: CF, cystic fibrosis; AAT, arylaminothiazole; CFTR, cystic fibrosis transmembrane conductance regulator; CoPo, corrector-
potentiator; DCM, dichloromethane; ER, endoplasmic reticulum; FRT, Fisher rat thyroid; YFP, yellow fluorescence protein; PBS, phosphate-
buffered saline; DMSO, dimethyl sulfoxide; VX-770, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide; VX-809,
3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid; VRT-532, pyrazole 4-methyl-2-(5-
phenyl-1H-pyrazol-3-yl)-phenol.
683

684
Phuan et al.
There is great interest in the development of drugs that
restore chloride permeability to CF cells (Riordan, 2008; Verk-
man and Galietta, 2009). An ideal drug for treatment of CF
caused by the ⌬Phe508 mutation would be a single, nontoxic
small molecule that normalizes defective ⌬Phe508-CFTR fold-
ing and cellular processing, fully restoring cell chloride perme-
ability. The drug discovery strategy used to date, as first intro-
duced by our laboratory, uses separate assays for the
identification of ⌬Phe508-CFTR “potentiators,” which normal-
ize defective ⌬Phe508-CFTR chloride channel gating, and “cor-
Materials and Methods
Cell Lines
Fisher rat thyroid (FRT) epithelial cells were stably transfected
with ⌬Phe508, G551D, or wild-type CFTR as reported previously
(Pedemonte et al., 2005b). A549 cells stably expressing ⌬Phe508-
CFTR (Pedemonte et al., 2010) were provided by Dr. Luis Galietta
(Genoa, Italy). Each of the CFTR-expressing cell lines (and the
nontransfected parental cells) was also transfected with halide-sen-
sitive green fluorescent protein YFP-H148Q/I152L/F46L. FRT cells
were cultured in Coon’s modified Ham’s F-12 medium and A549 cells
rectors,” which correct defective ⌬Phe508-CFTR protein pro- in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1). All media
were supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
100 U/ml penicillin, and 100 ␮g/ml streptomycin. For primary
screening, ⌬Phe508-CFTR-expressing FRT cells were plated in
black, 96-well microplates (Corning Life Sciences, Lowell, MA) at
50,000 cells/well. For short-circuit current measurements, cells were
cultured on Snapwell permeable supports (Corning Life Sciences) at
500,000 cells/insert.
cessing and promote ER-to-plasma membrane targeting. Using
cell-based high-throughout screens, we reported small-molecule
⌬Phe508-CFTR potentiators (Yang et al., 2003; Pedemonte et
al., 2005b; benzothiophenes, phenylglycines, and sulfonamides)
and correctors (Pedemonte et al., 2005a; Yu et al., 2008; Ye et
al., 2010; aminoarylthiazoles and bithiazoles). Subsequent
small-scale screening by several groups identified additional
candidate potentiators (Van Goor et al., 2006; Pedemonte et al.,
2007) and correctors (Noe¨l et al., 2008; Robert et al., 2008, 2010;
Kalid et al., 2010; Sampson et al., 2011). A potentiator, N-(2,4-
di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-
carboxamide (VX-770) (Van Goor et al., 2009), and a corrector,
VX-809 (Van Goor et al., 2010), identified by Vertex Pharma-
Compounds
A total of 110,000 diverse drug-like synthetic compounds (Ͼ90%
with molecular masses of 250–500 Da; ChemDiv Inc., San Diego, CA,
and Asinex Inc., Moscow, Russia) were used for initial screening. For
optimization, ϳ180 commercially available cyanoquinoline analogs
were tested. Structures of active compounds were confirmed by 1H
ceuticals are in clinical trials (Accurso et al., 2010). In a recent NMR and liquid chromatography/mass spectrometry.
proof-of-concept study, we synthesized a hybrid bithiazole-
Screening Procedures
phenylglycine corrector-potentiator which, when cleaved by in-
testinal enzymes, yielded an active bithiazole corrector and
phenylglycine potentiator (Mills et al., 2010). Pedemonte et al.
(2011) have reported that aminoarylthiazoles, which were iden-
tified in our original corrector screen (Pedemonte et al., 2005a),
had corrector activity, as reported previously, and the ability to
correct defective ⌬Phe508-CFTR gating when incubated with
cells over many hours. However, a bona fide potentiator should
exert its effect within minutes (see below).
Screening was carried out using a Beckman Coulter platform
equipped with FLUOstar fluorescence plate readers (Optima; BMG
Labtech, Durham, NC) with dual syringe pumps and 500 Ϯ 10 nm
excitation and 535 Ϯ 15 nm emission filters (Chroma Corporation,
McHenry, IL). For corrector assay, FRT cells were grown at 37°C/5%
CO2 for 18 to 24 h and then incubated for 18 to 24 h with 100 ␮l of
medium containing test compounds (25 ␮M final concentration). At
the time of the assay, cells were washed with PBS and then incu-
bated for 10 min with PBS containing forskolin (20 ␮M) and
genistein (50 ␮M). For potentiator assay, FRT cells were grown at
37°C/5% CO2 for 18 to 24 h and then for 18 to 24 h at 27°C. At the
time of the assay, cells were washed with PBS and then incubated for
10 min with PBS (50 ␮l) containing forskolin (20 ␮M) and test
compound (0–25 ␮M final concentration). For both of the corrector
and potentiator assays, 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 addition of 165 ␮l of PBS,
in which 137 mM ClϪ was replaced by IϪ. Initial IϪ influx rate was
computed by fitting the final 11.5 s of the data to an exponential for
extrapolation of initial slope, which was normalized for background-
subtracted initial fluorescence. All compound plates contained neg-
ative controls (DMSO vehicle) and positive controls (10 ␮M Corr-4a
for corrector assay; 50 ␮M genistein for potentiator assay).
The goal of this study was to identify dual-acting com-
pounds with independent ⌬Phe508-CFTR potentiator and
corrector activities. Potentiator activity is defined as com-
pound efficacy in increasing ⌬Phe508-CFTR chloride conduc-
tance at the cell plasma membrane. Operationally, potentiator
activity is assayed in low-temperature rescued ⌬Phe508-
CFTR-expressing cells in which ⌬Phe508-CFTR is targeted
to the plasma membrane by Ͼ12-h incubation at reduced
temperature, and test compound (together with cAMP ago-
nist) is added just before or at the time of fluorescence or
electrophysiological assay (Yang et al., 2003). Corrector ac-
tivity is defined as compound efficacy in increasing ⌬Phe508-
CFTR cell surface expression. Corrector activity is assayed in
⌬Phe508-CFTR-expressing cells by Ͼ12 h incubation with
test compound at 37°C, followed by washout and incubation
with a potentiator such as genistein (together with cAMP
agonist) just before or at the time of assay (Pedemonte et al.,
2005a). We report here the identification a cyanoquinoline
class of compounds having independent corrector and poten-
tiator activities, termed CoPo. Although the corrector and
potentiator activities of CoPos depended on their chemical
structure and cell type, the cyanoquinolines are the first class
of compounds with bona fide corrector and potentiator activ-
ities, providing rationale for their further chemical optimiza-
tion and for additional screening to identify other compound
classes with dual corrector and potentiator activities.
Short-Circuit Current Measurements
⌬Phe508-CFTR-expressing FRT cells were cultured on Snapwell in-
serts for 7 to 9 days. For corrector testing, test compounds were incu-
bated with FRT cells for 18 to 24 h at 37°C before measurements. For
potentiator testing, the FRT cells were incubated for 18 to 24 h at 27°C
before measurements. 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 main-
tained at 37°C. The basolateral membrane was permeabilized with 250
␮g/ml amphotericin B. Hemichambers were connected to a DVC-1000
voltage clamp (World Precision Instruments, Inc., Sarasota, FL) via

Cyanoquinoline ⌬Phe508-CFTR Activators
685
Ag/AgCl electrodes and 1 M KCl agar bridges for recording of short- 37°C in the absence or presence of forskolin (20 ␮M) or forskolin ϩ
circuit current.
isobutyl methylxanthine (500 ␮M). Cells were lysed by freeze-thaw,
centrifuged to remove cell debris (600g, 10 min, 4°C), and assayed for
cAMP using a parameter cAMP immunoassay kit (R&D Systems,
CFTR Immunoblot
⌬Phe508-CFTR-expressing FRT cells grown on six-well plates were Minneapolis, MN).
treated with Corr-4a (10 ␮M), N-(2-((3-Cyano-5,7-dimethylquinolin-2-
yl)amino)ethyl)-3-methoxybenzamide (CoPo-22; 20 ␮M), or vehicle
(DMSO) at 37°C for 24 h. After treatment, cells were washed with PBS
CoPo-22 Synthesis
N-(3,5-Dimethylphenyl)acetamide (1). Acetic anhydride (2.84
and lysed in 20 mM HEPES, pH 7, 150 mM NaCl, 1 mM EGTA, and 1%
Igepal containing complete protease inhibitor cocktail (Roche Diagnos-
tics, Indianapolis, IN). After preclearing, lysates were subjected to SDS-
polyacrylamide gel electrophoresis and analyzed by immunoblot. Pro-
teins were immunodetected using a mouse monoclonal anti-CFTR
antibody (M3A7; Millipore Corporation, Billerica, MA) followed by
horseradish peroxidase-conjugated anti-mouse IgG, and visualized by
chemiluminescence (ECL Plus; GE Healthcare, Chalfont St. Giles,
Buckinghamshire, UK).
ml, 30 mmol) was dissolved in dry tetrahydrofuran (10 ml), purged
with N2, and brought to 0°C. 3,5-Dimethylaniline (1.25 ml, 10 mmol)
was then added drop-wise. After the addition of the aniline, the
reaction was allowed to warm to room temperature and stirred for an
additional hour. The solution was then poured over ice and 1 M
NaOH (aqueous) was added until the pH was between 12 and 14. The
precipitate was filtered, dissolved in dichloromethane (DCM), and
dried over Na2SO4. The drying agent was filtered and solvent was
removed under reduced pressure to afford pure product in 99% yield
as a white solid (Moussaoui et al., 2002).
Cyclic Nucleotide Assay
FRT cells expressing ⌬Phe508-CFTR were grown in six-well
2-Chloro-5,7-dimethylquinoline-3-carbaldehyde (2). Phos-
plates and incubated with CoPo-22 (0, 5, and 10 ␮M) for 10 min at phorous oxychloride (6.52 ml, 70 mmol) and dry dimethylformamide
Fig. 1. Dual-acting ⌬Phe508-CFTR CoPo identified by high-throughput screening. A, screening procedure. Left, corrector assay: FRT cells coexpress-
ing human ⌬Phe508-CFTR and a halide-sensing YFP were incubated with test compounds at 37°C for 24 h. ⌬Phe508-CFTR function was assayed in
a plate reader from YFP fluorescence quenching in response to iodide addition in the presence of forskolin (20 ␮M) plus genistein (50 ␮M). Right,
potentiator assay: cells were incubated at 27°C for 24 h before assay (temperature rescue) to target ⌬Phe508-CFTR to the plasma membrane. Test
compounds were added for 10 min at room temperature in presence of forskolin (20 ␮M) before iodide addition. B, structures of novel ⌬Phe508-CFTR
correctors identified in the primary screen. C, representative traces showing iodide influx at different [CoPo-22] for corrector assay (left) and
potentiator assay (right). D, dose-response data of CoPo-22 in corrector (left) and potentiator (right) assays (S.E.M., n ϭ 3). Fits to single-site activation
model shown.

686
Phuan et al.
(1.94 ml, 25 mmol) were refluxed for 2 h under N2. Acetamide 1 pure product as an orange solid in 95% yield (Moussaoui et al.,
(1.632 g, 10 mmol) was added to the reaction solution as a solid and
stirred at room temperature for an additional 3 h. The solution was
2002).
2-Chloro-5,7-dimethylquinoline-3-carbonitrile (3). Aldehyde
poured slowly over ice, diluted with water (200 ml), and neutral- 2 (1.095 g, 5 mmol), hydroxylamine hydrochloride (0.365 g, 5.25
ized with solid K2CO3. The precipitate was then filtered, dissolved mmol), and triethylamine (1.00 ml, 7 mmol) were combined in eth-
in chloroform, and dried over Na2SO4. The drying agent was anol (50 ml). The solution was refluxed for 3 h, and then the ethanol
filtered and solvent was removed under reduced pressure to afford
was removed under reduced pressure. HCl (1 M aqueous, 100 ml)
TABLE 1
Corrector and potentiator activities of selected CoPo analogs
Corrector
Potentiator
Compound
EC50
Vmax
EC50
Vmax
␮M
␮M/s
␮M
␮M/s
CH3
CN
H
N
CoPo-22
2.2
300
14
306
CH3
H3C
N
N
H
O
O
CN
O
O
CH3
CH3
H
N
CoPo-01
CoPo-02
3.8
3.9
223
281
15
15
250
297
N
N
H
CH3
O
H3C
CN
O
O
CH3
CH3
H
N
N
N
H
O
CH3
CN
H
CoPo-03
CoPo-05
14
288
102
140
14
289
72
N
S
H3C
H3C
N
N
N
H
O
CN
CH3
O
N
5.0
4.2
3.8
N
S
O
O
O
CN
N
H3C
N
CoPo-08
CoPo-20
CoPo-09
11
13
4.6
195
261
280
N
O
F
CN
N
S
Inactive
Inactive
H3C
H3C
N
N
N
O
O
O
CN
N
N
O
Cl
CN
H3C
N
N
CoPo-10
CoPo-14
Inactive
Inactive
5.0
6.0
235
275
N
O
Br
CN
O
CH3
H
N
H
N
N
N
H
CH3
S

Cyanoquinoline ⌬Phe508-CFTR Activators
687
was added to the crude material, and product was extracted with
DCM (100 ml). The organic layer was separated and dried over
dissolved in dry DCM (10 ml). The reaction was stirred at room
temperature for 30 min. Carbonitrile 4 (0.201g, 1 mmol) dissolved
Na2SO4, the drying agent was removed by filtration, and the solvent in 5 ml of dry DCM was added drop-wise to the solution, and the
was removed under reduced pressure. The crude product was then reaction was stirred for 18 h. The reaction was diluted with DCM
dissolved in 50 ml of dry benzene. Thionyl chloride (0.73 ml, 10 (50 ml) and washed with 1 M NaHSO4 (aqueous, 2 ϫ 100 ml).
mmol) was added drop-wise to the solution, and the reaction was
Organics were dried over Na2SO4, filtered, and solvent was re-
refluxed for 4 h under N2. The solution was allowed to cool to room moved under reduced pressure. The crude product was then pu-
temperature, then the benzene and excess thionyl chloride were rified by flash chromatography (4:1 hexane/ethyl acetate mobile
removed under reduced pressure to afford the known product 3 in phase) to produce a light yellow solid (CoPo-22) in 73% yield. 1H
93% yield as a light brown solid [1H NMR (400 MHz, CDCl3) ␦ 8.62 NMR (400 MHz, CDCl3) ␦ 8.35 (s, 1H), 8.13 (s, 1H), 7.32 (s, 1H),
(s, 1H), 7.62 (s, 1H), 7.33 (s, 1H), 2.66 (s, 3H), 2.54 (s, 3H)], which was
used directly in the next reaction.
2-((2-Aminoethyl)amino)-5,7-dimethylquinoline-3-carbo-
7.28 (s, 1H), 7.20 (d, J ϭ 7.5, 1H), 7.13 (t, J ϭ 7.8, 1H), 6.98 (s, 1H),
6.95 (d, J ϭ 8.1, 1H), 5.82 (s, 1H), 3.91 (q, J ϭ 5.3, 2H), 3.79 to 3.68
(m, 5H), 2.55 (s, 3H), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3) ␦
nitrile (4). Carbonitrile 3 (1.083 g, 5 mmol) and 1,2-aminoethane 168.10, 159.87, 155.10, 144.27, 140.97, 136.27, 135.41, 129.42,
(1.00 ml, 15 mmol) were refluxed in dioxane (50 ml). The reaction 127.13, 124.48, 119.35, 119.23, 117.72, 116.87, 112.46, 94.30,
was allowed to cool to room temperature, and dioxane was re- 55.49, 43.39, 41.25, 29.92, 22.21, 18.57. IR (neat): 3380, 3357,
moved under reduced pressure. The crude product was suspended 2957, 2925, 2217, 1605, 1583, 1535, 1258. Electrospray ionization-
in saturated NH4Cl (aqueous) and filtered. The solids were liquid chromatography/mass spectrometry, m/z [M ϩ H]ϩ
washed with diethyl ether and allowed to dry on filter paper under 375.18.
ϭ
vacuum to give the known product 4 in 80% yield [1H NMR (400
MHz, CDCl3) ␦ 8.31 (s, 1H), 7.32 (s, 1H), 6.92 (s, 1H), 5.64 (t, J ϭ
4.8, 1H), 3.65 (q, J ϭ 5.7, 2H), 3.01 (t, J ϭ 6.0, 2H), 2.52 (s, 3H),
2.42 (s, 3H)], which was used directly in the next reaction.
Results
Identification of Cyanoquinoline Correctors/Poten-
CoPo-22. EDC hydrochloride (0.192 g, 1 mmol), m-anisic acid
(0.152 g, 1 mmol), and triethylamine (0.14 ml, 2.5 mmol) were tiators of ⌬Phe508-CFTR. Screening of ϳ110,000 small
Fig. 2. Structure-activity relationship analysis of CoPo analogs. A, structural determinants of CoPo corrector (left) and potentiator (right) activities.
(see Table 1 for activity data of CoPo analogs). B, concentration-dependence of corrector (left) and potentiator (right) activities of CoPo-01, CoPo-02,
CoPo-05, and CoPo-08. C, CoPo-22 synthesis scheme.

688
Phuan et al.
molecules was done to identify new ⌬Phe508-CFTR corrector below, the maximal efficacy of CoPo-22 corrector and poten-
scaffolds having high correction efficiency or having indepen- tiator actions were comparable with those of the bithiazole
dent potentiator activity. As diagrammed in Fig. 1A (left), Corr-4a and the flavone genistein, respectively.
primary screening for corrector activity was done using a
Synthesis and Structure-Activity Relationship Anal-
cell-based fluorescence assay of iodide influx in which FRT ysis of CoPo-22. In an attempt to identify CoPo analogs
cells expressing ⌬Phe508-CFTR and an iodide-sensitive YFP with improved potency as well as to establish initial struc-
were incubated with test compounds at 10 ␮M for 18 to 24 h ture-activity relationship data of the core cyanoquinoline
before assay. Iodide influx was measured by the addition of scaffold, we screened 180 commercially available analogs of
extracellular iodide in the presence of maximal concentra- CoPo-22. Table 1 summarizes corrector and potentiator ac-
tions of a potentiator (50 ␮M genistein) and cAMP agonist (20 tivities (EC50 and Vmax from concentration-dependence stud-
␮M forskolin). Compound efficacy and potency (from concen- ies) of active compounds, and Fig. 2A summarizes the struc-
tration-dependence studies) in the corrector assay were tural requirements for corrector and potentiator activities.
compared with reference bithiazole Corr-4a (10 ␮M) and to Figure 2B shows representative concentration-dependence
low-temperature-rescued cells. Active compounds were coun- data for four analogs. The majority of the 180 cyanoquinoline
ter-screened for potentiator activity (Fig. 1A, right), in which analogs were inactive. Six compounds showed both corrector
iodide influx was measured in the ⌬Phe508-CFTR expressing and potentiator activities, with CoPo-22 being the most po-
FRT cells after low-temperature rescue (to target ⌬Phe508- tent corrector. CoPo-01 and CoPo-02 are structurally similar
CFTR to the plasma membrane) and in the presence of 20 ␮M to CoPo-22 and have corrector and potentiator activities com-
forskolin.
parable with those of CoPo-22. Compounds containing het-
Screening yielded three novel scaffolds with corrector ac- erocycles, such as the thiophene CoPo-03 and the benzosul-
tivity (Fig. 1B), which was verified by CFTRinh-172 inhibition fonamide CoPo-05, also show dual activities, albeit of lower
of corrector-dependent iodide influx and inability to increase potency and/or maximum efficacy. Interestingly, several
iodide influx in FRT null cells (data not shown). Although compounds, such as CoPo-14, showed potentiator-only activ-
none of the three compounds had greater corrector potency ity. Replacing the ethylene bridge with a piperazine or 1,4-
than Corr-4a, the cyanoquinoline CoPo-22 showed indepen- diazepane ring diminished or abolished corrector activity
dent potentiator activity. Representative iodide influx data (comparing CoPo-03 and CoPo-20). No compounds were iden-
from the corrector (left) and potentiator (right) fluorescence tified with corrector-only activity. These data suggest that
plate reader assays of CoPo-22 are shown in Fig. 1C. We have the dual corrector-potentiator activity is not a general fea-
not observed potentiator activity previously by a ⌬Phe508- ture of cyanoquinolines but is dependent on the particular
CFTR corrector, in which compound is added only a few scaffold and substituents.
minutes before assay. Concentration-dependence data are
For further characterization studies, we resynthesized
shown in Fig. 1D. As shown by electrophysiological analysis CoPo-22 in Ͼ98% purity in six steps with an overall yield of
Fig. 3. Characterization of CoPo-22 corrector activity. A, ⌬Phe508-CFTR expressing FRT cells were incubated at 37 or 27°C with or without CoPo-22
(20 ␮M) or Corr-4a (10 ␮M). Iodide influx (S.E.M., n ϭ 4) shown in the presence of forskolin (20 ␮M) or forskolin (20 ␮M) plus genistein (50 ␮M). ء,
P Ͻ 0.01 compared with control. B, forskolin dose-response for experiments as in A, measured in the presence of genistein (50 ␮M). C, representative
short-circuit measurements showing apical membrane chloride current after incubation for 24 h at 37°C with indicated [CoPo-22]. Incubation with
Corr-4a (5 ␮M) shown as reference (right). Forskolin (20 ␮M), genistein (50 ␮M), and CFTRinh-172 (10 ␮M) added where indicated. D, CoPo-22
concentration-dependence deduced from experiments as in C (S.E.M., n ϭ 3–4). Fits to single-site binding model shown.

Cyanoquinoline ⌬Phe508-CFTR Activators
689
52% (Fig. 2C). Acetylation of commercially available 3,5-
dimethylaniline produced the corresponding acetamide in
near quantitative yield. Formation of the quinoline ring was
achieved through reaction with phosphorous oxychloride,
giving 2-chloroquinoline carbaldehyde. Condensation of the
carbaldehyde with hydroxylamine followed by dehydration
using thionyl chloride gave the cyanoquinoline core in 93%
yield. Displacement of the chloride by 1,2-di-aminoethane
gave the aminocyanoquinoline, which was coupled with m-
anisic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide to give CoPo-22 in 73% yield after purification.
Characterization of CoPo-22 Corrector Activity. To
investigate whether CoPo-22 corrector activity is additive
with low-temperature rescue, as found previously for Corr-4a
(Pedemonte et al., 2005a), ⌬Phe508-CFTR expressing FRT
cells were incubated with CoPo-22 (20 ␮M) or Corr-4a (10
␮M) for 24 h at 27 or 37°C. As shown in Fig. 3A, CoPo-22 and
Corr-4a each increased iodide influx at 37°C, with the need
for inclusion of genistein. Both compounds substantially in-
creased iodide influx at 27°C in an approximately additive
manner with low-temperature rescue (control, 27°C), sug-
gesting different mechanisms for low-temperature rescue
and corrector action. To further investigate the forskolin
requirement to increase ⌬Phe508-CFTR conductance in the
corrector assay, a forskolin concentration-dependence was
done in the ⌬Phe508-CFTR-expressing FRT cells after cor-
rector incubation and/or low-temperature rescue. Figure 3B
shows that substantial increase in iodide influx by each of the
rescue/corrector maneuvers required relatively high concen-
trations of forskolin compared with that of Ͻ1 ␮M needed for
activation of wild-type CFTR.
Short-circuit current was measured as a definitive electro-
physiological assay to verify CoPo-22 corrector action. Apical
membrane chloride current was measured in ⌬Phe508-CFTR-
expressing FRT cells after basolateral membrane permeabili-
zation and in the presence of a transepithelial chloride gradient
(apical, 65 mM; basolateral, 130 mM). Figure 3C shows in-
creased apical membrane current when cells were incubated for
18 to 24 h with increasing [CoPo-22] before short-circuit current
assay. The increased apical membrane current was fully inhib-
ited by CFTRinh-172. The increase in apical membrane current
conferred by 5 and 10 ␮M CoPo-22 was comparable with that
conferred by 5 ␮M Corr-4a (Fig. 3C, right). Figure 3D summa-
rizes CoPo-22 concentration dependence data from short-circuit
current studies.
Fig. 4. Additive corrector efficacy of CoPo-22 and Corr-4a. A, Corr-4a
concentration-dependence of iodide influx (measured with 20 ␮M forsko-
lin ϩ 50 ␮M genistein) in the presence of indicated (submaximal) [CoPo-
22] (S.E.M., n ϭ 4). B, additivity studies showing iodide influx after
incubation with maximal CoPo-22 and Corr-4a (S.E.M., n ϭ 4, ء, P Ͻ 0.01
compared with CoPo-22 or Corr-4a alone). C, immunoblot (anti-CFTR
antibody) after 24-h incubation at 37°C of ⌬Phe508-CFTR expressing
FRT cells with Corr-4a or CoPo-22 (or DMSO vehicle). Bands B (core-
glycosylated) and C (complex-glycosylated) indicated. For comparison,
data shown for (untreated) FRT cells expressing wild-type CFTR.
To investigate possible synergy between CoPo-22 and
Corr-4a in corrector efficacy, a Corr-4a concentration-depen- the absence of corrector, but band C was visualized after 24-h
dence was done at submaximal [CoPo-22] of 0.3 and 1 ␮M, incubation at 37°C with CoPo-22 or Corr-4a. Band B, which
which did not by itself increase iodide influx significantly. corresponds to core-glycosylated ⌬Phe508-CFTR, was also
Figure 4A shows a small but significant increase in iodide seen.
influx at relatively high [Corr-4a] for 1 versus 0 ␮M CoPo-22.
Characterization of CoPo-22 Potentiator Activity.
We further investigated additivity from measurements of Short-circuit current measurements were done to character-
iodide influx done after incubation with maximal concentra- ize CoPo-22 potentiator activity, in which apical membrane
tions of CoPo-22 and Corr-4a, alone or in combination. Figure chloride current was measured in ⌬Phe508-CFTR-expressing
4B shows significant additivity of CoPo-22 and Corr-4a ac- FRT cells, after low-temperature rescue, in response to
tion, supporting the possibility of independent actions of CoPo-22 additions. Figure 5A shows CoPo-22 concentration-
these correctors.
dependent increases in apical membrane current seen in the
The action of CoPo-22 as a corrector of defective ⌬Phe508- presence of forskolin. The lack of CoPo-22 effect in the ab-
CFTR cellular processing was verified by CFTR immunoblot sence of forskolin indicates the need for ⌬Phe508-CFTR
analysis. Wild-type CFTR was detected as a strong band at phosphorylation, as has been found for other potentiators.
170 kDa (band C), corresponding to complex glycosylated Genistein produced a small increase in chloride current after
CFTR. Little or no band C for ⌬Phe508-CFTR was detected in maximal CoPo-22. CFTRinh-172 abolished all chloride cur-

690
Phuan et al.
Fig. 5. Characterization of CoPo-22 potenti-
ator activity. Short-circuit current measured
in FRT cells expressing ⌬Phe-CFTR (A) and
wild-type (B), showing responses to indicated
forskolin and CoPo-22 concentrations.
⌬Phe508-CFTR expressing cells were incu-
bated at 27°C for 24 h before measurement.
Where indicated, genistein (50 ␮M) and
CFTRinh-172 (10 ␮M) were added. Represen-
tative of two to four sets of measurements.
rent, as expected. Apparent EC50 for CoPo-22 potentiator 23 Ϯ 2 pmol/ml for CoPo-22 plus forskolin. Addition of the
activity as measured by short-circuit current was ϳ10 ␮M. phosphodiesterase inhibitor isobutyl methylxanthine (500
To further investigate CoPo-22 potentiator action, short- ␮M) together with forskolin increased cAMP levels to 192 Ϯ
circuit current was measured in FRT cells expressing wild- 10, 214 Ϯ 18, and 207 Ϯ 26 pmol/ml.
type CFTR (Fig. 5B). Studies were done as in Fig. 5A, except
Characterization of CoPo-22 Activity in Human A549
that low concentrations of forskolin (0–0.5 ␮M) were used Cells. To test whether CoPo-22 is active in a different cell
because higher concentrations fully activate wild-type CFTR background, potentiator and corrector assays were done in
and thus would mask CoPo-22 potentiator action. As found ⌬Phe508-CFTR-transfected A549 cells, which are of human
for ⌬Phe508-CFTR, there was little effect of CoPo-22 in the lung epithelial origin. Figure 6B (top) shows that CoPo-22 had
absence of forskolin. In each experiment, after CoPo-22 ad- potentiator activity in the A549 cells comparable with that in
ditions, 10 ␮M forskolin was added to fully activate wild-type FRT cells. Apparent EC50 for CoPo-22 potentiator activity was
CFTR, followed by 50 ␮M genistein, which had little effect, ϳ8 ␮M (Fig. 6B). However, CoPo-22 showed little corrector
followed by 10 ␮M CFTRinh-172, which inhibited all chloride activity compared with Corr-4a when incubated for 24 h at 37°C
current. CoPo-22 partially activated wild-type CFTR when (Fig. 6C, top). We further tested CoPo-22 for corrector activity in
added after 0.25 or 0.5 ␮M forskolin, with EC50 ϳ10 ␮M.
A549 cells under the low-temperature synergy condition; how-
Potentiator studies were also done in FRT cells expressing ever, we found that ⌬Phe508-CFTR was fully activated by
G551D-CFTR, a CF-causing CFTR mutation with defective forskolin (20 ␮M) and genistein (50 ␮M) in this cell model,
channel gating but not plasma membrane trafficking. Figure 6A precluding analysis of possible synergy (Fig. 6C, bottom). Cell-
shows that CoPo-22 functioned as a weak potentiator of specific corrector activity is well described (Pedemonte et al.,
G551D-CFTR, producing a smaller increase in chloride cur- 2010); however, the mechanism(s) responsible for cell type-
rent than that produced by genistein. Fluorescence plate specificity are not clear.
reader assays in Fig. 6B confirmed that CoPo-22 activated
G551D-CFTR in the presence of forskolin, albeit with lower
maximal efficacy than genistein. Apparent EC50 for CoPo-22
Discussion
activation of G551-CFTR was ϳ5 ␮M (Fig. 6A, bottom), with
maximum efficacy much lower than that of genistein.
We report here the identification of a novel class of cyano-
quinolines, some of which have independent corrector and
CoPo-22 did not affect cellular cAMP levels in assays done potentiator activities for normalization of defective ⌬Phe508-
on the FRT cells after 10-min incubation with CoPo-22 alone CFTR folding/ER retention and chloride channel gating, re-
or in the presence of 20 ␮M forskolin. cAMP levels were 4.1 Ϯ spectively. The dual corrector-potentiator activity of CoPo-22
0.4, 4.2 Ϯ 0.5 and 4.3 Ϯ 0.4 pmol/ml (S.E., n ϭ 4) for 0, 5, and is consistent with the possibility of binding to site(s) on
10 ␮M CoPo-22 alone, respectively, and 26 Ϯ 1, 22 Ϯ 2, and the CFTR protein. The rapid normalization of defective

Cyanoquinoline ⌬Phe508-CFTR Activators
691
Fig. 6. CoPo-22 activity in G551D-CFTR
expressing FRT cells and ⌬Phe508-CFTR-
expressing A459 cells. A, top, short-circuit
current measured in FRT cells expressing
G551D-CFTR, showing responses to indi-
cated forskolin and CoPo-22 concentra-
tions. Where indicated, genistein (100 ␮M)
and CFTRinh-172 (10 ␮M) were added. Rep-
resentative of three sets of measurements.
Bottom, plate reader assay of G551D-
CFTR chloride conductance showing repre-
sentative fluorescence quenching curves
(inset) and deduced concentration depen-
dence of CoPo-22 and genistein potentiator
action (S.E.M., n ϭ 4). Measurements were
made in the presence of 20 ␮M forskolin.
B, potentiator assay done in ⌬Phe508-
CFTR expressing A549 cells by YFP/iodide
fluorescence quenching as in Fig. 1. Repre-
sentative fluorescence quenching curves
(top) shown with deduced CoPo-22 and
genistein concentration-dependence (bot-
tom S.E.M., n ϭ 4). C, corrector assay done
in ⌬Phe508-CFTR expressing A549 cells by
YFP/iodide fluorescence quenching, in which
cells were incubated with vehicle or indi-
cated correctors at 37°C (top) or 27°C
(bottom) for 24 h before iodide influx mea-
surement. Representative of three sets of
measurements.
⌬Phe508-CFTR channel gating by CoPo-22 is probably a a non-⌬Phe508 CFTR mutant. Mutagenesis studies using a
consequence of direct binding, as is its efficacy in the rapid double-cysteine CFTR mutant, in which cross-linking occurs
activation of chloride conductance in wild-type and G551D- only when protein folds into native structure, suggested that
CFTR. The mechanism of CoPo-22 correction action is less Corr-4a interacts with ⌬Phe508-CFTR in the ER to promote
clear.
its folding (Loo et al., 2008), although this could be also an
The molecular mechanisms remain largely unknown by indirect consequence of Corr-4a effect via proteostasis. Al-
which ⌬Phe508-CFTR correctors partially promote ⌬Phe508- though the peripheral stabilization of ⌬Phe508-CFTR in the
CFTR escape from the ER, allowing Golgi and plasma mem- presence of Corr-4a could be explained by a more native-like
brane targeting. The general possibilities include corrector structure, recent evidence suggests that Corr-4a may inter-
action as a pharmacological chaperone by direct ⌬Phe508- fere with the ubiquitination machinery (Jurkuvenaite et al.,
CFTR binding to improve its folding efficiency and stability 2010) that is involved in disposal of rescued ⌬Phe508-CFTR
at the endoplasmic reticulum and plasma membrane and/or from the cell surface (Okiyoneda et al., 2010). Direct compel-
by influencing activity of the proteostasis network (Powers et ling evidence is lacking for the mechanisms by which correc-
al., 2009). The latter may entail transcriptional, transla- tors promote ⌬Phe508-CFTR plasma membrane targeting.
tional, and/or posttranslational modulations to enhance
An interesting observation was the apparent dissociation
⌬Phe508-CFTR biogenesis and/or to impede degradation. For of corrector and potentiator activities of the cyanoquinolines,
example, ⌬Phe508-CFTR escape from the ER could be facil- in which some compounds have dual activities whereas oth-
itated by extending its folding time and delaying degradation ers have only potentiator activity. SAR analysis indicated
by inhibiting Rma1 and C terminus of Hsc70-interacting that replacing the flexible ethylene bridge, as found in
protein (CHIP), E3 ubiquitin ligases (Grove et al., 2009) and CoPo-22 or CoPo-03, with a constrained six- or seven-mem-
the 90-kDa heat shock protein cochaperone Aha1 (Wang et bered ring diminished or abolished corrector activity. Several
al., 2006a). Initial studies of Corr-4a mechanism support the mechanisms could account for dissociation between corrector
possibility of direct bithiazole-⌬Phe508-CFTR interaction and potentiator activities. One possibility is distinct com-
(Pedemonte et al., 2005a), because Corr-4a was ineffective in pound binding sites on ⌬Phe508-CFTR, or other secondary
correcting other mutant membrane proteins for which phar- targets, for corrector and potentiator activities. Alternatively,
macological or low-temperature rescue is possible, including the differential sensitivity of the protein quality control mech-

692
Phuan et al.
Authorship Contributions
anism and CFTR functional responsiveness may account for
their discordant potentiator and corrector activities.
Participated in research design: Phuan, Yang, and Verkman.
Conducted experiments: Phuan and Yang.
Contributed new reagents or analytic tools: Knapp, Wood, and
Kurth.
Wrote or contributed to the writing of the manuscript: Phuan,
Lukacs, Knapp, Kurth, and Verkman.
Although there are no prior published data on CoPo-22 or
the cyanoquinoline analogs tested here, there are several
reports in the patent literature on biological data for other
compounds containing the cyanoquinoline core. Cyanoquino-
lines containing a six- or seven-membered ring with struc-
ture similar to CoPo-09 and CoPo-20 have been reported to
inhibit neuronal degeneration and stimulate neurogenesis
(Kelleher, 2007). Cyanoquinolines have also been reported to
have B-raf kinase inhibition activity for the potential treat-
ment of cancer (Gahman et al., 2006). Compounds containing
only the cyanoquinoline core but with different bridging
substituents have been described as orexin antagonists
(Branch et al., 2002) and adenosine A2A receptor antago-
nists (Kosakata et al., 2005). The patent literature thus
suggests little cyanoquinoline toxicity. To our knowledge,
ion channel-modulating effects of cyanoquinolines have
not been reported.
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Address correspondence to: Dr. Alan S. Verkman, 1246 Health Sciences
East Tower, University of California, San Francisco, CA 94143-0521. E-mail:
alan.verkman@ucsf.edu