From the Departments of Medicine and Physiology,
Cardiovascular Research Institute, University of California, San
Francisco, California, 94143-0521 and the ¶ Department of
Chemistry, University of California, Davis, California
95616-5295
Received for publication, March 1, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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The flavonoid genistein and the
benzo[c]quinolizinium MPB-07 have been shown to
activate the cystic fibrosis transmembrane conductance regulator
(CFTR), the protein that is defective in cystic fibrosis. Lead-based
combinatorial and parallel synthesis yielded 223 flavonoid,
quinolizinium, and related heterocyclic compounds. The compounds were
screened for their ability to activate CFTR at 50 µM
concentration by measurement of the kinetics of iodide influx in Fisher
rat thyroid cells expressing wild-type or G551D CFTR together with the
green fluorescent protein-based halide indicator YFP-H148Q. Duplicate
screenings revealed that 204 compounds did not significantly affect
CFTR function. Compounds of the 7,8-benzoflavone class, which are
structurally intermediate between flavones and
benzo[c]quinoliziniums, were effective CFTR activators
with the most potent being
2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate
(UCCF-029). Compounds of the novel structural class of
fused pyrazolo heterocycles were also strong CFTR activators with the
most potent being
3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCCF-180). A CFTR inhibitor was also identified. The
active compounds did not induce iodide influx in null cells deficient
in CFTR. Short-circuit current measurements showed that the CFTR
activators identified by screening induced strong anion currents in the
transfected cell monolayers grown on porous supports. Compared with
genistein, the most active compounds had up to 10 times greater potency
in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP
concentration or inhibit phosphatase activity, suggesting that
CFTR activation may involve a direct interaction. These results
establish an efficient screening procedure to identify CFTR activators
and inhibitors and have identified 7,8-benzoflavones and pyrazolo
derivatives as novel classes of CFTR activators.
The most common lethal genetic disease, cystic fibrosis
(CF),1 is caused by mutations
in the cystic fibrosis transmembrane conductance regulator protein CFTR
(1). CFTR is a cAMP-regulated epithelial cell membrane Cl Activators of CFTR chloride permeability can function by a number of
direct and indirect mechanisms including increased cAMP production, inhibition of phosphodiesterase or phosphatase activities, or direct interactions with CFTR. Several chemical classes of activators have been identified including flavones/isoflavones (e.g. genistein, Refs. 3 and 4),
benzo[c]quinoliziniums (MPB-07, Ref. 5), xanthines
(isobutylmethylxanthine and 8-cyclopentyl-1,3-dipropylxanthine, Refs.
6-8), and benzimidazolones (NS004, Ref. 9).
Flavones/isoflavones such as genistein and apigenin are thought to
interact directly with the nucleotide binding fold regions of CFTR and
not by the inhibition of tyrosine kinases (10, 11), although the exact mechanism has not been determined (12). There has been considerable interest in developing improved flavone-type CFTR activators because of
their consistent and strong activation of wild-type and mutant CFTR in
different cell types (13-15). The benzo[c]quinolizinium MBP-07 also seems to activate CFTR without elevating intracellular cAMP
or ATP concentrations or affecting the activities of several known
protein phosphatases (5).
The purpose of this study was to apply a high throughput screening
assay for the discovery of novel CFTR activators. Preliminary combinatorial libraries were synthesized based on the flavone and
benzo[c]quinolizinium structures. We chose these lead
compounds because they are the most likely of the known activators to
interact directly with CFTR, and they share a common structural motif
amenable to the design of hybrid structures. In addition, as reported
separately (4, 16-21), we have developed solution and solid-phase
synthesis and purification methods for the efficient large scale
production of test compounds. We report here the screening of purified
test compounds for CFTR-activating potency by a cell-based halide
transport assay that utilized fluorescent epithelial cells stably
expressing wild-type or G551D CFTR together with the green fluorescent
protein halide indicator YFP-H148Q. YFP-H148Q fluorescence is decreased by halides by a rapid shift in pKa upon halide
binding to a site near its tri-amino acid chromophore (22). The screen revealed new classes of CFTR activators and a CFTR inhibitor, which
were characterized further in terms of potency, CFTR specificity, activation mechanism, and the ability to induce transepithelial chloride currents in polarized epithelial cells.
Generation of Combinatorial Compound Library--
(See
Supplemental Material for a complete list of compounds and their
structures.) Unless otherwise indicated, the flavonoids screened in the
present study were prepared using the recently reported modification
(23) of the conventional Baker-Venkataraman flavone synthesis (24, 25).
The flavones UCCF-016-UCCF-21 were synthesized
using a different modification of the Baker-Venkataraman flavone
synthesis (26). All 2-aryl-4-quinolones and 2-aryl-quinoline-4-thiones were prepared using our previously described protocol (19). The flavone
UCCF-045 was prepared as described (4), and the flavones
UCCF-027, UCCF-028, and
UCCF-034-UCCF-038 were prepared from
5-hydroxy-4'-iodoflavone via Suzuki coupling (27) using the following
representative experimental procedure: 3-thiopheneboronic acid (1.5 eq,
15 mg), Pd(PPh3)4 (2.5 mol %, 1.7 mg, 0.00157 mmol), and K2CO3 (2.7 eq, 23.1 mg, 0.254 mmol)
were added to a stirred solution of the 5-hydroxy-4'-iodoflavone (1.0 eq, 25 mg, 0.0628 mmol) in a mixture of dimethoxyethane (0.75 ml) and
water (0.1 ml). The mixture was refluxed at 85 °C for 9 h. After cooling, the solvent was removed by rotary evaporation. The
solid residue was dissolved in dichloromethane and extracted twice with
30 ml of water. The combined organic layers were washed with brine and dried using anhydrous sodium sulfate. After concentration, the solid
residue was redissolved with a minimum quantity of hot ethyl acetate
followed by the slow addition of hexane to allow crystallization. The
solids were then filtered and washed with excess 10:1 hexane/ethyl acetate to obtain 10.5 mg of UCCF-037 (52% yield) as a
light cream-colored solid. Compound identity and purity were confirmed
by 1H and 13C NMR and thin layer
chromatography. Quinolizinium salts were synthesized using the
procedure described by Becq et al. (5). The
( Cell Culture and Transfection--
Fischer rat thyroid (FRT)
cells expressing the human wild-type CFTR or CFTR-G551D were
transfected with the plasmid pcDNA3.1 (Invitrogen) containing the
cDNA encoding YFP-H148Q and selected in G418 (0.75 mg/ml).
Clonal populations were obtained by repeated limiting dilution and
cloning rings. Cells were cultured in Coon's modified F-12 medium
supplemented with 5% fetal calf serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Halide Transport Assay--
Cells were plated in 96-well black
microplates (Corning Costar) at a density of 20,000 cells/well using a
Labsystems multidrop apparatus. After 24-48 h, the cells were washed
three times with phosphate-buffered saline using a Labsystems cell-wash
apparatus and were incubated at 37 °C for 30 min. The residual
volume of phosphate-buffered saline was 40 µl/well. The assay was
performed in a FLUOstar Galaxy microplate reader (BMG LabTechnologies,
Inc.) equipped with HQ500/20X (500 ± 10 nm) excitation and
HQ535/30M (535 ± 15 nm) emission filters (Chroma Technology
Corp.) and syringe pumps. YFP fluorescence in each well was monitored
continuously for 100 s, and data were binned in 0.5-s intervals.
At 5 s after the start of fluorescence recording, a syringe pump
delivered 110 µl of a modified phosphate-buffered saline in which 137 mM NaI replaced NaCl to give an extracellular
[I Cell Toxicity, Intracellular cAMP, and Phosphatase
Assays--
The cell toxicity of all synthesized compounds was
assessed using the sulforhodamine incorporation method (28). Briefly, cells grown in 96-well plates were exposed for 24 h to a 50 µM concentration of each compound in the culture medium.
Cells were then fixed for 1 h at 4 °C by adding 10 µl of 50%
trichloroacetic acid/100 µl of culture medium and subsequently washed
five times with water. The cells were stained with 100 µl of 0.4%
sulforhodamine B in 1% acetic acid for 30 min at room temperature.
Excess dye was removed by washing with 1% acetic acid. After the
removal of residual solution and drying, the bound dye was solubilized in 100 µl of 10 mM unbuffered Tris for measurement of
solution absorbance at 564 nm.
cAMP activity was measured using the BIOTRAK enzymatic immunoassay
(Amersham Pharmacia Biotech). FRT cells expressing CFTR were cultured
in 96-well plates. After washing and incubation in phosphate-buffered
saline, the cells were incubated for 10 min with specified
concentrations of forskolin, UCCF-029, or
UCCF-180 and then lysed with the reagent provided by the
kit. The lysates were assayed for cAMP content in triplicate according
to manufacturer's instructions. Phosphatase activity was determined
with a nonradioactive assay kit (Promega). Briefly, 17 Petri dishes
containing CFTR-expressing FRT cells were washed and scraped using
ice-cold phosphate-free buffer. After centrifugation, the cells were
resuspended in 3 ml of a buffer containing 250 mM imidazole
(pH 7.2), 1 mM EGTA, 1 mM MgCl2,
0.1% Short-circuit Current Measurements--
FRT cells stably
expressing human wild-type or G551D CFTR were cultured on Snapwell
inserts (Costar) at a density of 500,000 cells/insert. After 8-15
days, when the transepithelial electrical resistance was 3-4
kilo-ohm/cm2, the inserts were mounted in an Ussing chamber
system (Vertical diffusion chamber, Costar). Apical and basolateral
hemichambers were filled with 5 ml of a solution containing 75 mM NaCl and 75 mM sodium gluconate (apical
solution) and 150 mM NaCl (basolateral solution) (both pH
7.3). The chambers were connected to a DVC-1000 voltage clamp (World
Precision Instruments) via silver/AgCl electrodes and 1 M
KCl agar bridges. The electrode offset potential and the fluid
resistance were corrected using an insert not containing cells. During
the experiments, the transepithelial potential was clamped at 0 mV, and
the short-circuit current was recorded by a 16-bit analog-to-digital
converter (PC-516 DAQ board, National Instruments) using a
collection/display program written using LabView 6I software (National
Instruments). At the beginning of the experiment, the basolateral
membrane was permeabilized by adding 250 µg/ml amphotericin B. Permeabilization (developing over ~30 min) was monitored by changes
in transepithelial resistance. Forskolin was added at indicated
concentrations in both hemichambers, and test compounds were added on
the apical side only. In these measurements the basolateral membrane
was permeabilized with amphotericin B, and a Cl Fig. 1 shows core structures of the
classes of compounds that were synthesized. The compounds are referred
to as UCCF-01 (University of
California-cystic fibrosis) through
UCCF-223 and have been grouped in the following structural
classes: flavonoids, quinoliziniums, pyridiniums, azacyanines,
isoxazoles, and fused pyrazole heterocycles. Fig. 1 (bottom
row) shows the structures of the reference compound genistein and
two CFTR activators that were investigated in detail.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel that seems also to regulate the activities of other membrane proteins (2). Normally Cl
-permeable epithelial cells in
airways, pancreas, and other tissues become
Cl
-impermeable in CF, resulting in defective salt, water,
and protein transport. Although the exact mechanism by which decreased
CFTR Cl
permeability produces lung and pancreatic disease
in CF remains unclear, it is generally believed that restoration of
CFTR Cl
permeability will be clinically beneficial. An
important goal in CF research is thus the identification of small
molecule activators of CFTR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxy)alkylpyridinium and azacyanine analogs were prepared using
our previously published procedures (16, 17). The fused pyrazolo
heterocycles were synthesized as reported (18). Tetrazines and
isoxazole heterocyles were prepared as described (20, 21). Compound
homogeneity was verified by thin layer chromatography. A subset of
compounds was examined by 1H and 13C NMR and
found to be >90% pure.
] of 100 mM. After an additional 15 s, a second syringe pump delivered 50 µl of a 100 mM
I
-containing solution including specified concentrations
of forskolin. The assays were performed at 37 °C.
-mercaptoethanol, and a mixture of protease inhibitors
(Roche). Cells were lysed with 30 strokes of a Dounce homogenizer, and
the supernatant was recovered after centrifugation at 14,000 rpm for 15 min and frozen. Phosphatase activity was determined by incubating the
homogenate (5 µg of protein) with the phosphorylated peptide
substrate (provided in the kit) for 30 min at 30 °C, which was shown
to be in the linear region. The reaction was terminated by the addition
of the dye/additive mixture, and absorbance was measured at 620 nm.
gradient
was established to measure CFTR-mediated Cl
transport
directly as reported (29, 30). This maneuver eliminates possible
contributions of basolateral membrane channels. A Cl
gradient was established to generate a driving force for basolateral to
apical Cl
transport at zero transepithelial potential difference.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structures of the compounds screened for CFTR
activation. The core structures of indicated compound classes are
shown. The bottom row shows the structures of genistein and
two novel CFTR activators. See Supplemental Material for the structures
of all compounds synthesized and screened.
Each compound was screened individually for its potency as a CFTR
activator/inhibitor. Fig. 2A
shows the fluorescence assay. FRT cells coexpressing human wild-type
CFTR or G551D CFTR (which causes CF) and YFP-H148Q were cultured on
96-well plates for the monitoring of YFP fluorescence in a plate
reader. The FRT cells and assay conditions were chosen to minimize
CFTR-independent halide transport and basal (prior to cAMP simulation)
CFTR halide transport. After recording baseline fluorescence, an
osmotically matched I-containing solution was added to
the Cl
-containing solution bathing the cells to establish
a 100 mM inwardly directed I
gradient (Fig.
2A, left). There was little I
influx (decrease in fluorescence) prior to the activation of wild-type
CFTR under the experimental conditions used here. Subsequent activation
by forskolin resulted in dose-dependent I
influx and decreased cell fluorescence. Neither activation of G551D
CFTR nor of null cells that do not express CFTR was observed under
these conditions (data not shown). As depicted schematically (Fig.
2A, middle and right panels), a test
compound could (a) have no effect, (b) activate
CFTR directly (prior to forskolin addition), (c) activate
CFTR in synergy with forskolin (after forskolin addition), or
(d) inhibit CFTR.
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In the initial screen, test compounds were added to the cell bathing
solution at 50 µM concentration just prior to the assay. For screening of the cells expressing wild-type CFTR, a submaximal concentration of forskolin (250 nM) was added during the
assay to probe for synergy with the test compounds with cAMP
activation. Fig. 2B summarizes the data from duplicate
screening of the compound library. The ordinate is the maximum
normalized slope of the fluorescence time course, representing
I influx (in mM/s) measured after
I
addition. Although most of the compounds were inactive,
several compounds activated CFTR strongly in the absence of forskolin, as seen by the prompt fluorescence decrease upon I
addition. Fig. 2B (inset) provides representative
original curves from the screen showing activation of wild-type CFTR by
the compounds UCCF-027, UCCF-029,
UCCF-031, and UCCF-180. The novel activators included compounds of the novel 7,8-benzoflavone and fused pyrazole heterocycle classes. Interestingly, compound UCCF-019,
which contains a t-butyl substituent, inhibited
forskolin-stimulated CFTR activity. None of the compounds synergized
forskolin-stimulated CFTR activity without increasing CFTR activity in
the absence of forskolin. Several of the novel activating compounds
were characterized further (see below).
A full screen was also carried out in FRT cells expressing G551D CFTR.
Halide transport by G551D CFTR is not increased by cAMP elevation
alone. For screening, cells were treated with 50 µM of
each test compound, and then a high forskolin concentration (5 µM) was added during the assay to identify compounds that
potentiated the effect of cAMP elevation. With the exception of
UCCF-029, none of the 222 remaining compounds activated
CFTR prior to forskolin addition. Fig. 2C summarizes the
maximum normalized fluorescence slope, representing I
influx after forskolin addition. Many of the same compounds that activated wild-type CFTR also activated G551D CFTR, although relative activating potencies differed, and a few compounds activated
preferentially wild-type CFTR (e.g. UCCF-180) or
G551D CFTR (e.g. UCCF-023 and UCCF-030). Representative original fluorescence curves are
shown in Fig. 2C (inset).
Further analysis was done on compounds with apparent CFTR-activating
potency. Except for compound UCCF-152, repeat assays of the
putative CFTR activators confirmed the results of the original screens.
Additional screening using a different cell line (Chinese hamster ovary
cells expressing CFTR and YFP-H148Q) confirmed the results in FRT cells
(data not shown). None of the putative CFTR activators induced halide
flux in cells not expressing CFTR. Preliminary dose-response data
(concentration range 1-50 µM) were generated to identify
those compounds warranting further investigation. Fig.
3A depicts a
dose-dependence for the activation of wild-type CFTR by
UCCF-029 and UCCF-180, showing substantially
better potency than the reference compound genistein. Fig.
3B shows a similar comparison for the activation of G551D
CFTR. Genistein was active at >50 µM, whereas
UCCF-023, UCCF-027, UCCF-028, and
UCCF-030 induced significant iodide influx at 6.25-12.5
µM.
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Electrophysiological measurements of Cl current were done
to confirm CFTR activation and to compare activation potencies. Short-circuit current was measured in polarized monolayers of FRT cells
expressing wild-type or G551D CFTR. Fig.
4A shows short-circuit current
in response to the activation of wild-type CFTR by genistein versus UCCF-029 and UCCF-180. The
induced Cl
current was inhibited by the CFTR blocker
diphenylamine-2-carboxylate. The effect of forskolin is shown for
comparison. UCCF-029, the most potent activator of
wild-type CFTR in the initial screening, induced a strong
Cl
current at 5-10 µM with a significant
effect at 1-2 µM, whereas genistein was not effective at
the same concentrations. At 5 and 10 µM concentrations,
UCCF-029 was 10 and 16 times more effective than genistein
in increasing short-circuit current (in µA/cm2,
n = 5; S.E. = 61 ± 16 versus 5.7 ± 3 [5 µM]; 115 ± 28 versus 7 ± 2 [10 µM]). As found in the plate-reader assay,
UCCF-180 induced significant short-circuit current at
concentrations lower than those required for genistein. Similar
experiments showed no increase in short-circuit current in
nontransfected FRT cells, supporting the conclusion that these
compounds activate CFTR. The CFTR inhibitor UCCF-019
identified in the screening was confirmed by short-circuit current
analysis (data not shown). We found that compounds of the
benzoquinolizinium class including the lead compound MPB-07 (freshly
synthesized and dissolved) were not effective in activating CFTR in the
plate-reader assay or by short-circuit current analysis. It is unclear
whether cell-type differences or other factors account for the absence
of MPB-07 activation of CFTR in our experiments.
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Fig. 4B shows short-circuit current analysis of G551D CFTR activation by genistein versus UCCF-030. UCCF-030 was remarkably more potent than genistein, which is in agreement with the fluorescence plate-reader data. Interestingly, the activator of wild-type CFTR, UCCF-180, was ineffective in activating G551D CFTR. The compounds UCCF-023, UCCF-027, and UCCF-028 were also effective in inducing short-circuit current in cells expressing G551D CFTR (data not shown).
All compounds were screened for cell toxicity using a dihydrorhodamine
accumulation assay. At 50 µM concentrations for 24 h, the reference compound genistein caused ~20% growth inhibition. All compounds with activating potency on wild-type and/or G551D CFTR
were nontoxic (cell growth >90% of control) except for
UCCF-027, which was similar to genistein. To investigate
the CFTR-activating mechanism, the CFTR activators were tested for
their ability to elevate intracellular cAMP concentration and inhibit
cell phosphatase activity. Using an enzymatic immunoassay, no
significant elevation in cAMP concentration was found for 10-min
incubations with the activators UCCF-029 at 5 µM and UCCF-180 at 25 µM (Fig.
5A), concentrations that
induced short-circuit currents of 61 ± 16 and 53 ± 12 µA/cm2, respectively. These values were significantly
higher than that induced by 1 µM forskolin (19 ± 4 µA/cm2), which produced a substantial elevation in
intracellular cAMP concentration. Fig. 5B summarizes the
results of an assay of cell phosphatase activity, showing strong
inhibition by okadaic acid and NaF. Total cell phosphatase activity was
not significantly inhibited at concentrations of UCCF-029
and UCCF-180 that markedly stimulated CFTR. Together the
data suggest that the CFTR-activation mechanism may involve direct
compound-CFTR interactions.
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DISCUSSION |
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This study was designed to identify new CFTR activators that
interact directly with CFTR. Structure-based drug design is not yet
possible for CFTR because of the very limited knowledge about CFTR
tertiary structure. We chose to generate a combinatorial compound
library based on two lead compounds, flavones and
benzo[c]quinoliziniums, which are believed to activate
CFTR Cl conductance by direct interaction with the CFTR
molecule. A secondary purpose of this study was to establish the
utility of a new cell-based screening assay to efficiently quantify the
activity of putative CFTR activators and inhibitors. A number of
technical challenges were encountered including the synthetic organic
chemistry, cell line development, and transport assay optimization.
Several novel CFTR activators were identified as well as a CFTR
inhibitor. Interestingly, the structures of a potent class of
compounds, the 7,8-benzoflavones (e.g. UCCF-027
and UCCF-029), contained features of both flavones and
benzo[c]quinoliziniums. Activators of the novel class
of fused pyrazole heterocycles were also identified. Although these
compounds (such as UCCF-180) were less potent than the most
potent benzoflavones, they represent a new class of CFTR activators.
The CFTR activators did not induce Cl currents in null
cells, and short-circuit current analysis showed that they had
activating potencies for wild-type and G551D CFTR that were
substantially better than the existing lead compounds. The CFTR
activators were nontoxic in the cell culture model and activated CFTR
without measurable elevation in intracellular cAMP concentration or
inhibition of total cell phosphatase activity. Single channel
measurements will be required to establish whether these compounds
interact directly with CFTR.
Interestingly, the order of activating potencies of the CFTR activators
on wild-type versus G551D CFTR was different. For example,
UCCF-029 was the most potent compound in activating
wild-type CFTR but had less effect on G551D CFTR. In contrast,
UCCF-023, UCCF-028, and UCCF-030,
which were substantially less potent than UCCF-029 in
activating wild-type CFTR, were very potent in activating G551D CFTR in
synergy with forskolin. As additional structural information on CFTR
becomes available, the compounds with differential potencies discovered
here may be useful in identifying critical structural motifs important
for CFTR activation. For the most common CFTR mutant causing cystic
fibrosis in humans, F508 CFTR, the activators identified here may be
useful in synergy with compounds that correct the intracellular
processing defect of this mutant.
Although our main aim was to identify CFTR activators, the assay permits the identification of CFTR inhibitors. It was found that the flavone UCCF-019 significantly reduced CFTR-mediated halide transport. Further studies are needed to determine whether the reduced halide transport involves direct CFTR binding or inhibition of the CFTR-activating pathway. CFTR inhibitors are important in basic cystic fibrosis research in terms of CFTR functional studies and may be useful clinically in the treatment of secretory diarrheas such as cholera.
The fluorescence assay reported here should be useful in high
throughput screening of other lead-based compound libraries as well as
random combinatorial libraries. The assay permits the rapid
quantitative measurement of cAMP-independent and -dependent halide permeability using a commercial automated fluorescence plate
reader. The goal in identifying CFTR activators is to discover clinically useful drugs that will activate mutant CFTR molecules in
cystic fibrosis. The underlying assumption in CF drug discovery is that
activation of CFTR in cell culture models is a good surrogate marker
for in vivo efficacy in improving clinical outcome in cystic fibrosis patients. Although this assumption is supported by a considerable body of data on the biology and function of CFTR, rigorous
validation will require animal and human testing of potent nontoxic
CFTR activators.
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ACKNOWLEDGEMENT |
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We thank Dr. Horst Fischer for helpful discussions regarding flavone targets.
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FOOTNOTES |
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* This work was supported by a program grant from the Cystic Fibrosis Foundation for CF drug discovery and National Institutes of Health Grants DK43840, HL60288, HL59198, and DK35124.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains an appendix.
§ Present address: Instituto Gaslini, Genoa, Italy.
Cardiovascular Research Institute, 1246 Health Sciences E. Tower, University of California, San Francisco, CA 94143-0521. Tel.:
415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101892200
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ABBREVIATIONS |
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The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; YFP, yellow fluorescent protein; FRT, Fischer rat thyroid.
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