Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel

Article abstract of DOI:10.1124/mol.107.043208

Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput scree

Full text of DOI:10.1124/mol.107.043208

0026-895X/08/7303-758–768$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 73:758–768, 2008
Vol. 73, No. 3
43208/3310644
Printed in U.S.A.
Small-Molecule Screen Identifies Inhibitors of a Human
Intestinal Calcium-Activated Chloride Channel
Ricardo De La Fuente, Wan Namkung, Aaron Mills, and A. S. Verkman
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California
ABSTRACT
Calcium-activated chloride channels (CaCCs) are widely ex- out inhibition of calcium elevation, calcium-calmodulin kinase II
pressed in mammalian tissues, including intestinal epithelia, activation, or cystic fibrosis transmembrane conductance reg-
where they facilitate fluid secretion. Potent, selective CaCC ulator chloride channels. These compounds also inhibited cal-
inhibitors have not been available. We established a high- cium-dependent chloride secretion in T84 human intestinal ep-
throughput screen for identification of inhibitors of a human ithelial cells. Patch-clamp analysis indicated inhibition of CaCC
intestinal CaCC based on inhibition of ATP/carbachol-stim- gating, which, together with the calcium-calmodulin data, sug-
ulated iodide influx in HT-29 cells after lentiviral infection with gests that the inhibitors target the CaCC directly. Structure-
the yellow fluorescent halide-sensing protein YFP-H148Q/ activity relationships were established from analysis of more
I152L. Screening of 50,000 diverse, drug-like compounds than 1800 analogs, with IC50 values of the best analogs down
yielded six classes of putative CaCC inhibitors, two of which, to ϳ1 ␮M. Small-molecule CaCC inhibitors may be useful in
3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhib- pharmacological dissection of CaCC functions and in reducing
ited by Ͼ95% iodide influx in HT-29 cells in response to mul- intestinal fluid losses in CaCC-mediated secretory diarrheas.
tiple calcium-elevating agonists, including thapsigargin, with-
There are at least five distinct classes of mammalian ClϪ et al., 2005, 2006; Thiagarajah and Verkman, 2005; Far-
channels, including the cystic fibrosis transmembrane con-
ductance regulator (CFTR), CLC-type voltage-sensitive ClϪ
channels, ligand-gated (GABA and glycine) ClϪ channels,
volume-sensitive ClϪ channels, and calcium-activated ClϪ
channels (CaCCs) (reviewed in Eggermont, 2004; Hartzell et
al., 2005a). The molecular identities of epithelial cell CaCCs
remain unclear, with potential candidates including bestro-
phins (Best1-Best4), CLCs of the CKCA family, and products
of the recently described tweety gene (Hartzell et al., 2005b;
Loewen and Forsyth, 2005; Suzuki, 2006). Evidence has been
reported suggesting that CaCC(s) in intestinal epithelial
cells provide an important route for ClϪ and fluid secretion in
secretory diarrheas caused by certain drugs (antiretrovirals,
chemotherapeutics) and viruses (Morris et al., 1999, Barrett
and Keely, 2000; Kidd and Thorn, 2000; Takahashi et al.,
2000; Gyo¨mo¨rey et al., 2001; Rufo et al., 2004; Schultheiss
thing, 2006; Lorrot and Vasseur, 2007). Smooth-muscle
CaCCs have been implicated in the pathophysiology of
asthma (Bolton, 2006). Airway CaCCs are found in some
model systems to be up-regulated in cystic fibrosis (Tarran
et al., 2002), providing an alternative chloride conductance
to compensate for missing or defective CFTR.
A significant limitation in studying CaCCs has been the
lack of potent or selective inhibitors. Available compounds,
including fenamates, anthracene-9-carboxylic acid, inday-
loxyacetic acid, ethacrynic acid, and tamoxifen have low po-
tency and inhibit multiple types of ClϪ channels and trans-
porters and in some cases cause activation of BKCa Kϩ
channels (Hartzell et al., 2005a). Small-molecule CaCC in-
hibitors are predicted to be of potential utility in the therapy
of certain secretory diarrheas and of excess mucus secretion
in asthma and cystic fibrosis (Morris et al., 1999; Rufo et al.,
2004; Barnes, 2005; Cuthbert, 2006; Hegab et al., 2007).
The purpose of this study was to identify small-molecule
inhibitors of a human intestinal CaCC that target the chan-
nel itself or site(s) distal to calcium elevation. Because the
molecular identity of intestinal epithelial CaCC(s) is not
known, we developed a phenotype-based screening assay us-
ing a human intestinal epithelial cell line (HT-29) that grew
This work was supported by grants DK72517, HL73854, EB00415,
EY13574, DK35124, and DK43840 from the National Institutes of Health, and
Drug Discovery and Research Development Program grants from the Cystic
Fibrosis Foundation.
W.N. and A.M. contributed equally to this work.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.043208.
ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; CaCC, calcium-activated ClϪ channel; YFP, yellow fluorescent
protein; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; NMDG, N-methyl-D-glucamine
chloride; CaMKII, calcium-calmodulin protein kinase 2.
758

Small-Molecule CaCC Inhibitors
759
well after lentiviral expression of a yellow fluorescent protein solutions in dimethyl sulfoxide (DMSO). For the primary screen,
compounds were tested in groups of four compounds per well.
(YFP)-type halide sensor (YFP-H148Q/I152L; Jayaraman et
al., 2000). As diagrammed in Fig. 1A, the cell-based assay
involved measurement of iodide influx in a monolayer of
YFP-expressing HT-29 cells in response to a combination of
calcium-elevating agonists. A combination of agonists was
used to prevent or at least minimize the identification of
compounds that target site(s) proximal to cell calcium eleva-
tion. CaCC function was quantified from iodide influx as
measured from the kinetics of YFP fluorescence quenching
after extracellular iodide addition. YFP-expressing epithelial
cells have been used in our lab in similar screens to identify
small-molecule activators and inhibitors of wild-type and
mutant CFTRs (Ma et al., 2002a,b; Yang et al., 2003; Muan-
prasat et al., 2004; Pedemonte at al. 2005). Compounds with
apparent CaCC inhibition activity were prioritized, resynthe-
sized, analyzed for mechanism of action, and subject to struc-
ture-activity analysis.
Screening Procedures
Screening was done using a customized screening system (Beckman-
Coulter, Inc., Fullerton, CA) consisting of the SAGIAN Core system
integrated with SAMI software, and equipped with an ORCA arm for
labware transport, a 96-channel head Biomek FX, CO2 incubator, plate
washer, bar code reader, delidding station, and two FLUOstar Optima
fluorescence plate readers (BMG Labtechnologies, Durham, NC), each
equipped with syringe pumps and custom excitation/emission filters
(500/544 nm; Chroma, Brattleboro, VT). HT-29 cells expressing YFP-
H148Q/I152L were plated in 96-well plates at 70% confluence. Plates
were incubated overnight at 37°C, 5% CO2, and then the growth me-
dium was replaced with fresh growth medium. Cells were incubated
further until 95% confluence (12–18 h), washed three times with PBS,
and incubated with the test compounds at 32.5 ␮M final concentration
for 5 min in PBS, in a final volume of 60 ␮l/well. YFP fluorescence was
measured for 1 s before and 30 s after addition of a PBS-iodide solution
(PBS with 100 mM chloride replaced by iodide) containing carbachol
and ATP (100 ␮M each). Each 96-well plate contained “positive” con-
trols (DMSO vehicle without agonists or test compounds) and “nega-
tive” controls (DMSO vehicle with agonists but without test com-
pounds). In some experiments, solutions included (individually or in
combination): carbachol, ATP, or histamine (at 50, 100, 150, or 200
␮M), calcimycin (10 ␮M), thapsigargin (2 ␮M), and CFTRinh-172 (20
␮M). All chemicals were purchased from Sigma-Aldrich (St. Louis,
MO). For experiments with thapsigargin or calcimycin, cells in 96-
well plates at 100% confluence were washed three times with PBS,
incubated with test compounds at 32.5 ␮M for 5 min in PBS in a final
volume of 60 ␮l/well, and then incubated with 2 ␮M thapsigargin for
7 min or 10 ␮M calcimycin for 3 min before assay of iodide influx.
Iodide influx (d[IϪ]/dt at t ϭ 0) was computed from fluorescence time
course data by single exponential regression, as described previously
(Ma et al., 2002a). Percentage inhibition was computed as: percent-
Materials and Methods
Cell Culture and Infection
HT-29 cells were obtained from the American Type Culture Col-
lection (Manassas, VA) and grown in DMEM supplemented with
10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 ␮g/ml
streptomycin. T84 cells (American Type Culture Collection) were
maintained in DMEM/Ham’s F-12 (1:1) medium containing 10%
FBS, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Fisher rat
thyroid (FRT) cells expressing human CFTR and YFP-H148Q/I152L
were generated as described previously (Ma et al., 2002a), and grown
in Ham’s F-12-modified Coon’s medium supplemented with 10%
FBS, 2 mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml strep-
tomycin. All cells were grown at 37°C in 5% CO2/95% air.
For infection of HT-29 cells with lentivirus encoding YFP-H148Q/
I152L, cells were cultured in 90-mm diameter plates in McCoy’s 5a
medium supplemented with 1.5 mM L-glutamine, 10% FBS, and 2.2
g/l sodium bicarbonate. Cells were cultured until ϳ80% confluence.
Cells were washed three times with PBS, and then 1 ml of a high-
titer lentiviral supernatant was added to each well in the presence of
8 ␮g/ml Polybrene. Cells were incubated at 37°C in 5% CO2/95% air
for 6 h, and medium was then replaced with the regular DMEM
growth medium described above. YFP expression was detected in
Ͼ90% of cells 48 h after infection.
age inhibition ϭ 100 ϫ (Slope
Ϫ Slope
compound)/
negative control
test
(Slope
Ϫ Slope positive control).
negative control
CFTR Functional Assay
As described previously (Ma et al., 2002a), FRT cells expressing
human wild-type CFTR and YFP-H148Q/I152L at 100% confluence
were washed three times with PBS, leaving 60 ␮l. CFTR was acti-
vated by a cocktail containing forskolin (20 ␮M), genistein (50 ␮M)
and 3-isobutyl-1-methylxanthine (100 ␮M). Iodide influx was mea-
sured at 10 min after addition of test compounds by measuring YFP
fluorescence for 2 s before and 20 s after addition of 165 ␮l of
PBS-iodide.
Compounds
Compounds for primary screening were purchased from ChemDiv
(San Diego, CA). The compound collection contained 50,000 diverse,
drug-like compounds with Ͼ90% of compounds in the molecular size
Synthesis Procedures
CaCCinh-A01. For synthesis of 6-t-butyl-2-(furan-2-carboxamido)-
range 200 to 450 Da. Compounds for the secondary screening were 4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylic acid 1, a mixture
purchased from ChemDiv and Asinex. Compounds were prepared in of 4-(t-butyl) cyclohexanone (0.154 g, 1.00 mmol), methyl-2-cyanoac-
96-well plates (Costar; Corning Life Sciences, Acton, MA) as 10 mM etate (0.109 g, 1.1 mmol), morpholine (0.104 g, 1.2 mmol), and ele-
Fig. 1. Assay for CaCC inhibitor screening. A, principle of
cell-based, fluorescence high-throughput screening assay.
CaCC-facilitated iodide influx is measured from the kinet-
ics of decreasing YFP-H148Q/I152L fluorescence in re-
sponse to iodide addition to the extracellular solution.
CaCC is activated by a mixture of calcium-elevating ago-
nists. CCh, carbachol; m3AChR, carbachol receptor; P2Y2
,
purinergic receptor. B, fluorescence micrograph of lentivi-
rus-infected HT-29 cells stably expressing the YFP iodide
sensor.

760
Fuente et al.
mental sulfur (0.048 g, 1.5 mmol) in methanol (5 ml) were reacted for [Ca2؉]i Measurements
10 min at 120°C using a Biotage microwave reactor. Purification by
[Ca2ϩ]i was measured in confluent monolayers of HT-29 cells after
flash chromatography afforded 1 (0.242 g, 0.91 mmol) in 91% yield
(Gewald and Hofmann, 1969; Sridhar et al., 2007). Acylation of the
amine with furfuryl chloride (0.186 mg, 0.70 mmol) gave compound
2 in 87% yield. Ester hydrolysis of 2 (0.179 g, 0.500 mmol) was
accomplished with NaOH in methanol, giving t-butylbenzothiophene
3 (CaCCinh-A1). CaCCinh-A01was purified by column chromatogra-
phy (160 mg, 91% yield). 1H NMR (400 MHz, CDCl3): ␦ 12.88 (s,
1H),7.59 (s, 1H), 7.30 (d, J ϭ 3.2 Hz, 1H), 6.59 (dd, J ϭ 7.6 Hz and 2.0
Hz, 1H), 3.18 (d, 2H), 2.72 (m, 2H), 2.43 (t, 1H), 2.05 (m, 1H), 1.51 (m,
1H), 1.35 (m, 1H), 0.95 (s, 9H); 13C NMR (CDCl3): ␦ 179.2, 170.6,
154.8, 149.1, 146.8, 145.4, 131.8, 128.3, 116.6, 112.9, 94.5, 45.1, 32.6,
27.4, 26.0, 24.5; LC-MS: m/z 348.16 [Mϩ H]ϩ (Nova-Pak C18 column,
99%, 200–400 nm).
CaCCinh-B01. The synthesis of 2-hydroxy-4-(4-p-tolylthiazol-2-
ylaminobenzoic acid 4 began with the synthesis of thiourea 5. Reac-
tion of thiophosgene (2.7 g, 23.890 mmol) with 4-amino-2-hydroxy-
benzoic acid 2 (3.06 g, 20 mol) in aqueous HCl (41 ml) gave
thioisocyanate 4 by crystallization in 83% yield (Seligman et al.,
1953). Treatment of thioisocyanate 4 with ammonium hydroxide
gave thiourea 5 in 84% yield. Thiazole cyclization of thiourea 5 (0.300
g, 1.415 mmol) with 2-bromo-1-p-tolylethanone (0.298 g, 1.415 mmol)
in EtOH (15 ml) gave aminothiazole 6. The aminothiazole was puri-
fied by column chromatography to give CaCCinh-B01 (406 mg, 88%
yield). 1H NMR (400 MHz, CDCl3): ␦ 10.67 (s, 1H), 7.80 (d, J ϭ 8.0
Hz, 2H), 7.71 (d, J ϭ 8.1 Hz, 1H), 7.58 (d, J ϭ 2.0 Hz, 1H), 7.36 (s,
1H), 7.25 (d, J ϭ 8.0, 2H), 7.04 (dd, J ϭ 8.1 Hz and 2.0 Hz, 1H), 2.49
(s, 1H), 2.31 (s, 3H); 13C NMR (CDCl3): ␦ 171.7, 162.7, 162.0, 150.3,
147.2, 137.2, 131.7, 131.3, 129.3, 125.6, 108.5, 105.4, 103.6, 103.1,
20.8; LC-MS: m/z 327.11 [Mϩ H]ϩ (Nova-Pak C18 column, 97%,
200–400 nm).
loading with Fura-2 (2 ␮M Fura-2-AM, 30 min, 37°C). Labeled cells
were mounted in a perfusion chamber on the stage of an inverted
epifluorescence microscope. Cells were superfused with 140 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM D-glucose, and
10 mM HEPES, pH 7.4, initially without and then with ATP/carba-
chol. Test compounds were present in some experiments for 10 min
before agonist addition. Fura-2 fluorescence was recorded at excita-
tion wavelengths of 340 nm and 380 nm using standard procedures.
Immunoblotting
Calcium/calmodulin-dependent protein kinase II (CaMKII) was
activated by 2-min treatment of HT-29 cells with ATP/carbachol
(each 100 ␮M). Cells were then lysed with cell lysis buffer (20 mM
Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 50
mM ␣-glycerol phosphate, 1 mM Na3VO4, 1 mM dithiothreitol, and
protease inhibitor mixture (Roche Applied Science). Cell debris was
removed by centrifugation, and proteins in the supernatant were
resolved by SDS-polyacrylamide gel electrophoresis and immuno-
blotted using standard procedures (transfer to polyvinylidene diflu-
oride membrane, 1 h blocking in 5% nonfat dry milk, primary/
secondary antibody incubations, enhanced chemiluminescence
detection). Rabbit polyclonal antibodies for anti-phospho-CaMKII
(Thr286) and ␤-actin were purchased from Cell Signaling Technology
(Danvers, MA).
Results
High-Throughput Screening Assay Development and
Validation. As diagrammed in Fig. 1A, our assay used a hu-
man intestinal cell line (HT-29) having robust CaCC activity. In
the presence of calcium-elevating agonist(s), extracellular io-
dide addition results in CaCC-facilitated iodide influx, which is
detected by fluorescence quenching of a cytoplasmic YFP-based
halide sensor (YFP-H148Q/I152L). After lentiviral infection,
HT-29 cells stably expressing YFP-H148Q/I152L were brightly
fluorescent, with nearly all cells showing fluorescence (Fig. 1B).
Alternative human intestinal epithelial cells lines, including
T84 and Caco-2 cells, were found to be inadequate for screening
because of slow growth, lack of growth to confluent monolayers,
poor YFP expression, and/or weak CaCC activity.
Several CaCC agonists were assayed to establish the cel-
lular model for CaCC inhibitor screening. Histamine (100
␮M), calcimycin (10 ␮M), ATP (100 ␮M), carbachol (100 ␮M),
and forskolin (10 ␮M) were tested individually (Fig. 2A) and
in combinations (Fig. 2B). The CFTR inhibitor CFTRinh-172
(20 ␮M) was also tested. Of the agonists tested individually,
carbachol and ATP produced the strongest responses as seen
from the slopes of the fluorescence decrease after extracellu-
lar iodide addition. In combination, carbachol and ATP pro-
duced as large a response as seen for any of the combinations.
Increased iodide influx was not found after forskolin addi-
Short-Circuit Current Measurements
T84 cells were seeded at a density of 105 cells/cm2 on permeable
supports (Snapwell; 1.12 cm2 surface area) and grown until conflu-
ent. Supports containing confluent cell monolayers were mounted in
Snapwell inserts. Cells were bathed for a 30-min stabilization period
in HCO3Ϫ-buffered solution containing 120 mM NaCl, 5 mM KCl, 1
mM MgCl2, 1 mM CaCl2, 10 mM D-glucose, 5 mM HEPES, and 25
mM NaHCO3, pH 7.4, aerated with 95% O2/5% CO2 at 37°C. Mono-
layers were voltage-clamped at 0 mV (EVC4000 MultiChannel V/I
Clamp; World Precision Instruments, Sarasota, FL), and short-cir-
cuit current (Isc) was recorded continuously with agonists/inhibitors
added at specified times.
Patch-Clamp
Whole-cell recordings were done on HT-29 cells at room tempera-
ture. The pipette solution contained 30 mM CsCl, 100 mM Cs-
aspartate, 1 mM MgCl2, 0.5 mM EGTA, 2 mM Tris-ATP, and 10 mM
HEPES, pH 7.2 with CsOH, and the bath solution contained 140
N-methyl-D-glucamine chloride (NMDG-Cl), 1 mM CaCl2, 1 mM
MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.2. In one set of
studies, symmetric NMDG-Cl solutions contained 140 mM NMDG-
Cl, 1 mM MgCl2, 0.5 mM EGTA, 2 mM Tris-ATP, and 10 mM tion, and CFTRinh-172 did not inhibit iodide influx in re-
HEPES, pH 7.2. Pipettes were pulled from borosilicate glass and had
resistances of 3 to 5 M⍀ after fire polishing. Seal resistances were
typically between 3 to 10 G⍀. After establishing the whole-cell con-
figuration, CaCCs were activated by 1 ␮M ionomycin. Whole-cell
currents were elicited by applying hyperpolarizing and depolarizing
voltage pulses from a holding potential of 0 mV to potentials between
Ϫ120 mV and ϩ120 mV in steps of 20 mV. Currents were filtered at
5 kHz, and digitized and analyzed using an AxoScope 10.0 system
with Digidata 1440A converter (Molecular Devices, Sunnyvale, CA).
Mean currents were expressed as current densities (picoampere per
picofarad).
sponse to calcium agonists, indicating that the cell clone used
in our assay expressed little or no functional CFTR.
A combination of carbachol and ATP, each at high concen-
tration, was selected for compound screening, reasoning that
any inhibitors should act beyond the receptor binding step.
Iodide influx measurements were done to establish agonist
concentrations and addition times. Concentration depen-
dence studies for carbachol (Fig. 2C) and ATP (not shown)
indicated maximal responses at 100 ␮M. Figure 2D shows
reduced iodide influx as a function of time between addition

Small-Molecule CaCC Inhibitors
761
of 100 ␮M carbachol/ATP and extracellular iodide, which is screening to identify putative CaCC inhibitors. The presence of
consequence of the transient elevation in cytoplasmic cal- DMSO (from compound addition) did not affect assay quality.
cium produced by these agonists. The greatest iodide influx
Based on initial small-scale screenings at different compound
was found when agonists were added at ϳ10 s before iodide, concentrations, which showed a low “hit” rate for discovery of
although the influx was not much greater than when ago- active compounds, we screened 50,000 compounds at a concen-
nists were added at the time of iodide addition. For high- tration 32.5 ␮M. Figure 3B, left, shows examples of data from
throughput screening, to simplify assay conditions, agonists single wells for compounds with “strong,” “weak,” and no inhi-
bition activity. Figure 3B, right, summarizes percentage inhi-
bition data as a frequency histogram. Of 50,000 small molecules
screened (in groups of four), 300 compound groups had CaCC
inhibitory activity as defined arbitrarily by a 70% cutoff (verti-
cal dashed line). Retesting of these 300 compound groups indi-
cated a false-positive rate of ϳ40%.
(carbachol and ATP, each 100 ␮M) were added together with
iodide.
CaCC Inhibitor Identification by High-Throughput
Screening. To characterize the assay as used for high-through-
put screening, iodide influx was measured in the YFP-express-
ing HT-29 cells in a 96-well format using an automated work-
station capable of assaying more than 10,000 compounds
overnight. Figure 3A, left, shows representative YFP fluores-
cence kinetics measured in single wells of 96-well plates. Each
curve consisted of recording of baseline fluorescence for 1 s,
followed by 30 s of continuous recording of fluorescence after
rapid addition of a solution containing iodide and the CaCC
agonist combination (carbachol and ATP, each 100 ␮M). After a
small solution addition artifact, there was little decrease in
fluorescence in the absence of activators, compared with a ro-
bust reduction in fluorescence with agonists. Figure 3A (right)
shows frequency histograms of iodide influx rates, d[IϪ]/dt, in
individual wells for “positive” (no agonists, equivalent to 100%
inhibition) and “negative” (with agonists, 0% inhibition) con-
trols. The computed ZЈ-factor (Verkman, 2004) for the assay
was very good, 0.74, indicating adequacy of a single compound
Further analysis was done on 15 compound groups that
produced greatest inhibition when retested at 10 and 30 ␮M
concentrations. The compound responsible for activity in
each group was determined by testing of individual com-
pounds. In each case, a single active compound was identified
in the groups of four. Figure 4A shows structures of the most
active of six classes of putative CaCC inhibitors (classes A–F)
identified by single compound testing; in some cases, partic-
ularly for classes A and B, similar structures were seen
several times. These structures are unrelated to all known
ClϪ channel inhibitors. Percentage inhibition of these com-
pounds at 30 ␮M was in the range 60 to Ͼ90%. Based on
multiple criteria, including potency, water solubility, drug-
like properties, identification of active analogs, chemical sta-
bility, and CaCC targeting, we focused attention on com-
pounds of classes A and B. As shown below, the class E
compound did not inhibit iodide influx in response to thap-
sigargin, suggesting that its target is proximal in the calcium
signaling pathway, and thus not of interest here. The class D
compound was determined to have poor stability in aqueous
solutions, probably because of retro-Diels-Alder reaction.
Fig. 2. Functional characterization of YFP-expressing HT-29 cells. Time
course of YFP fluorescence after extracellular iodide addition. As indi-
cated, solutions contained histamine (100 ␮M), calcimycin (10 ␮M), ATP
(100 ␮M), carbachol (100 ␮M), carbachol (100 ␮M) ϩ CFTRinh-172 (20
␮M), or forskolin (20 ␮M), individually (A), and in various combinations
(at same concentrations) (B). The scale-bar on the y-axis indicates the
percentage reduction in fluorescence relative to baseline fluorescence
(before iodide addition). C, carbachol concentration-response study. D,
initial negative fluorescence curve slope (after extracellular iodide addi-
tion) as a function of time after carbachol/ATP (each 100 ␮M) addition
and extracellular iodide addition.
Fig. 3. CaCC inhibitor screening. A, screen validation. Left, time course
of YFP fluorescence in HT-29 cells after iodide addition in the absence or
presence of carbachol (100 ␮M) and ATP (100 ␮M). Right, histogram
distribution of iodide influx rates (d[IϪ]/dt) determined from initial fluo-
rescence slopes. B, left, examples of fluorescence data for individual
compounds in the primary screen. Right, histogram distribution of per-
centage inhibition from primary compound screening. Dashed vertical
line denotes selection criteria for further evaluation.

762
Fuente et al.
Class F compounds were excluded from further study be- rified by chromatography and recrystallization. Synthesis of
cause of their small size and relatively low potency. CaCCinh-B01 also involved three steps (Fig. 4B, bottom). Reac-
Synthesis and Characterization of Class A and B Com- tion of 4-amino-2-hydroxybenzoic acid with thiophosgene in
pounds. Before further analysis of their biological activities HCl gave isothiocyanate 4 in good yield. Treatment of 4 with
and mechanisms of action, we synthesized the class A and B ammonium hydroxide gave thiourea 5, which was then reacted
compounds to high purity and verified their structures and with 2-bromo-1-p-tolylethanone to give CaCCinh-B01 6.
chemical stability in aqueous solutions. The synthesis of CaC-
C
CaCCinh-A01 and CaCCinh-B01 were confirmed by 1H-
inh-A01 was accomplished in three steps (Fig. 4B, top), involv- NMR, 13C-NMR and mass spectrometry (analytical data pro-
ing Knovenagle condensation of methyl cyanoacetate with t- vided under Materials and Methods). The aqueous solubility
butylcyclohexanone followed by cyclization on elemental sulfur. of CaCCinh-A01 and CaCCinh-B01 in PBS was Ͼ500 ␮M, as
Purification by flash chromatography produced the 2-amino- measured by optical absorbance of a saturated solution after
thiophene Gewald product. Acyclation of the aminothiophene appropriate dilution. The high aqueous solubility of CaCCinh
-
gave the N-acyl methyl ester 2 in good yield. Ester 2 was A01 and CaCCinh-B01 is a consequence of their polarity and
hydrolyzed with NaOH to give CaCCinh-A01 3, which was pu- negative charge at physiological pH. By liquid chromatogra-
A
O
H
OH
NH
N
OH
CO H
N
N
N
S
O
S
S
2
O
Ph
NH HN
Et
O
class A
class B
O
class C
Et
O F C
O
O
3
CF
N
H
3
S
N
N
N
H
NH
CF
H
F
Ph
N
O
N
N
H
H
3
class D
CO H
2
class E
class F
C
B
O
Cl
O
O
O
100
80
60
40
20
0
NC
O
O
O
NH
2
DIEA
DCM
O
S
S
8
NH
MeOH
CaCC -B2
;
1 91%
inh
O
S
HO
O
O
NaOH
MeOH
NH
NH
CaCC -A2
S
inh
O
O
O
O
2;
;
3 91%
87%
1
10
Concentration (µM)
100
S
H2N
OH
N
OH
Cl
Cl
NH4OH
H2O
C
S
HCl, O oC
H2O
CO2H
CO2H
;
4 83%
O
Br
H
N
H
OH
H2N
N
OH
N
S
S
EtOH
CO2H
CO2H
;
;
6 88%
5 84%
Fig. 4. Structures and synthesis of putative CaCC inhibitors. A, structures of compounds of classes A–F identified from the primary screen. B,
synthesis of CaCCinh-A01 and CaCCinh-B01. See Materials and Methods for details. C, concentration-inhibition data for CaCCinh-A01 and CaCCinh-B01
determined by plate reader fluorescence assay.

Small-Molecule CaCC Inhibitors
763
phy, purity was 97% and 99% for CaCCinh-A01 and CaCCinh
B01, respectively.
-
Target Identification. Several targets are possible based
on the screening strategy in Fig. 1A, including ligand-recep-
Testing of the purified compounds using the fluorescence tor binding, calcium elevation, calcium-calmodulin (CaMKII)
plate-reader assay verified their activities, with IC50 values signaling, and the CaCC itself. To distinguish between pre-
ϳ10 ␮M (Fig. 4C). These apparent IC50 values are only and postcalcium signaling targets, compounds of each class
semiquantitative because of the multistep nature of the were tested after stimulation of HT-29 cells by thapsigargin,
screening assay and because of the ϳ3-fold compound dilu- which produces calcium elevation without ligand-receptor
tion at the time of iodide addition during the assay. Accurate binding or phosphoinositide signaling. Figure 5A shows that
IC50 values were determined by electrophysiological assays each of the compounds inhibited iodide influx after thapsi-
(see below).
gargin, except for the class E compound, whose target is thus
Fig. 5. Target dissection studies. A, time course of YFP fluorescence in HT-29 cells after iodide addition in the absence or presence of thapsigargin
in cells pretreated with 30 ␮M concentrations of the indicated compounds. B, YFP fluorescence in FRT cells expressing human wild-type CFTR after
iodide addition in cells pretreated with 30 ␮M concentrations of the indicated compounds. CFTRinh-172 (20 ␮M) was present where indicated. C,
calcium signaling measured by fura-2 fluorescence in response to indicated agonists and test compounds. A, CaCCinh-A01. B, CaCCinh-B01.
Representative data shown on the left, with averaged data on the right (SE, n ϭ 3–4). Differences not significant. D, ATP/carbachol-induced CaMKII
phosphorylation determined by immunoblot analysis (representative of three separate experiments).

764
Fuente et al.
likely upstream of calcium signaling. Experiments were also
Whole-cell patch-clamp was done to investigate the inhibi-
done to rule out compound effects on CFTR, because CFTR is tion of calcium-dependent chloride current in HT-29 cells by
a major intestinal chloride channel, and there is evidence for CaCCinh-A01 and CaCCinh-B01. Measurements were carried
cross-talk between cAMP and calcium signaling in intestinal out under Kϩ-free conditions (Csϩ in pipette) to ensure that
cell chloride secretion (Chao et al., 1994; Takahashi et al., chloride currents were being measured. Treatment with 1
2000; Schultheiss et al., 2006). Using our well established ␮M ionomycin produced large currents of 28 Ϯ 3 pA/pF (Vm
FRT-CFTR assay, Fig. 5B shows no CFTR inhibition by any ϩ100 mV) in NMDG-Cl solutions, with an outwardly rectify-
of the compounds at 30 ␮M, with CFTRinh-172 inhibition ing current-voltage relationship (Fig. 6, A and B). Ionomycin-
shown as positive control.
stimulated currents were measured when the current was
Measurements of cytoplasmic calcium were done to inves- maximally activated at a Vm of Ϫ40 mV and current density
tigate whether CaCCinh-A01 and CaCCinh-B01 interfered data were analyzed at Vm of ϩ100 mV. As summarized in
with agonist-induced calcium signaling in HT-29 cells. Fig- Fig. 6C, calcium-dependent chloride current was reduced by
ure 5C shows [Ca2ϩ]i elevations in response to 1 ␮M ATP, 38 Ϯ 14, 66 Ϯ 10, and 91 Ϯ 1% by 0.1, 1, and 10 ␮M
100 ␮M ATP, and 100 ␮M carbachol (left), or 1 ␮M ionomycin CaCCinh-A01, respectively, and by 11 Ϯ 7, 34 Ϯ 12, and 77 Ϯ
(middle). Pretreatment with CaCCinh-A01 or CaCCinh-B01 at 4% by 0.1, 1, and 10 ␮M CaCCinh-B01. As a control to exclude
high concentration before agonist addition did not signifi- interference by transient receptor potential and nonselective
cantly reduce the ATP- or carbachol-induced [Ca2ϩ]i eleva- cation channels, ionomycin-induced chloride currents were
tions (right).
also recorded in the whole-cell configuration using symmetric
Regulation of CaCC activation by CaMKII occurs in a cell NMDG-Cl solutions (Fig. 6D). As predicted, pretreatment
type-dependent manner (Hartzell et al., 2005a). CaMKII has with CaCCinh-A01 and CaCCinh-B01 reduced ionomycin-in-
been reported to regulate calcium-activated chloride currents duced currents.
in HT-29 and T84 cells (Worrell and Frizzell, 1991; Morris
Chloride secretion was also measured in T84 cells to inves-
and Frizzell, 1993; Chan et al., 1994; Braun and Schulman, tigate whether the CaCC inhibitors identified from screening
1995). To determine whether the CaCC inhibitors interfered of HT-29 cells also inhibit the CaCC in a different human
with ATP/carbachol-induced CaMKII activation, CaMKII intestinal cell line. ATP/carbachol were used to induce calci-
phosphorylation in HT-29 cells was measured by immunoblot um-dependent chloride secretion in well differentiated T84
analysis. CaMKII was activated by a 2 min incubation with cell monolayers. The increase in short-circuit current in re-
100 ␮M ATP/carbachol. Figure 5D shows strong pCaMKII sponse to carbachol (without inhibitors added) was similar in
immunoreactivity after agonist treatment. Pretreatment all experiments (Fig. 7A). Subsequent application of 100 ␮M
with 30 ␮M CaCCinh-A01 or CaCCinh-B01 did not signifi- ATP ϳ30 min later produced a large response (top curve),
cantly affect agonist-induced CaMKII phosphorylation. ␤-Ac- which was reduced in the presence of inhibitors (lower four
tin is shown as a loading control.
curves). Carbachol and ATP-induced short-circuit currents
Fig. 6. Patch-clamp analysis of CaCC inhibitors. CaCC channel activity in the whole-cell configuration was measured HT-29 cells. A, ionomycin-
induced currents in asymmetrical solutions in the absence or presence of CaCCinh-A01 or CaCCinh-B01 (each 10 ␮M) recorded at a holding potential
at 0 mV, and pulsing to voltages between Ϯ 120 mV in steps of 20 mV (see Materials and Methods). B, current/voltage (I/V) plot of mean currents at
the end of each voltage pulse from experiments as in A. C, summary of current density data measured at Vm of ϩ 100 mV (S.E., n ϭ 6–8). D, top,
ionomycin-induced chloride currents in the whole-cell configuration with symmetrical NMDG-Cl solutions. Bottom, current/voltage plot of mean
currents at the end of each voltage pulse.

Small-Molecule CaCC Inhibitors
765
are summarized in Fig. 7B. ATP-induced short-circuit cur- CaCC inhibition activity based on consideration of data for
rents were reduced by 38 Ϯ 7 and 78 Ϯ 3% at 10 and 30 ␮M all analogs tested. An essential moiety for strong activity in
CaCCinh-A01, respectively, and by 29 Ϯ 11 and 64 Ϯ 3% by 10 both compound classes was the presence of a carboxylic acid
and 30 ␮M CaCCinh-B01, respectively.
functional group, as has been found in most chloride channel
Structure-Activity Analysis. An important characteris- inhibitors.
tic of small molecules for drug development is the existence of
For class A compounds, t-butyl at position R1 conferred the
many active chemical analogs (“evolvable” SAR) to allow greatest inhibition; inhibition was reduced or lost when R1
optimization of drug properties. SAR analysis was performed was methyl or H, as with analogs CaCCinh-A12-A15 (many
on 936 commercially available aminothiophenes (class A an- inactive methyl analogs not included in Table). Modification
alogs) and 944 aminothazoles (class B analogs). Tables 1 and of the cyclohexyl ring system to cyclopentyl and cycloheptyl
2 provide semiquantitative CaCC inhibition data (from plate (varying n) indicated greatest activity with six membered
reader assay) for 18 active aminothiophenes (class A) and 14 rings. Several cycloheptylthiophenes were found to be active
active aminothiazoles (class B). Figure 8 summarizes the but much less potent than the six membered ring analogs. At
SAR analysis in terms of the functional groups conferring R2, hydroxy, methoxy, ethoxy, and substituted phenyl amides
Fig. 7. Inhibition of chloride secretion
in T84 cells by short-circuit current
analysis. A, After carbachol stimula-
tion, ATP-induced Isc was measured
in the absence or presence of inhibi-
tors (representative of three or four
separate experiments). B, summary of
short-circuit currents induced by car-
bachol and ATP for experiments as in
A
(S.E.,
n ϭ 3–4). (Inhibitors not
present for carbachol stimulation).
TABLE 1
Structure-activity of Class A CaCC inhibitors
See Fig. 8 for locations of R1, R2, and R3 and meaning of n.
Compound
R1
R2
R3
n
Inhibition at 25 ␮M
%
CaCCinh-A01
CaCCinh-A02
CaCCinh-A03
CaCCinh-A04
CaCCinh-A05
CaCCinh-A06
CaCCinh-A07
CaCCinh-A08
CaCCinh-A09
CaCCinh-A10
CaCCinh-A11
CaCCinh-A12
CaCCinh-A13
CaCCinh-A14
CaCCinh-A15
CaCCinh-A16
CaCCinh-A17
CaCCinh-A18
t-butyl
t-butyl
t-butyl
t-butyl
t-butyl
t-butyl
t-butyl
t-butyl
t-butyl
EtMe2C-
t-butyl
H
OH
OH
OH
OH
OH
OEt
OEt
OEt
OMe
OEt
OMe
OH
OH
OH
OH
CO-furanyl
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
100
95
50
85
75
75
98
80
45
75
81
15
27
18
20
42
7
CO-2-methylphenyl
CO-3-chlorophenyl
CO-phenyl
CO-2-chlorophenyl
CO-CH2CH2CO2H
(trans)COCH ϭ CHCO2H
CO-2-carboxycyclohexyl
CO-CH2CH2CH2CO2H
CO-2-carboxycyclohexyl
CO-CH2CH2CO2H
CO-4-methylphenyl
CO-phenyl
CO-2-chlorophenyl
CO-3-methoxyphenyl
CH2CH2CO2H
CO-3-methylphenyl
CH2CH2CO2H
H
H
H
t-butyl
H
t-butyl
NH-3-MePhCO-
OH
NH-4-MeOPh CO-
35
Ph, phenyl; Br, bromo; Cl, chloro; F, fluoro; OMe, methoxy; NO2, nitro.

766
Fuente et al.
were tolerated. If R2 is OH, as in the carboxylic acid, then R3 2-(cyclopenta-1,3-dienecarboxamido)-4,5,6,7-tetrahydrobenzo-
can be a substituted phenyl ring or heterocycle as seen in [b]thiophene-3-carboxylic acid (CaCCinh-A01).
CaCCinh-A01-A05, -A15, and -A17. Methyl and ethyl esters
For class B, at position R1 methyl substitution at the 4
were tolerated at R2 if an alkyl carboxylic acid was present at R3 position of the aromatic ring was present in most active
as seen in CaCCinh-A06-A11. Substituted phenyl amides at R2 compounds as seen in CaCCinh-B01-B06. Additional methyl
had weak activity as seen in CaCCinh-A16 and CaCCinh-A18. substitution at the 2 position gave active analogs CaCCinh
-
For R3, activity was found for substituted phenyl rings, het- B07-B09. Inhibition was greatly reduced when the phenyl
erocycles, and alkyl carboxylic acids such as 3-carboxypro- ring was substituted at the 2 or 3 positions. As with class A,
panamido, trans-3-carboxyacrylamido, and 2-carboxycyclo- a carboxylic acid functional group was essential for activity,
hexane-carboxamido, but only when R2 was OMe, OEt, or either with an alkyl substitution at R2 or a phenyl substitu-
NHPh. Of the alkyl substituted carboxylic acids at R3, the tion at R3. Loss of this carboxylic acid functionality reduced
most active analog was the trans-3-carboxyacrylamido in activity as seen in CaCCinh-B09-B13. At position R2, most
CaCCinh-A07. CO-phenyl substitutions at R3 were tolerated active compounds contained either hydrogen substitution or
in active compounds with the 2-position favored. Electron- an acetic acid group. For position R3, a variety of phenyl
donating substituents led to substantially reduced activity. A substitutions were tolerated, the majority being alkyl or
variety of heterocycles were screened at R3 with only CO- halogen substitution at the 3 and 4 positions. Electron do-
furfuryl active, The most potent compound was 6-tert-butyl- nating substituents produced inactive compounds. The SAR
TABLE 2
Structure-activity of Class B CaCC inhibitors
See Fig. 8 for locations of R1, R2, and R3 and meaning of n.
Compound
R1
R2
R3
Inhibition at 25 ␮M
%
CaCCinh-B01
CaCCinh-B02
CaCCinh-B03
CaCCinh-B04
CaCCinh-B05
CaCCinh-B06
CaCCinh-B07
CaCCinh-B08
CaCCinh-B09
CaCCinh-B10
CaCCinh-B11
CaCCinh-B12
CaCCinh-B13
CaCCinh-B14
4-Me
-H
3-Carboxylate-4-hydroxyphenyl
3-Chloro-4-methylphenyl
3-Bromophenyl
100
40
60
65
60
100
70
75
65
55
65
3
4-Me
-CH2CO2H
-CH2CO2H
-CH2CO2H2,
-CH2CO2H
-Pr
4-Me
4-Me
4-Dichlorophenyl
4-Me
3-Trifluoromethylphenyl
4-Carboxylatephenyl
4-Bromophenyl
3-Trifluoromethylphenyl
4-Isobutylphenyl
4-Cyclohexylphenyl
4-Ethoxyphenyl
CO-3-Methylphenyl
Ethyl
4-Methoxyphenyl
2,4-DiMe
2, 4-DiMe
2,4-DiMe
4-PhO
3-CF3
4-Me
-CH2CO2H
-CH2CO2H
-H
-H
-CH2CO2H
-Ph
4-Me
4-Me
-H
20
18
4-MeO
-CH2CH2CO2H
Ph, phenyl; Br, bromo; Cl, chloro; F, fluoro; OMe, methoxy; NO2, nitro.
Fig. 8. Structure-activity analysis of CaCC
inhibitors. Summary of SAR conclusions for
class A and B CaCC inhibitors. See Structure-
Activity Analysis under Results, for explanations.

Small-Molecule CaCC Inhibitors
767
study thus indicates the existence of many active class A and tifying current-voltage relationships (Arreola et al., 1995;
B CaCC inhibitors having a wide range of potencies.
Braun and Schulman, 1995; Nilius et al., 1997), as well as
ionomycin-induced calcium-dependent chloride current in
T84 cells. Chloride currents in both cell types were strongly
inhibited by the aminothiophenes and aminothazoles discov-
Discussion
Our phenotype-based small-molecule screen identified ered here, suggesting common CaCC(s) in these cell types
novel chemical classes of inhibitors of CaCC conductance, and indicating little or no inhibitor-insensitive calcium-de-
two of which seem to target the CaCC itself rather than pendent chloride current.
upstream signaling mechanisms. The aminothiophenes and
In summary, we report the development and validation of
aminothazoles did not interfere with agonist-induced cyto- an efficient screen for identification of inhibitors of intestinal
plasmic calcium elevation or calmodulin (CaMKII) phosphor- CaCCs, along with the identification and characterization of
ylation, and by patch-clamp analysis inhibited CaCC gating. several classes of active compounds from a screen of 50,000
The data here do not distinguish between an external versus compounds. These or other compounds identified from simi-
internal site of compound action, nor do they provide infor- lar screens, with follow-on work, may be useful for molecular
mation about the molecular identity of the intestinal CaCC identification of CaCCs and for therapeutic applications in
responsible for the observed chloride conductance, though certain secretory diarrheas and in lung diseases associated
determination of comparative compound potencies on candi- with mucus overproduction.
date CaCCs, as mentioned in the Introduction, may offer
indirect evidence about CaCC identity. The verified “hit” rate
for our small-molecule screen, ϳ1 per 10,000 compounds
tested, is in the range generally found in ion channel inhib-
itor screens. Therefore, further screening of larger collections
of diverse compounds is likely to succeed in identifying new
chemical classes of CaCCs.
Our high-throughput screening assay used a kinetic fluo-
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