Chemical inhibitors of the calcium entry channel TRPV6

Article abstract of DOI:10.1007/s11095-010-0249-9

Purpose: Calcium entry channels in the plasma membrane are thought to play a major role in maintaining cellular Ca²⁺ levels, crucial for growth and survival of normal and cancer cells. The calcium-selective channel TRPV6 is expressed in prostate, breast, and other cancer cells. Its expression coincides with cancer progression, suggesting that it drives cancer cell growth. However, no specific inhibitors for TRPV6 have been identified thus far. Methods: To develop specific TRPV6 inhibitors, we synthesized molecules based on the lead compound TH-1177, reported to inhibit calcium entry channels in prostate cancer cells in vitro and in vivo. Results: We found that one of our compounds (#03) selectively inhibited TRPV6 over five times better than TRPV5, whereas TH-1177 and the other synthesized compounds preferentially inhibited TRPV5. The IC ₅₀ value for growth inhibition by blocking endogenous Ca²⁺ entry channels in the LNCaP human prostate cancer cell line was 0.44 ± 0.07 μM compared to TH-1177 (50 ± 0.4 μM). Conclusions:These results suggest that compound #03 is a relatively selective and potent inhibitor for TRPV6 and that it is an interesting lead compound for the treatment of prostate cancer and other cancers of epithelial origin.

Full text of DOI:10.1007/s11095-010-0249-9

Pharm Res (2011) 28:322–330
DOI 10.1007/s11095-010-0249-9
RESEARCH PAPER
Chemical Inhibitors of the Calcium Entry Channel TRPV6
Christopher P. Landowski & Katrin A. Bolanz & Yoshiro Suzuki & Matthias A. Hediger
Received: 26 April 2010 /Accepted: 13 August 2010 /Published online: 6 November 2010
#
Springer Science+Business Media, LLC 2010
ABSTRACT
preferentially inhibited TRPV5. The IC50 value for growth
inhibition by blocking endogenous Ca2+ entry channels in the
LNCaP human prostate cancer cell line was 0.44 0.07 μM
compared to TH-1177 (50 0.4 μM).
Conclusions These results suggest that compound #03 is a
relatively selective and potent inhibitor for TRPV6 and that it is
an interesting lead compound for the treatment of prostate
cancer and other cancers of epithelial origin.
Purpose Calcium entry channels in the plasma membrane are
thought to play a major role in maintaining cellular Ca2+ levels,
crucial for growth and survival of normal and cancer cells. The
calcium-selective channel TRPV6 is expressed in prostate, breast,
and other cancer cells. Its expression coincides with cancer
progression, suggesting that it drives cancer cell growth. However,
no specific inhibitors for TRPV6 have been identified thus far.
Methods To develop specific TRPV6 inhibitors, we synthe-
sized molecules based on the lead compound TH-1177,
reported to inhibit calcium entry channels in prostate cancer
cells in vitro and in vivo.
.
.
KEY WORDS breast cancer calcium channel chemical
.
.
inhibitors prostate cancer TRPV6
Results We found that one of our compounds (#03)
selectively inhibited TRPV6 over five times better than TRPV5,
whereas TH-1177 and the other synthesized compounds
ABBREVIATIONS
CRAC Ca2+ release-activated Ca2+
FBS
fetal bovine serum
LNCaP lymph node carcinoma of the prostate
:
:
:
NFAT
NMM
Orai1
nuclear factor of activated T-cells
N-methylmorpholine
calcium release-activated calcium channel protein 1
C. P. Landowski K. A. Bolanz Y. Suzuki M. A. Hediger (*)
Institute of Biochemistry and Molecular Medicine, University of Bern
Bern CH-3012, Switzerland
e-mail: matthias.hediger@mci.unibe.ch
PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium
SCID
SOC
severe combined immunodeficiency
store-operated calcium channel
M. A. Hediger
Swiss National Center of Competence in Research, NCCR-
TransCure, University of Berne
Switzerland
STIM1 stromal interaction molecule 1
THF
TRP
Tetrahydrofuran
transient receptor potential
Present Address:
C. P. Landowski
VTT Technical Research Center of Finland
Espoo, Finland
INTRODUCTION
Present Address:
K. A. Bolanz
Agroscope, Swiss Federal Office for Agriculture
Bern, Switzerland
Prostate cancer is the most common form of cancer in men
in the Western world (accounting for one-third of diag-
nosed cancers) and the second overall leading cause of
death from cancer in Europe and the United States. Tumor
invasion and metastasis are the primary cause of death (1).
At the time of diagnosis, over 70% of men have lymph
Present Address:
Y. Suzuki
National Institute for Physiological Sciences
Okazaki, Japan

Chemical Inhibitors of the Calcium Entry Channel TRPV6
323
node metastases, which subsequently spread to bone in the
pelvic and lumbar vertebral column (2–4). Epidemiologic
studies have linked diets rich in calcium to a higher risk of
prostate cancer (5–7). However, the biological basis for this
link is still unknown. The evidence linking calcium and
prostate cancer suggests a critical role for calcium channels
in the progression of the disease. Understanding this role
may lead to the development of novel therapeutics to
improve prostate cancer treatment.
known inhibitors include two antifungal compounds econa-
zole and miconazole and the staining dye ruthenium red.
Some evidence that it is possible to target TRPV6 in
prostate cancer cells by chemical inhibitors comes from studies
conducted by Haverstick et al. (35), who developed calcium
channel inhibitors structurally similar to dihydropyridines.
One of these compounds, TH-1177, effectively blocked
store-operated calcium entry in LNCaP prostate cancer cells
and inhibited fractional survival of LNCaP cells. When
tested in SCID mice carrying prostate cancer cells, TH-1177
treatment prolonged the mean life span of mice by up to
38%. Healthy mice that received 18 times the therapeutic
dose showed no signs of toxicity. In these studies, the
molecular target of TH-1177 was not identified, although
TRPV6 is a likely candidate.
In this study, we make the first directed attempt to create
selective inhibitors for TRPV6. Based on the above-
mentioned findings, we synthesized compounds that have
similar structures to TH-1777 and performed biological assays
to screen for TRPV6 inhibition. We identified both TRPV5-
and TRPV6-selective inhibitors that were effective at reducing
the proliferation of prostate and breast cancer cells. Thus,
these inhibitors might be interesting lead compounds in the
development of a TRP-targeted cancer treatment.
In non-excitable cell types and some excitable cells,
store-operated calcium channels (SOCs) in the plasma
membrane serve to replenish intracellular calcium stores
in the endoplasmic reticulum following agonist-elicited
calcium release (8–10). Besides playing a major role in
store-operated calcium entry, these channels are also
involved in regulating numerous important physiological
processes such as cell growth, proliferation, and apoptosis.
The best functionally and biophysically characterized SOC
subtypes are the Ca2+ release-activated Ca2+ (CRAC)
channels, originally described in immune cells (11–13) and
later in various exocrine cells (e.g., prostate) involved in
secretion (14,15). STIM1 and Orai1 have been shown to
mediate CRAC currents and store-operated calcium entry
in a variety of cells (16–19), but not in prostate cancer cells.
CRAC-like activities have been extensively described for
the human prostate carcinoma cell lines PC-3 and LNCaP
(14,20). The TRPC1 and TRPV6 members of the transient
receptor potential (TRP) ion channel family are the most
likely molecular candidates for prostate-specific endogenous
SOC or CRAC-like activities (21,22). Numerous studies
have linked enhanced endoplasmic reticulum Ca2+ accu-
mulation to proliferation in prostate cancer (23–25).
Moreover, endogenous SOCs play an important role in
the apoptosis of LNCaP prostate cancer cells (26,27).
TRPV6 is strongly expressed in advanced stages of prostate
cancer, whereas there is little to no expression evident in
healthy tissue and benign prostate hyperplasia (28). TRPV6
transcripts are highly expressed in lymph node metastases
originating from the prostate (28), and their expression
correlates to the Gleason score and pathological stage (29).
There is also evidence that TRPV6 plays a role in breast
cancer, being strongly expressed in malignant tissue com-
pared to normal mammary gland tissue (30). We have
demonstrated that the breast cancer cell line T47D displays
decreased viability after siRNA knockdown of TRPV6 (31).
Several studies have shown that TRPV6 is involved in
highly calcium-selective currents in prostate cancer cells and is
regulated by intracellular calcium levels (21,32,33). TRPV6-
mediated calcium entry has been reported to directly control
the proliferation of LNCaP cells and promote apoptosis
resistance (34). These studies suggest that TRPV6 would be
a promising therapeutic target, although there have been no
previous directed efforts to develop inhibitors. The few
MATERIALS AND METHODS
Materials
T47D cells and LNCaP cells were obtained from NIH
Culture Collection. Plastic six-well cell culture dishes,
96-well cell culture dishes, and T75 cultivation flasks were
purchased from BD Biosciences. RPMI-1640 medium from
Invitrogen was used to culture the cells. Fetal bovine serum
(FBS), trypsin, and penicillin/streptomycin for cell culture,
and TRIzol reagent for total RNA purification were also
purchased from Invitrogen. TaqMan Universal Master Mix
and Primer Express for real-time PCR were from Applied
Biosystems. siRNAs and HiPerFect transfection reagent
were obtained from Qiagen. Chemicals used for synthesis
were from Sigma and Novabiochem.
Cell Culture
The human breast cancer cell line T47D and the human
prostate cancer cell line LNCaP were used in this study.
Tumor cells were grown in T75 culture flasks in RPMI-
1640 medium supplemented with 10% FBS and antibiotics
(100 U/ml penicillin and 100 μg/ml streptomycin) in a
humidified atmosphere with 5% CO2. Cells used in these
studies were between passages 30 to 45. The cells were
routinely passaged twice per week.

324
Landowski, Bolanz, Suzuki and Hediger
siRNA Treatment
with log dilutions of the compound (10–0.001 mM). After
incubation, the number of living cells was determined using
Cell Proliferation Kit II (XTT; Roche) following the
manufacturer’s instructions. The absorbance was measured
by an ELISA reader (Vmax, Molecular Devices) at 450 nm,
with a reference wavelength at 650 nm. Data points were
measured in triplicates.
Two different siRNA molecules were used to knockdown
TRPV6 expression in the LNCaP cells. The siRNA1 (target
sequence): CTG CAT GTC AGA GCA CTT TAA,
siRNA2 (target sequence): AAC CTG CTG CAG CAG
AAG AGG, and the siRNA control: AAT CAT CTA AGC
TGG CTT TGC were used to transfect into cancer cells.
The cells were seeded in 2 ml of culture medium at 400,000
cells per well in six-well plates. After 24 h, siRNA was
diluted in phenol red-free medium and delivered with
HiPerFect reagent using a final siRNA concentration of
5 nM. The cells were incubated with siRNAs for 72 h
before the total RNA was isolated using the TRIzol RNA
isolation method, according to the manufacturer’s protocol.
TRPV Ca2+ Uptake Inhibition
Xenopus oocytes were injected with TRPV5 or TRPV6
cRNA and expressed for 48 h before assaying 45Ca2+
uptake, as described previously (36). Uptake of 45Ca2+ was
measured in the presence of diluted inhibitor in order to
calculate IC50 values.
Real-Time PCR
Ca2+ Uptake Activity Measurement
cDNA was prepared for each sample by reverse transcription
of total RNA using TaqMan reverse transcription reagents
according to the manufacturer’s manual, as described previ-
ously (31). For all experiments, mRNA expression was
measured by real-time PCR using an Applied Biosystems
7500 Real-Time PCR System. Reactions consisted of 1x
Master Mix, 0.9 μM forward and reverse primers, and
0.2 μM dual-labeled fluorescent probes each for TRPV6 and
β-actin. The sequences of the forward and reverse primers for
TRPV6 were 5′-GGT TCC TGC GGG TGG AA-3′ and
5′-CCT GTG CGT AGC GTT GGA T-3′, respectively, with
the resulting amplicon being 62 bp with a Tm of 60°C. The
probe sequence for TRPV6 was 5′-ACA GGC AAG ATC
TCA ACC GGC AGC-3′. The forward and reverse primer
sequences for β-actin were 5′-CCT GGC ACC CAG CAC
AAT-3′ and 5′-GCC GAT CCA CAC GGA ATA CT-3′,
respectively, with the resulting amplicon being 69 bp with a
Tm of 60°C. The probe sequence for β-actin was 5′-ATC
AAG ATC ATT GCT CCT CCT GAG CGC-3′. Primer
Express was used to select primers for TRPV6 and β-actin. All
primers were designed to cross exon-exon boundaries of the
coding sequence. Primers were optimized and validated for
the comparative Ct method, as described in the manufac-
turer’s manual. ABI Prism SDS software version 1 was used
for the analysis of the amplification plots. The fold change
SD in TRPV6 expression was normalized to β-actin.
Five-thousand cells per well were seeded in a black 96-well
plate in 50 μl of culture medium per well. After 24 h, siRNA
treatment was performed with 5 nM siRNA as described
above. Subsequently, after 48 h, the calcium uptake activity
was observed in real time using the FLIPR Calcium 3 Assay
Kit (Molecular Devices), according to the manufacturer’s
manual. To the medium, 50 μl of calcium-free loading buffer
containing EGTA (0.1 mM) was added to decrease the
influence of FBS in the culture medium. Then 100 μl of
calcium 3 dye was added, and the plate was incubated for 1 h
at 37°C. The plate was put into the FlexStation. EGTA
(100 mM) was added to the cells to deplete intracellular
calcium stores, followed by the addition of calcium solution
(100 mM). Fluorescence change (counts) showed the calcium
uptake activity after siRNA.
Chemical Synthesis
A general synthesis scheme is shown in Fig. 1. Step 1 yields
the amide, step 2 reduces the amide, and step 3 forms the
ether bond. Step 1 in synthesis of Compound #01: L-
proline methyl ester was coupled with 4-
methoxyphenylacetic acid using benzotriazol-1-yl-oxytri-
pyrrolidinophosphonium (PyBOP) and 2 equivalents of N-
methylmorpholine (NMM) to produce methyl 1-[2-(4-
methoxyphenyl) acetyl] pyrrolidine-2-carboxylate. In step
2, the amide was reduced to the amino alcohol with LiAlH4
and AlCl3 in dry tetrahydrofuran (THF). In step 3, the
product was converted to the amine alcohol salt by
dissolving the step 2 product in ethyl acetate and 15%
HCl/ethyl acetate and evaporating the solvent with a
rotary vacuum. This salt was coupled with 4-
chlorobenzhydrol (or other alcohol groups as in subsequent
syntheses) under Williamson conditions, using catalytic
para-toluenesulfonic acid (p-TsOH) in refluxing toluene.
Cell Proliferation
Prostate cancer cell proliferation experiments were carried
out in 96-well plates. To determine the effect of TRPV6-
specific siRNAs on proliferation, 5,000 cells were seeded
per well, and after 24 h cells were treated with 5 nM (final
concentration) for 72 h. The effect of the synthesized
compounds was determined by treating 5,000 cells for 72 h

Chemical Inhibitors of the Calcium Entry Channel TRPV6
325
The final brownish oil was isolated using column chroma-
tography on silica gel with a 50:50 ethyl acetate:hexane
mixture. Structures were confirmed by NMR and mass
spectrometry. The three-step synthesis described here provides
a general method for synthesis of a large number of derivatives.
The following variations were made to the above procedure to
synthesize the other compounds. Compound #02: In step 3,
benzhydrol was coupled instead of 4-chlorobenzhydrol. Com-
pound #03: In step 3, 4-chlorophenylmethanol was used
instead of 4-chlorobenzhydrol. Compound #05: In step 1, 4-
dimethylaminophenylacetic acid was used instead of 4-
methoxyphenylacetic acid. Compound #06: Same as #05,
except the synthesis was ended after step 2. Compound #09: In
step 1, 4-chlorophenylacetic acid was used instead of 4-
methoxyphenylacetic acid.
Chemical Analysis
Compound #01: purity 90%; 1H NMR (300 MHz, DMSO-
d6): δ 7.25–7.15 (m, 9H), 6.95–6.82 (d, 2H), 6.65–6.58
(d, 2H), 5.36 (s, 1H), 3.62 (s, 3H), 3.31–3.25 (m, 2H), 2.90–
2.80 (m, 2H), 2.69–2.60 (m, 2H), 2.56–2.52 (m, 1H), 2.36–
2.30 (m, 2H), 1.77–1.60 (m, 2H), 1.56–1.44 (m, 2H); ESI-MS
436.1 (M + H)+.
Compound #02: purity 90%; 1H NMR (300 MHz,
DMSO-d6): δ 7.22–7.18 (m, 10H), 6.97–6.86 (d, 2H),
6.63–6.55 (d, 2H), 5.37 (s, 1H), 3.73 (s, 3H), 3.32–3.24
(m, 2H), 2.94–2.82 (m, 2H), 2.72–2.61 (m, 2H), 2.57–2.54
(m, 1H), 2.37–2.28 (m, 2H), 1.78–1.62 (m, 2H), 1.58–1.44
(m, 2H); ESI-MS 402.1 (M + H)+.
Fig. 1 General scheme for synthesis of chemical compounds.
RESULTS
Chemical Synthesis
Compound #03: purity 90%; 1H NMR (300 MHz,
DMSO-d6): δ 7.41–7.30 (m, 4H), 7.18–7.06 (d, 2H), 6.82–
6.79 (d, 2H), 4.47–4.44 (s, 2H), 3.71 (s, 3H), 3.40–3.38
(m, 2H), 2.98–2.85 (m, 2H), 2.64–2.59 (m, 2H), 2.51–2.49
(m, 1H), 2.36–2.28 (m, 2H), 1.79–1.64 (m, 2H), 1.62–1.51
(m, 2H); ESI-MS 360.1 (M + H)+.
To develop inhibitors of TRPV6 we synthesized a group of
compounds similar to TH-1177 using a three-step method,
as shown in Fig. 1. The structures of the five resulting
compounds (#02, #03, #05, #06 and #09) are shown in
Fig. 2 and were confirmed by ESI-MS and NMR analysis.
Compound #01 is TH-1177. While TH-1177 has been
established as an inhibitor of Ca2+ entry in prostate cancer
cells (35), it has not been previously shown to be a TRPV6
inhibitor. The compounds and controls were tested for
their inhibitory potential on TRPV5 and TRPV6, and for
their antiproliferative effect in in vitro assays using prostate
and breast cancer cells.
Compound #05: purity 90%; 1H NMR (300 MHz,
DMSO-d6): δ 7.41–7.32 (m, 9H), 6.94–6.92 (d, 2H), 6.65–
6.59 (d, 2H), 5.45 (s, 1H), 3.37–3.33 (m, 2H), 2.92–2.89
(m, 2H), 2.86–2.83 (m, 6H), 2.72–2.65 (m, 2H), 2.57–2.54
(m, 1H), 2.37–2.28 (m, 2H), 1.75–1.63 (m, 2H), 1.58–1.45
(m, 2H); ESI-MS 449.2 (M + H)+.
Compound #06: purity 95%; 1H NMR (300 MHz,
DMSO-d6): δ 7.14–7.11 (d, 2H), 6.90–6.81 (d, 2H), 4.30 (s,
1H), 3.39–3.35 (d, 2H), 2.98–2.95 (m, 2H), 2.87–2.83 (m,
6H), 2.68–2.63 (m, 2H), 2.40 (s, 1H), 2.35–2.25 (m, 2H),
1.79–1.62 (m, 2H), 1.59–1.45 (m, 2H); ESI-MS 249.2 (M +
H)+.
Inhibition of TRPV5 and TRPV6
To test the potency and selectivity of our compounds for
the calcium channels TRPV5 and TRPV6, we performed
45Ca2+ uptake assays using TRPV5- or TRPV6-expressing
Xenopus oocytes. This assay allows for high protein expres-
sion and to separately express different isoforms. For each
compound and control, IC50 values were calculated and are
plotted in Fig. 3. The most potent TRPV6 inhibitor was
Compound #09: purity 90%; 1H NMR (300 MHz,
DMSO-d6): δ 7.35–7.13 (m, 13H), 5.49 (s, 1H), 3.41–3.34
(m, 2H), 2.98–2.86 (m, 2H), 2.83–2.68 (m, 2H), 2.59–2.54
(m, 1H), 2.34–2.25 (m, 2H), 1.75–1.59 (m, 2H), 1.56–1.44
(m, 2H); ESI-MS 440.1 (M + H)+.

326
Landowski, Bolanz, Suzuki and Hediger
Fig. 2 Structures of synthesized compounds that were tested in in vitro assays. Compound #01 is TH-1177. Econazole and miconazole are controls.
compound #03 (IC50=90 μM), which was over five times
less effective at inhibiting TRPV5. Compound #09, which
has a chlorinated benzhydrol group, had the strongest
inhibitory effect on TRPV5 (IC50=109 μM), with roughly
four times less inhibition of TRPV6. Compound #06,
which does not have an ester-linked phenolic side group,
did not inhibit either channel and demonstrates that the
presence of an ester-linked side group is critical for activity.
Overall, the least effective inhibitors of both channels were
compounds #01 and #02. Both control compounds, econa-
zole and miconazole, inhibited TRPV6 more than TRPV5.
IC50 values and the compound selectivity index (TRPV5/
TRPV6) are displayed in Table I and reveal that compound
#03 is the most potent and selective for TRPV6.
1000
900
Growth Inhibition in Cancer Cell Lines
TRPV6
TRPV5
800
700
600
500
400
300
200
100
0
To analyze the effectiveness of our compounds on cancer
cell growth, we selected the LNCaP human prostate cancer
cell line, since TRPV6 is endogenously expressed and has
been reported to play a predominant role in the Ca2+ entry
pathway in this cell line (21). To confirm this finding, two
distinct siRNAs were used to knock down TRPV6
expression in LNCaP cells. In Fig. 4A, the effectiveness of
the siRNA treatments can be seen in their ability to reduce
TRPV6 mRNA expression. The siRNA1 reduced expres-
sion by 55% and siRNA2 by 50%, whereas the control
Fig. 3 TRPV-mediated 45Ca2+ uptake inhibition. IC50 values of tested
compounds on TRPV5 and TRPV6 expressed in Xenopus oocytes.

Chemical Inhibitors of the Calcium Entry Channel TRPV6
327
Table I IC50 Inhibition Values and Selectivity Index (TRPV5/TRPV6) of
Compounds Tested in TRPV5- or TRPV6-expressing Xenopus Oocytes
pattern to the uptake inhibition data in the oocytes.
Compound #03 was the best growth inhibitor of T47D
cells (IC50=38 μM) and the second best inhibitor of
LNCaP cells (IC50=0.44 μM). Likewise, compounds #01
and #02 had the weakest effect in both the prostate and
breast cancer cells. Compound #06, which did not show
calcium uptake inhibition activity in the oocytes, also
showed no effect on reducing cell growth in either cancer
cell line (Fig. 5A, B and Table II).
Compound IC50 TRPV6 (μM) IC50 TRPV5(μM) TRPV5/TRPV6a
#01
675
456
0.68
#02
869
712
0.82
#03
90
503
5.56
#05
386
246
0.64
#06
No effect
428
No effect
109
No effect
0.25
#09
Econazole
Miconazole
190
296
1.56
201
442
2.20
DISCUSSION
a Larger (TRPV5/TRPV6) ratios indicate a more TRPV6-selective compound
Calcium is a central signaling ion that is crucial for
controlling growth, proliferation, and survival of normal
and malignant cells. Recent studies have revealed that the
calcium entry channel TRPV6 is involved in the growth
control of prostate cancer cells via Ca2+/NFAT-dependent
pathways (34). We have also shown that it plays a role in
breast cancer, since siRNA knockdown of TRPV6 reduces
proliferation in the T47D breast cancer cell line (31).
In this study, we created the first set of chemical
compounds directed at TRPV6 inhibition and demonstrat-
ed their inhibitory potential in in vitro studies. Compound
#01, TH-1177, was previously shown to inhibit Ca2+ influx
into LNCaP human prostate cancer cells (35). However, the
molecular target for TH-1177 in LNCaP cells was not
siRNA treatment did not affect TRPV6 mRNA. This
expression knockdown in turn led to a reduction in LNCaP
cell proliferation by 57% and 52%, respectively (Fig. 4B).
Control siRNA treatment did not affect prostate cancer cell
growth.
Next, the synthesized compounds were tested for their
ability to inhibit the growth of LNCaP prostate cancer
cells as well as a breast cancer cell line endogenously
expressing TRPV6. Their effectiveness was similar in
A
100
80
60
40
20
0
siRNA1
siRNA2
siRNA
control
untreated
B
100
80
60
40
20
0
siRNA1
siRNA2
siRNA
control
untreated
Fig. 4 siRNA knockdown treatments in LNCaP cells. (A) Reduction in
TRPV6 expression (%) after treatment with TRPV6 siRNAs compared to
control siRNA. TRPV6 mRNA level standardized to β-actin. (B) Effect of
TRPV6 siRNA treatments on cell viability compared to control siRNA,
determined by XTT assay.
Fig. 5 Viability of LNCaP (A) and T47D (B) cells after treatment with the
different synthesized compounds and control (XTTassay), log scale in μM.

328
Landowski, Bolanz, Suzuki and Hediger
Table II IC50 Inhibition Values of Compounds Tested in LNCaP Prostate
Cancer Cells and T47D Breast Cancer Cells
assay. Our compounds might also be inhibiting other calcium
entry channels present in the cancer cells. However, we
believe the major effect is through TRPV6 inhibition.
Compound
LNCaP
T47D
IC50 (μM)
IC50 (μM)
Our experiments showed that the presence of an ester-
bonded phenolic group in the synthesized compounds was
critical for the inhibition against TRPV5 or TRPV6, since
compound #06 was unable to inhibit either channel. The
control compounds, econazole and miconazole, two other
known inhibitors of TRPV5 and TRPV6, also contain this
ester-bonded phenolic group at the center of the molecule
(Fig. 2). This suggests that the benzhydrol or phenolic group
is structurally important, probably reflecting the structure of
binding pockets in TRPV5 and TRPV6 channels. Addition-
ally, all the compounds with benzhydrol side groups were
more TRPV5-selective, with compound #09 being the best
inhibitor of TRPV5 (Table I). Interestingly, the most
TRPV6-selective compound, #03, contained only an ester-
linked chlorinated phenolic side group. Therefore, the
nature of the ester-bonded side group seems to play a role
in determining the channel selectivity of the compounds.
One can speculate that changing the π density of the
aromatic groups by introducing different electron releas-
ing/withdrawing groups could be a good strategy for
further TRPV5-or TRPV6-targeted drug design, as de-
scribed for a potassium channel (38). Although we
investigated this in a limited way here, we can make a few
noteworthy observations. Comparing compounds #01 and
#02, it is evident that chlorinating the benzhydrol group
produced a stronger TRPV-channel inhibitor, since both
TRPV5 and TRPV6 were inhibited better by compound
#01. Replacing the methoxyphenolic group (compound
#01) with a chlorinated phenol (compound #09) further
increased the effectiveness of the compound in inhibiting
both channels. Compound #09, containing two chlorine-
substituted phenolic groups, was the best inhibitor of
TRPV5. When the chlorinated phenol was replaced with
a dimethylaniline, the resulting compound (#05) became a
slightly weaker TRPV5 inhibitor, but was the second best
TRPV6 inhibitor. Compound #05 was the best
benzhydrol-containing TRPV6 inhibitor tested. These
compounds point the way to developing a larger group of
inhibitors to probe deeper into the structure-activity
relationships.
#01
50 0.4
220.5 0.9
198.2 0.4
38.8 0.2
44.5 0.5
No effect
#02
61 0.2
#03
0.44 0.07
0.31 0.05
No effect
1.0 0.1
#05
#06
#09
49.1 0.2
40.9 0.4
Miconazole
0.18 0.02
identified. Our experiments using the Xenopus oocyte
expression system indicated that TH-1177 inhibited 45Ca2+
influx derived from TRPV6 channel activity (Fig. 3).
Moreover, it also inhibited TRPV5, the closest homolog of
TRPV6 (Fig. 3 and Table I).
To develop more potent and selective inhibitors for
TRPV6, we synthesized several compounds based on different
chemical modifications of TH-1177 (Fig. 2) and checked their
biological activities. Compound #03 was found to be the
most potent and selective for TRPV6 (Fig. 3 and Table I),
significantly inhibiting prostate and breast cancer cell
proliferation (Fig. 5). Likewise, TRPV6-specific siRNAs were
also able to reduce proliferation (Fig. 4), in agreement with
previously published studies (34). In LNCaP cells, TRPV6 is
the most abundant calcium entry channel and was expressed
at levels around 45-fold higher than TRPV5 (37). The
calcium channel expression data for T47D cells shows that
TRPV6 is among the highest channels expressed (31). The
expression of TRPV5 relative to TRPV6 is also dramatically
lower. Therefore, we do not expect that TRPV5 plays a
major role in calcium uptake in either LNCaP or T47D
cells. It has been clearly demonstrated that TRPV6 controls
calcium entry and proliferation in LNCaP cells (34).
Similarly, in T47D cells, TRPV6 has been shown to play a
major role in these processes (31).
The compounds that best inhibited TRPV6-mediated
calcium uptake in oocytes were also the best inhibitors of
cancer cell growth. A clear correlation was seen between
the uptake inhibition and cell viability data. TH-1177 was
previously shown to inhibit calcium entry into LNCaP cells,
leading to reduced cell proliferation (35). These results
strongly suggest that our compounds, especially #03,
inhibit the calcium transport activity of endogenously
expressed TRPV6 in prostate and breast cancer cells, and
thus lead to lower proliferative ability. The benzyhdrol-
containing compounds #05 and #09 were also effective in
reducing cancer cell proliferation. While the TRPV6
affinity of these compounds is lower than #03, it is high
enough to produce a significant effect in the proliferation
The selectivity and potency of compound #03 suggest
that it could be a better compound for in vivo studies than
TH-1177. TH-1177 treatment was shown to extend the
average life span of mice carrying prostate tumors by up to
38% without toxicity (34). Therefore, compound #03 could
be expected to perform as well or better. The systemic
toxicity of compound #03 would be expected to be low
because of its structural similarity to TH-1177. Studies of
the systemic side effects of our compounds, as well as their
effectiveness in vivo, are the next step.

Chemical Inhibitors of the Calcium Entry Channel TRPV6
329
15. Zweifach A, Lewis RS. Rapid inactivation of depletion-activated
calcium current (ICRAC) due to local calcium feedback. J Gen
Physiol. 1995;105:209–26.
16. Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK,
Trebak M. Stim1 and Orai1 mediate CRAC currents and store-
operated calcium entry important for endothelial cell prolifera-
tion. Circ Res. 2008;103:1289–99.
17. Gwack Y, Feske S, Srikanth S, Hogan PG, Rao A. Signalling to
transcription: store-operated Ca2+ entry and NFAT activation in
lymphocytes. Cell Calcium. 2007;42:145–56.
18. Ng SW, di Capite J, Singaravelu K, Parekh AB. Sustained
activation of the tyrosine kinase Syk by antigen in mast cells
requires local Ca2+ influx through Ca2+ release-activated Ca2+
channels. J Biol Chem. 2008;283:31348–55.
19. Stiber J, Hawkins A, Zhang ZS, Wang S, Burch J, Graham V, et al.
STIM1 signalling controls store-operated calcium entry required for
development and contractile function in skeletal muscle. Nat Cell
Biol. 2008;10:688–97.
20. Perret S, Cantereau A, Audin J, Dufy B, Georgescauld D.
Interplay between Ca2+ release and Ca2+ influx underlies
localized hyperpolarization-induced [Ca2+]i waves in prostatic
cells. Cell Calcium. 1999;25:297–311.
21. Vanden Abeele F, Lemonnier L, Thebault S, Lepage G, Parys JB,
Shuba Y, et al. Two types of store-operated Ca2+ channels with
different activation modes and molecular origin in LNCaP human
prostate cancer epithelial cells. J Biol Chem. 2004;279:30326–
37.
CONCLUSION
We synthesized TRPV5 and TRPV6 inhibitors and identified
one selective inhibitor for TRPV6. Several of our compounds
were effective in inhibiting prostate and breast cancer cell
proliferation. We propose that this growth inhibition may be
caused by a reduction in TRPV6-mediated calcium uptake.
Although we did not synthesize sufficient compounds to fully
investigate structure-activity relationships, our study provides
a starting point for the synthesis of further inhibitory
compounds. Future studies will examine the effectiveness of
our compounds in in vivo cancer models.
ACKNOWLEDGMENTS
This study was supported by the Swiss National Science
Foundation (Hediger), Novartis Foundation (Hediger), NIH/
NCI (Hediger), Marie Curie Reintegration Grant (Hediger)
and Bernese Cancer League (Landowski). We thank Leah
Witton for her helpful comments on the manuscript.
22. Vanden Abeele F, Roudbaraki M, Shuba Y, Skryma R,
Prevarskaya N. Store-operated Ca2+ current in prostate cancer
epithelial cells. Role of endogenous Ca2+ transporter type 1. J
Biol Chem. 2003;278:15381–9.
REFERENCES
1. Mettlin C, Jones GW, Murphy GP. Trends in prostate cancer care in
the United States, 1974–1990: observations from the patient care
evaluation studies of the American College of Surgeons Commission
on Cancer. CA Cancer J Clin. 1993;43:83–91.
2. Mundy GR. Mechanisms of bone metastasis. Cancer.
1997;80:1546–56.
3. Orr FW, Sanchez-Sweatman OH, Kostenuik P, Singh G. Tumor-bone
interactions in skeletal metastasis. Clin Orthop Relat Res 19–33 (1995).
4. Saitoh H, Hida M, Shimbo T, Nakamura K, Yamagata J, Satoh
T. Metastatic patterns of prostatic cancer. Correlation between
sites and number of organs involved. Cancer. 1984;54:3078–84.
5. Chanand JM, Giovannucci EL. Dairy products, calcium, and vitamin
D and risk of prostate cancer. Epidemiol Rev. 2001;23:87–92.
6. Clinton SK, Giovannucci E. Diet, nutrition, and prostate cancer.
Annu Rev Nutr. 1998;18:413–40.
7. Tavani A, Gallus S, Franceschi S, La Vecchia C. Calcium, dairy
products, and the risk of prostate cancer. Prostate. 2001;48:118–21.
8. Putney Jr JW. A model for receptor-regulated calcium entry. Cell
Calcium. 1986;7:1–12.
9. Putney Jr JW. Type 3 inositol 1, 4, 5-trisphosphate receptor and
capacitative calcium entry. Cell Calcium. 1997;21:257–61.
10. Putney Jr JW. Capacitative calcium entry in the nervous system.
Cell Calcium. 2003;34:339–44.
11. Hoth M, Penner R. Depletion of intracellular calcium stores
activates a calcium current in mast cells. Nature. 1992;355:353–6.
12. Lewis RS, Cahalan MD. Mitogen-induced oscillations of cytosolic
Ca2+ and transmembrane Ca2+ current in human leukemic T
cells. Cell Regul. 1989;1:99–112.
23. Legrand G, Humez S, Slomianny C, Dewailly E, Vanden Abeele
F, Mariot P, et al. Ca2+ pools and cell growth. Evidence for
sarcoendoplasmic Ca2 + -ATPases 2B involvement in human
prostate cancer cell growth control. J Biol Chem. 2001;276:47608–
14.
24. Tombal B, Weeraratna AT, Denmeade SR, Isaacs JT. Thapsigargin
induces a calmodulin/calcineurin-dependent apoptotic cascade
responsible for the death of prostatic cancer cells. Prostate.
2000;43:303–17.
25. Wertz IE, Dixit VM. Characterization of calcium release-
activated apoptosis of LNCaP prostate cancer cells. J Biol Chem.
2000;275:11470–7.
26. Skryma R, Mariot P, Bourhis XL, Coppenolle FV, Shuba Y,
Vanden Abeele F, et al. Store depletion and store-operated Ca2+
current in human prostate cancer LNCaP cells: involvement in
apoptosis. J Physiol. 2000;527(Pt 1):71–83.
27. Vanden Abeele F, Skryma R, Shuba Y, Van Coppenolle F,
Slomianny C, Roudbaraki M, et al. Bcl-2-dependent modulation
of Ca(2+) homeostasis and store-operated channels in prostate
cancer cells. Cancer Cell. 2002;1:169–79.
28. Wissenbach U, Niemeyer BA, Fixemer T, Schneidewind A, Trost
C, Cavalie A, et al. Expression of CaT-like, a novel calcium-
selective channel, correlates with the malignancy of prostate
cancer. J Biol Chem. 2001;276:19461–8.
29. Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H. Expression
of the Ca2+ -selective cation channel TRPV6 in human prostate
cancer: a novel prognostic marker for tumor progression.
Oncogene. 2003;22:7858–61.
13. Zweifach A, Lewis RS. Mitogen-regulated Ca2+ current of T
lymphocytes is activated by depletion of intracellular Ca2+ stores.
Proc Natl Acad Sci U S A. 1993;90:6295–9.
14. Shuba YM, Prevarskaya N, Lemonnier L, Van Coppenolle F,
Kostyuk PG, Mauroy B, et al. Volume-regulated chloride
conductance in the LNCaP human prostate cancer cell line. Am
J Physiol Cell Physiol. 2000;279:C1144–54.
30. Zhuang L, Peng JB, Tou L, Takanaga H, Adam RM, Hediger
MA, et al. Calcium-selective ion channel, CaT1, is apically
localized in gastrointestinal tract epithelia and is aberrantly
expressed in human malignancies. Lab Invest. 2002;82:1755–
64.
31. Bolanz KA, Hediger MA, Landowski CP. The role of TRPV6 in
breast carcinogenesis. Mol Cancer Ther. 2008;7:271–9.

330
Landowski, Bolanz, Suzuki and Hediger
32. Bodding M, Flockerzi V. Ca2+ dependence of the Ca2+-selective
TRPV6 channel. J Biol Chem. 2004;279:36546–52.
model by a compound synthesized to block Ca2+ entry. Cancer
Res. 2000;60:1002–8.
33. Schindl R, Kahr H, Graz I, Groschner K, Romanin C. Store
depletion-activated CaT1 currents in rat basophilic leukemia mast
cells are inhibited by 2-aminoethoxydiphenyl borate. Evidence for
a regulatory component that controls activation of both CaT1 and
CRAC (Ca(2+) release-activated Ca(2+) channel) channels. J Biol
Chem. 2002;277:26950–8.
34. Lehen’kyi V, Flourakis M, Skryma R, Prevarskaya N. TRPV6
channel controls prostate cancer cell proliferation via Ca(2+)/
NFAT-dependent pathways. Oncogene. 2007;26:7380–5.
35. Haverstick DM, Heady TN, Macdonald TL, Gray LS. Inhibition
of human prostate cancer proliferation in vitro and in a mouse
36. Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H,
Brown EM, et al. Molecular cloning and characterization of a
channel-like transporter mediating intestinal calcium absorption. J
Biol Chem. 1999;274:22739–46.
37. Peng JB, Zhuang L, Berger UV, Adam RM, Williams BJ, Brown
EM, et al. CaT1 expression correlates with tumor grade in prostate
cancer. Biochem Biophys Res Commun. 2001;282:729–34.
38. Imai YN, Ryu S, Oiki S. Docking model of drug binding to the
human ether-a-go-go potassium channel guided by tandem dimer
mutant patch-clamp data: a synergic approach. J Med Chem.
2009;52:1630–8.