Natural product inspired optimization of a selective TRPV6 calcium channel inhibitor

Article abstract of DOI:10.1039/d0md00145g

Transient receptor potential vanilloid 6 (TRPV6) is a calcium channel implicated in multifactorial diseases and overexpressed in numerous cancers. We recently reported the phenyl-cyclohexyl-piperazine cis-22a as the first submicromolar TRPV6 inhibitor. This inhibitor showed a seven-fold selectivity against the closely related calcium channel TRPV5 and no activity on store-operated calcium channels (SOC), but very significant off-target effects and low microsomal stability. Here, we surveyed analogues incorporating structural features of the natural product capsaicin and identified 3OG, a new oxygenated analog with similar potency against TRPV6 (IC50 = 0.082 ± 0.004 μM) and ion channel selectivity, but with high microsomal stability and very low off-target effects. This natural product-inspired inhibitor does not exhibit any non-specific toxicity effects on various cell lines and is proposed as a new tool compound to test pharmacological inhibition of TRPV6 mediated calcium flux in disease models.

Full text of DOI:10.1039/d0md00145g

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RESEARCH ARTICLE
Natural product inspired optimization of a
selective TRPV6 calcium channel inhibitor†
Cite this: DOI: 10.1039/d0md00145g
ab
c
a
Micael Rodrigues Cunha,
Rajesh Bhardwaj,
Aline Lucie Carrel,
*
*
Sonja Lindinger, d Christoph Romanin, d Roberto Parise-Filho,
b
a
*
Matthias A. Hediger
c and Jean-Louis Reymond
Transient receptor potential vanilloid 6 (TRPV6) is a calcium channel implicated in multifactorial diseases
and overexpressed in numerous cancers. We recently reported the phenyl-cyclohexyl-piperazine cis-22a
as the first submicromolar TRPV6 inhibitor. This inhibitor showed a seven-fold selectivity against the closely
related calcium channel TRPV5 and no activity on store-operated calcium channels (SOC), but very
significant off-target effects and low microsomal stability. Here, we surveyed analogues incorporating
structural features of the natural product capsaicin and identified 3OG, a new oxygenated analog with
Received 4th May 2020,
Accepted 18th June 2020
similar potency against TRPV6 (IC50 = 0.082
0.004 μM) and ion channel selectivity, but with high
microsomal stability and very low off-target effects. This natural product-inspired inhibitor does not exhibit
any non-specific toxicity effects on various cell lines and is proposed as a new tool compound to test
pharmacological inhibition of TRPV6 mediated calcium flux in disease models.
DOI: 10.1039/d0md00145g
rsc.li/medchem
expression was found to be abnormally upregulated in
numerous cancers of breast11–13 and prostate tissues,14
Introduction
TRPV6 is a Ca2+-selective member of the transient receptor
potential vanilloid (TRPV) family, referred to as the
gatekeeper of transepithelial Ca2+ transport.1–3 The channel is
primarily found in the human intestine, kidney and placenta
and in a number of exocrine organs such as pancreas,
prostate and mammary gland.4,5 It is known that TRPV6 has
an important contribution to multifactorial diseases.6,7 For
instance, TRPV6-deficient mice have diminished fertility,
osteopenia and reduced body weight,5,8 whilst human TRPV6
mutations, which render the channel less functional, cause
transient neonatal hyperparathyroidism (TNH) and skeletal
abnormalities.9,10 These pathological findings are related to
tissues in which TRPV6-expression at normal levels is
essential for Ca2+ homeostasis. On the other hand, TRPV6
compared to normal tissues.15,16
We recently reported cis-22a (1, Fig. 1) as the first
submicromolar small molecule inhibitor of hTRPV6 mediated
Ca2+ flux.17 Compound 1 was highly selective for hTRPV6
against other Ca2+ channels, and showed a 7-fold selectivity
against the closely related rTRPV5. Inhibitor 1 furthermore
a Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
3012 Bern, Switzerland. E-mail: jean-louis.reymond@dcb.unibe.ch
b Department of Pharmacy, University of São Paulo, Prof. Lineu Prestes Avenue 580,
05508‐000 São Paulo, Brazil. E-mail: roberto.parise@usp.br
c Department of Nephrology and Hypertension, University Hospital Bern,
Inselspital, 3010 Bern, Switzerland. E-mail: matthias.hediger@ibmm.unibe.ch
d Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020
Linz, Austria
† Electronic supplementary information (ESI) available: Tables S1–S3, Fig. S1–S4,
protocols for FLIPR assays, confocal imaging and antiproliferative assays,
preparation, crystal structure reports, HRMS results, 1H, 13C and 19F NMR
spectra of all new compounds, and purity of final compounds. CCDC 1997201–
1997205. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/d0md00145g
Fig. 1 Chemical structure and hTRPV6 inhibition potency of cis-22a
(1), capsaicin (2), and the photoswitchable inhibitor
3 (shown as
observed in X-ray structures).17–19
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decreased the cell viability of a tumor cell line overexpressing
TRPV6 as reported with siRNA knockdown experiments;
however the effect only occurred at high concentrations (IC50
≈ 25 μM). However, 1 also inhibited other targets such as
hERG, dopamine and muscarinic receptors, and was highly
unstable against microsomal degradation, implying that this
inhibitor only had limited applicability as a tool compound
to study hTRPV6 inhibition.
Herein, we set out to search for new analogs with similar
potency and selectivity but an improved pharmacological
profile. To this end, we introduced the structural features of
the natural product capsaicin (2),20–22 a well-known TRPV1
Scheme 1 Synthesis of TRPV6 inhibitors and capsaicin analogues. Reagents and conditions. (a) TBDMSCl, DMAP, imidazole, DCM, r.t., 2 h (quant.);
(b) (i) tert-butyl piperazine-1-carboxylate, AcOH, NaBHIJOAc)3, DCE, r.t., 48 h; (ii) TFA, DCM, r.t., 1 h (78–45%, over 2-steps); (c) 4-R-cyclohexanones,
NaBHIJOAc)3, Et3N, DCE, r.t., 48 h (6–45%); (d) TBAF, THF, r.t., 3 h (30–59%, over 2-steps); (e) (i) tert-butyl piperazine-1-carboxylate, EDCl, DMAP,
CH2Cl2, r.t., on; (ii) TFA, DCM, r.t., 1 h, (60%, over 2-steps); (f) 4-phenylcyclohexyl-piperazine, AcOH, NaBHIJOAc)3, DCE, r.t., 48 h (6–68%); (g) (i)
1-benzylpiperazine, NaBHIJOAc)3, DCE, r.t., 48 h; (ii) Pd/C, H2, AcOH, MeOH, r.t., on. (18–38%, over 2-steps); (h) 38, 15, AcOH, NaBHIJOAc)3, DCE, r.t.,
48 h (81%); (i) Mg, THF, Ar., r.t. to rf., 30 min; then 1,4-dioxaspiroij4.5]decan-8-one, THF, Ar., r.t. to rf., 30 min; (j) PPTS, acetone, H2O, 60 °C, 6 h (72–
90%); (k) 37, 15, NaBH3CN, Et3N, MeOH, r.t., 24 h (48%); (l) hexyl- or octyl-sulfonyl-hydrazide, MeOH, r.t., 2 h (56–73%).
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ligand also reported as a hTRPV6 modulator.23,24 Our aim
was to modify the 3-pyridine and m-phenyl appendages of 1,
which are frequent drug-type substituents and might be
responsible for the undesirable off-target effects of 1.
Previous structure–activity relationship studies had shown
that variations in both groups were often compatible with
hTRPV6 inhibition,17 such as introducing a phenyl diazo
group to obtain the photoswitchable hTRPV6 inhibitor 3.18
On the other hand, modifications of the central cis-1,4-
cyclohexane and piperazine groups mostly abolished the
activity.17 Therefore, we maintained this central core in all
derivatives of 1 investigated in the present study. We also
reinvestigated 2 itself to verify the claimed activity of this
natural product on hTRPV6 and extended the investigation to
a series of new analogues.
Inspired by a report that 2 itself inhibits TRPV6,23,24 we
finally prepared a series of capsaicin analogues reproducing
the essential pharmacophoric features of the natural product
(a methyl-catechol and a hydrophobic tail connected via an
amide bond linker) by connecting vanillin with aliphatic
chains via
a
sulfonylhydrazone (41 and 42),25 N-acyl
hydrazone (43 and 44)26 or sulfonylglycine hydrazone (45 and
46)27,28 as an amide replacement.29,30
2. X-ray crystallography
In all the above syntheses, the cis-1,4-cyclohexyl stereoisomer
was isolated either by column chromatography or by RP-
HPLC. All the compounds obtained as free bases were finally
precipitated as hydrochloride salts. In the case of a freshly
synthesized sample of the original inhibitor
1 and of
analogues 9, 19, 31 and 40, we obtained X-ray crystal
structures confirming the 1,4-cis-cyclohexane stereochemistry
(Fig. 2). In these structures, the cyclohexane and piperazine
Results and discussion
1. Design and synthesis
rings adopt
a
chair conformation. Furthermore, the
piperazinyl group is an axial substituent of the cyclohexane
ring for 1, 9, 19, and 40, but an equatorial substituent in 31
as observed previously with the photoswitchable inhibitor
3,18 possibly reflecting crystal packing effects (Fig. S1†).
The synthesis of all compounds investigated in this study is
presented in Scheme 1. First, we replaced the pyridine ring in
1 with the O-methyl-catechol group of capsaicin while either
preserving the phenyl appendage or substituting it with
aliphatic groups resembling the fatty acyl group of capsaicin.
Reductve alkylation of silyl protected vanillin 5 with Boc-
piperazine and Boc removal, followed by a second reductive
alkylation with cyclohexanone and subsequent deprotection
yielded analog 6. The same reaction sequence with 4-ethyl-,
4-tert-butyl- or 4-phenyl-cyclohexanone gave the corresponding
analogues 7–9. Condensation of vanillic acid (10) with N-Boc-
piperazine, Boc deprotection and reductive alkylation with
the same four cyclohexanones afforded analogues 11–14
including an amide linkage related to capsaicin. In a similar
approach starting with 6-hydroxynicotinaldehyde (15) and
6-hydroxynicotinic acid (18), we obtained 4-phenyl- and 4-tert-
butyl-cyclohexyl analogues 16–17 and 19–20 displaying a
pyridone group. Pyridone can be considered as a pyridine
analog containing a hydrogen-bond donor group related to
the phenolic hydroxyl group of capsaicin.
3. TRPV6 activity screening
We measured the possible modulation of TRPV6 activity by
detecting the uptake of Cd2+ into HEK293 cells stably
overexpressing human TRPV6 (HEK-hTRPV6) using the
fluorescence reporter Calcium-5 (Molecular Devices LLC) as
Due to the activity of 17 (see below), we prepared further
analogues of this compound. First, we modified the pyridone
carbonyl group with a methoxy (25) or bromo (26) substituent
by double reductive alkylation of the corresponding pyridine-
carboxaldehydes 21–23, and similarly prepared a new sample
of the previously reported meta-pyridine analog 24.17 We also
synthesized analogues 30–32 by combining the pyridone
group as a piperazine substituent with a meta-xylyl group as a
cyclohexyl substituent as in 1, or with an ortho- or meta-
trifluoromethyl phenyl group, by using a similar synthetic
route starting from the corresponding cyclohexanones. For
the particularly lipophilic trifluoromethyl derivatives, we
additionally synthesized analogues 39 and 40 preserving the
tertiary alcohol at the cyclohexane, which is present as an
intermediate for cyclohexanone synthesis, such as to enhance
water solubility.
Fig. 2 X-ray structures of compounds 1 (free base), 9 and 31 (di-HCl
salts), and 19 and 40 (mono-HCl salts).
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described previously.17 The HEK-hTRPV6 cells were previously
reported in our group31–33 and are routinely used for
screening campaigns. We first measured the inhibition
potency of reference compounds 1 and 2 in this assay
(Fig. 3). The assay confirmed the submicromolar activity of
our cyclohexylpiperazine inhibitor 1, showing an even slightly
stronger inhibition than the originally reported value. On the
other hand, we could not detect any modulation of hTRPV6
activity by 2 in this assay up to 100 μM, in contrast to the
reported activity, which was determined indirectly.23,24
We then proceeded to test the initial series of analogues
6–9, 11–14, 19–20, 24–26, and 41–46 (Fig. 4 and Table 1).
None of the capsaicin analogues 41–46 showed any activity,
in line with the lack of hTRPV6 inhibition observed with 2.
Nevertheless, the O-methyl-catechol group borrowed from
capsaicin was suitable as a replacement for the meta-pyridine
group of 1 when combined with a tert-butyl (8 and 13) or
phenyl (9 and 14) group as a cyclohexyl substituent, in
Fig. 4 Activity screening for hTRPV6 mediated Cd2+ uptake in HEK-
hTRPV6 cells. Data shown are mean + SEM (n = 3).
4. Optimization of pyridone 17
We investigated several changes around the phenyl group as
a means to improve the activity of 17. Inhibition increased
particular with 14 featuring
a vanillic amide group,
when adding a meta-CH3 group similar to 1 to form 30 (IC50
0.15 0.04 μM). The effect was even stronger with a meta-CF3
group (31, IC50 = 0.064 0.007 μM), while the inhibition
=
illustrating that a dibasic piperazine is not essential for
activity. We also observed good hTRPV6 inhibitory activity
with three of the four analogues for which the meta-pyridine
group was replaced by a pyridone group (16, 17, and 20). The
lower activity with 19 however showed that the tert-butyl
group, although potentially interesting as a non-aromatic
group, was not a very favourable replacement of the aromatic
group as a cyclohexane substituent. We selected pyridone 17
(IC50 = 0.37 μM) for further optimization due to its good
activity combined with a favorable ligand efficiency (LE =
0.34) and lipophilic ligand efficiency (LLE = 3.5)34 compared
to all other tested compounds.
decreased slightly with an ortho-CF3 group (32, IC50 = 0.69
0.18 μM). Introducing a hydroxyl group at the aromatic
attachment point on the cyclohexane caused a slight decrease
in potency for the meta-CF3 analog 39 (IC50 = 0.082 0.004
μM, Fig. 5A), but an increase in potency for the ortho-CF3
analog 40 (IC50 = 0.13 0.03 μM). In both cases, the addition
of the hydroxyl group also increased water solubility.
Analyzing all the analogues in terms of LE and LLE led us to
select 39, named 3OG, as a promising analog for further
investigation.
Table 1 Activity on hTRPV6
Cpd
IC50 (μM) (95% CI)a
HACb
LEc
LLEd
1
8
9
13
14
16
17
20
24
25
26
30
31
32
39 (3OG)
40
0.050 0.003
2.4 (1.4–4.2)
1.6 (1.1–2.3)
25
26
28
27
29
24
26
27
25
27
26
27
30
30
31
31
0.40
0.30
0.28
0.29
0.30
0.32
0.34
0.32
0.34
0.32
0.36
0.35
0.33
0.28
0.31
0.30
1.7
0.3
0.9
1.0
2.1
2.1
3.5
3.9
2.0
1.3
1.6
3.2
3.2
2.2
4.8
4.6
2.1 (1.5–2.9)
0.43 (0.31–0.59)
2.4 (0.30–19.0)
0.37 (0.25–0.57)
0.60 (0.45–0.80)
0.55 (0.35–0.87)
0.55 (0.40–0.77)
0.17 (0.14–0.22)
0.15 0.04
0.064 0.007
0.69 0.18
0.082 0.004
0.13 0.03
a
Data shown are mean SEM (n = 9/concentration) for at least 2
independent experiments or mean and 95% confidence interval (n =
b
6/concentration) of a single experiment. HAC: heavy atom count.
c
LE: ligand efficiency = (1.37 × pIC50)/HAC, where pIC50 = −logIJIC50
)
in the molar range.34 LLE: lipophilic ligand efficiency = pIC50
−
d
Fig. 3 Dose–response curves of 1 (A) and 2 (B) in HEK-hTRPV6 cells,
measuring Cd2+ influx.
clog P, where clog P was obtained from ChemDraw v. 19.0.1.28
(PerkinElmer Informatics, Inc.).34
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stained with the wheat germ agglutinin Alexa Fluor™ 594
conjugate to mark the cytoplasmic membrane.35,36 In control
cells (treated with the vehicle), the application of Cd2+ (50 μM)
revealed an increase in green fluorescence after 30 min,
indicating Cd2+ transport through hTRPV6 (Fig. 6A). When cells
were treated with 10 μM 39 prior to the addition of Cd2+, the
fluorescence was significantly less pronounced (Fig. 6B). Time-
lapse imaging of Cd2+ uptake over 30 minutes furthermore
showed that cells treated with inhibitor 39 had significantly
reduced uptake compared to non-treated cells over the first 12
minutes, after which a plateau was reached (Fig. S2†).
To assess whether pharmacological blockage of TRPV6
transport function by 39 could trigger a cellular effect, we
determined the ability of 39 to reduce the Cd2+ toxicity
towards HEK293 wt and HEK-hTRPV6 cell lines. Prolonged
Cd2+ exposure is known to produce toxic effects on human
cells, and eventually culminates in cell death.37 When we
tested the viability of these cells after 24 h of treatment, we
found that the Cd2+ dose–response in HEK-hTRPV6 was ∼2-
fold greater than that in HEK293 wt (4.6 0.1 μM and 8.3
0.3 μM, respectively). These results indicate that the
overexpression of hTRPV6 increased the transport of Cd2+
and consequently, the toxicity towards these cells (Fig. 7 and
S3†).38 While 39 at 10 μM or 1 μM had no effect on the Cd2+
sensitivity of HEK293 wt, treating HEK-hTRPV6 cells with 39
at 10 μM significantly reduced the toxic effect of Cd2+ (Fig. 7).
The reduction of Cd2+ toxicity occurring only in HEK-hTRPV6
but not in HEK293 wt indicates that 39 inhibits hTRPV6-
mediated Cd2+ transport.
Fig. 5 Evaluation of 39 (3OG) as a TRPV6 inhibitor. (A) Dose–response
curve of 39 on Cd2+ influx into HEK-hTRPV6 cells. Data shown are
mean SEM (n = 6/concentration) of 4 independent experiments. (B)
and (C) Electrophysiological characterisation of 39. (B) Averaged time
course of whole-cell current densities (mean SEM) from YFP-TRPV6
transfected HEK293 cells. The experiment started at 10 mM Ca2+
,
subsequently containing equivalent amounts of DMSO as the control
(green) or 0.1 and 10 μM 39 (blue) or 1 (black), followed by La3+. (C)
Bar graph (mean SEM and individual values) of inhibition by 0.1 and
10 μM 39 (blue) and 1 (black) on TRPV6 current densities. * indicates
significant p values < 0.05.
Electrophysiological experiments on YFP-TRPV6 transiently
transfected HEK293 cells confirmed the activity of 39 on TRPV6.
Initially, 10 mM Ca2+ solution was applied and after the
currents reached a plateau, the inhibitors at 0.1 and 10 μM were
added. Finally, the calcium currents were fully inhibited with
La3+ (Fig. 5B). 39 induced an inhibition of 30.8 5.1% and 88.2
2.0%, respectively, at 0.1 μM and 10 μM (Fig. 5C). In
comparison to the previously described 1, the inhibition by 39
occurred at a slower rate and was less pronounced.
5. Blocking TRPV6 transport function with pyridone 39
We further confirmed the TRPV6 inhibitory activity of 39 by
confocal microscopy of HEK-hTRPV6 cells preincubated with
Leadmium™ Green, which is a Ca2+-insensitive intracellular
green fluorogenic dye revealing Pb2+ and Cd2+ within the
cytosol, allowing TRPV6-mediated cellular uptake independent
of intracellular Ca2+ fluxes to be tracked. The cells were also
Fig. 6 HEK-hTRPV6 cells co-stained with Leadmium™ Green and
wheat germ agglutinin Alexa Fluor™ 594 conjugate. Images were
collected at time 0 min and 30 min after treatment with the vehicle (A)
or 10 μM 39 (B) followed by the addition of Cd2+ (50 μM) using
confocal microscopy (Nikon Eclipse TE2000-E, 100×). White bars
denote 25 μm scale.
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Fig. 9 A. Recording of store-operated Ca2+ entry (SOCE) in MDA-MB-
231 cells pretreated for 20 min with either DMSO or SOCE inhibitor
GSK-7975A, or 1 vs. 39. Ca2+ store-depletion was achieved by 10 min
treatment of cells with 1 μM thapsigargin (Tg) in a nominally Ca2+ free
buffer. B. SOCE quantification as the area under the curve (AUC) of the
2 mM CaCl2 (Ca2+) add-back trace is shown as normalized values to
Fig. 7 IC50 values of Cd2+ toxicity to HEK293 wt and HEK-hTRPV6
cells. Data shown are mean
+ SEM (n = 4/concentration) of 2
independent experiments. ****P < 0.0001; n.s., P > 0.05.
the DMSO control (mean
SD; n = 18). The P-value of the 1 vs. 39
pretreated cells is indicated above the respective bar as *** for p ≤
0.001 or non-significant (n.s.) for p > 0.05.
6. Ion channel selectivity and off-target profiling of pyridone
39
microsomes, which was relatively short for 1 (t1/2 = 6 min),
was significantly extended for 39 (t1/2 > 60 min), which we
attribute to the replacement of the aromatic meta-methyl
substituent with a trifluoromethyl group.42,43 Taken together,
these data showed that compound 39 had a much better
selectivity and stability profile compared to the original
hTRPV6 inhibitor 1.
To test if the higher polarity and LLE of 39 compared to
those of 1 translated into a better selectivity profile, we
investigated the activity of both compounds on the closely
related calcium channels. Similar to 1, 39 showed a 7-fold
selectivity for hTRPV6 (IC50 = 83 nM) against hTRPV5 (IC50
=
560 nM), low activity on L-type calcium channels at 10 μM
(Fig. 8), and no detectable activity on store-operated calcium
channels at 5 μM (Fig. 9). Similar to 1,17 39 did not activate
nor inhibit TRPV1, which is the only known TRP target of
capsaicin (Fig. S2†).39,40
We also characterized the off-targets predicted to be
potentially problematic for both compounds using the web-
based target prediction tool PPB2.41 Remarkably, 39 did not
show any significant off-target effects compared to 1,
indicating that its higher LLE translated into reduced
polypharmacology (Fig. 8). It is worth noting that the hERG
activity observed with 1 was completely abolished with 39.
Furthermore, we found that the half-life in human liver
7. Antiproliferative activity
In our initial discovery of 1, we reported that the inhibitor
significantly reduced the growth rate in T47D breast cancer
cells, which express high levels of TRPV6, at micromolar
concentration (IC50 = 25
10 μM), while SKOV3 ovarian
carcinoma cells, where TRPV6 expression was not detected,
were less affected (IC50 > 50 μM).17 However, these values
were much higher than the submicromolar levels sufficient
to block TRPV6, and occurred in the range of the off-target
effects of 1 (Fig. 8).
Here, we compared the effects of 1 and 39 on SKOV3 and
T47D, as well as on MCF-7 and MDA-MB-231 as two
additional breast cancer cell lines with low levels of
TRPV6.13,44 Previous studies have shown that RNA silencing
(siRNA) of TRPV6 reduces T47D cell proliferation.11,13 On the
other hand, siRNA knock-down of TRPV6 does not reduce the
proliferation of MCF-7 and MDA-MB-231 cells.12 We also
investigated the non-cancer derived HEK293 immortalized
cell line which does not express TRPV6,45,46 as well as the
HEK-hTRPV6 overexpressing cell line used for activity assays.
Inhibitor 1 over 6 days caused a significant reduction in
cell proliferation for all six cell lines at high concentrations
and not just limited to SKOV3, T47D and HEK-hTRPV6 cells
(Fig. 10A). 1 also induced changes in cell morphology for
T47D cells (Fig. S4†). By contrast, we did not observe any
significant decrease in proliferation with 39 (Fig. 10B) or
changes in cell morphology (Fig. S4†) under similar
conditions despite its comparable IC50 for TRPV6 channel
Fig.
8 In vitro polypharmacology for selected targets of 1 (blue
diamonds) and 39 (red diamonds). Data are shown for each replicate (n
= 2). The data for 1 were extracted from ref. 17. The experiments were
conducted by Eurofins Cerep SA, France.
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Fig. 10 Dose–response curves of 1 (A) and 39 (B) in different breast
and ovarian cancer cells and HEK293. Relative cell survival compared
to the DMSO control is shown as a function of inhibitor concentration.
Data shown are mean SEM (n = 8/concentration) of 3 independent
experiments.
function inhibition. The positive control doxorubicin (10
μM), as expected, significantly decreased the growth of all six
cell lines (data not shown). Note that HEK-hTRPV6 was more
susceptible to 1 toxicity than HEK293 wt cells, which might
be related to the fact that HEK-hTRPV6 overexpressing cells
proliferate faster than HEK293 wt cells in a Ca2+-dependent
manner (Table S3†).46 These results suggest that the cytotoxic
effects observed with 1 but not with 39 do not reflect TRPV6
inhibition but probably result from non-specific or off-target
effects. Our findings that the pharmacological inhibition of
TRPV6 channel function by 39 did not affect the viability of
TRPV6 expressing cell lines is intriguing. This highlights the
need for future studies to uncover the precise role of TRPV6
in cancer progression.
Fig. 11 Overview of TRPV6 inhibitor development. The interactive
version of the map is accessible at http://tm.gdb.tools/trpv6-inhibitors/
. For color-coding, IC50 values were used as reported, or estimated
from reported percentage inhibition at
5 μM or 10 μM. Inactive
compounds were assigned IC50 mM. Compound numbers in
=
1
parentheses are from ref. 17. The map was prepared using the public
website and instructions at https://try-tmap.gdb.tools/.
certain extent with substitutions on the aromatic cyclohexane
substituent. This suggests that further improvements in
inhibitory potency and in compound properties might be
achievable with further variations at these positions.
Conclusions
8. Overview of TRPV6 inhibitor development
The overall development of our inhibitors is illustrated here
with an interactive tree-map (TMAP)47 representing each
molecule as a point color-coded by TRPV6 inhibitory potency
To improve the properties of the previously reported TRPV6
inhibitor 1, we surveyed analogues incorporating structural
features inspired by the natural product capsaicin such as
aliphatic and oxygen-containing functional groups. Although
we found that, contrary to previous reports, capsaicin does
not have any inhibitory effect on TRPV6, our strategy led us
to identify the new inhibitor 39 (3OG), which incorporates a
pyridone group and a tertiary alcohol as typical natural
product-like features. Inhibitor 39 shows similar potency
against TRPV6 and ion channel selectivity to 1 but much
better microsomal stability and much lower off-target effects,
in particular suppressed hERG inhibition. Inhibitor 39 blocks
TRPV6 transport function in cells as assessed by the
reduction of Cd2+ toxicity in HEK-hTRPV6. However, even at
high concentration, 39 does not display any measurable
cellular toxicity on various cell lines, expressing TRPV6 or
not. Structural and mutagenesis studies based on the recently
published structure of hTRPV6 (ref. 3 and 49) and showing
how 1 and 39 inhibit TRPV6 will be reported in the near
future. This new tool compound should be useful to decipher
the role of TRPV6 mediated calcium flux in various disease
models.
(Fig. 11). In this map, molecules are connected by
a
minimum spanning tree to their most similar analogs as
measured by the extended connectivity fingerprint MHFP6.48
The lower left portion of the TMAP illustrates our initial
study (blue arrows),17 in which we searched for scaffold-
hopping analogues of known, weakly active TRPV6 inhibitors
including an L-prolinol derivative. We discovered a first hit
compound (cis-11a), which we optimized to 1 (cis-22a). The
upper right branch contains capsaicin and its analogs 41–48,
which were inactive. This branch also contains 14 which
combines elements from capsaicin and cis-22a. The further
optimization of 14 by introducing a pyridone to form 17 and
its further optimization to inhibitor 39 (3OG) appears as an
additional side branch.
Throughout these explorations, we found that
a
(4-arylcyclohexyl)-piperazine with cis-stereochemistry on the
cyclohexane was critical to give strong TRPV6 inhibition. On
the other hand, TRPV6 inhibition was compatible with
variations in the second piperazine substituent and to a
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Conflicts of interest
13 A. A. Peters, P. T. Simpson, J. J. Bassett, J. M. Lee, L. Da
Silva, L. E. Reid, S. Song, M.-O. Parat, S. R. Lakhani, P. A.
Kenny, S. J. Roberts-Thomson and G. R. Monteith, Mol.
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14 L. Zhuang, J.-B. Peng, L. Tou, H. Takanaga, R. M. Adam, M. A.
Hediger and M. R. Freeman, Lab. Invest., 2002, 82, 1755–1764.
15 J.-B. Peng, L. Zhuang, U. V. Berger, R. M. Adam, B. J.
Williams, E. M. Brown, M. A. Hediger and M. R. Freeman,
Biochem. Biophys. Res. Commun., 2001, 282, 729–734.
16 V. Lehen'kyi, M. Raphaël and N. Prevarskaya, J. Physiol.,
2012, 590, 1369–1376.
17 C. Simonin, M. Awale, M. Brand, R. Van Deursen, J.
Schwartz, M. Fine, G. Kovacs, P. Häfliger, G. Gyimesi, A.
Sithampari, R. P. Charles, M. A. Hediger and J. L. Reymond,
Angew. Chem., Int. Ed., 2015, 54, 14748–14752.
18 M. R. Cunha, R. Bhardwaj, S. Lindinger, C. Butorac, C.
Romanin, M. A. Hediger and J.-L. Reymond, ACS Med. Chem.
Lett., 2019, 10, 1341–1345.
There are no conflicts to declare.
Acknowledgements
J. L. R. acknowledges the financial support from the Swiss
National Science Foundation (SNF), NCCR TransCure. R. B.
and M. A. H. acknowledge the SNF Sinergia grants (grant
numbers CRSII5_180326 and CRSII3_160782) for funding
support of this work. R. P. F acknowledges the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional de Desenvolvimento Científico
e
Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP, grant numbers: 2013/19311-6
and 2017/00689-0) for financial support. MRC thanks the
Swiss Excellence Scholarship for Foreign Students and
Scholars (ESKAS – 2017.0670) for the academic exchange
program in Switzerland. The authors thank Dr. Daniel Probst
for help in assembling the TMAP in Fig. 11. This work was
also supported in part by the Austrian Science Funds FWF
P33283 (to C. R.).
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M. R. Cunha, R. Bhardwaj, Y. Cury, V. O. Zambelli, E. G.
Barbosa, M. A. Hediger and R. Parise-Filho, Bioorg. Med.
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