Photoswitchable Inhibitor of the Calcium Channel TRPV6

Article abstract of DOI:10.1021/acsmedchemlett.9b00298

Herein we report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a number of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, we identified a compound switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC₅₀ ? 10 μM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradiation at λ = 365 nm (Z/E = 3:1, IC₅₀ = 1.7 ± 0.4 μM), thereby allowing precise spatiotemporal control of inhibition. This new tool compound should be useful to deepen our understanding of TRPV6.

Full text of DOI:10.1021/acsmedchemlett.9b00298

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Letter
A Photoswitchable Inhibitor of the Calcium Channel TRPV6
Micael Rodrigues Cunha, Rajesh Bhardwaj, Sonja Lindinger, Carmen
Butorac, Christoph Romanin, Matthias A. Hediger, and Jean-Louis Reymond
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00298 • Publication Date (Web): 02 Aug 2019
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A Photoswitchable Inhibitor of the Calcium Channel TRPV6
Micael R. Cunha,† Rajesh Bhardwaj,± Sonja Lindinger,¥ Carmen Butorac,¥ Christoph Romanin,*¥
Matthias A. Hediger,*± Jean-Louis Reymond*†.
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†Department of Chemistry and Biochemistry, NCCR TransCure, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
±Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, Bühlstrasse 28, 3012 Bern,
Switzerland & Department of Nephrology and Hypertension, University Hospital Bern, Inselspital, 3010 Bern, Switzerland.
¥Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz, Austria.
KEYWORDS: Photoswitchable inhibitor, Photopharmacology, Transient Potential Receptor Vanilloid, Azobenzene.
ABSTRACT: Herein we report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective
calcium channel involved in a number of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6
inhibitor appended with a phenyl-diazo group, we identified a compound switching between a weak TRPV6 inhibitor in its dark, E-
diazo stereoisomer (Z/E = 3:97, IC50 >> 10 µM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV
irradiation at  = 365 nm (Z/E = 3:1, IC50 = 1.7 ± 0.4 µM), thereby allowing precise spatiotemporal control of inhibition. This new
tool compound should be useful to deepen our understanding of TRPV6.
TRPV6 is a member of the Transient Receptor Potential
Vanilloid (TRPV) family1–5 with affinity for divalent ions,
critically involved in Ca2+ uptake in intestine, placenta and
exocrine tissues.6,7 TRPV6 expression is altered in a diversity
of diseases, including Crohn’s-disease and tumors.8–10
Nevertheless the exact role of TRPV6 in these pathologies
remains to be clarified.11–14 Recently we reported the first
selective and potent TRPV6 inhibitors, e.g. 1 and 2, based on a
(cis-4-phenyl-cyclohexyl)piperazine scaffold (Figure 1). These
inhibitors were used to probe the pharmacological effect of
TRPV6 inhibition on cancer cell growth.15 Here we set out to
develop a photoswitchable version of our inhibitors to facilitate
further studies on the mechanism of TRPV6.
were allowed at the second piperazine substituent, the most
potent inhibitors containing a meta-substituted pyridine.15 We
therefore extended the pyridine ring in 1 or 2 to form a
diazobenzene group, hoping to observe differential TRPV6
inhibition between the Z-diazo and E-diazo isomers,
introducing a pyridine nitrogen atom where allowed by
synthesis (Figure 1).
N
N
N
N
N
9a
N
N
N
N
N
Photoswitchable inhibitors can be obtained by incorporating
a diazo group into their structure, which isomerizes between the
more stable E- and the less stable Z-isomer upon UV irradiation
at 365 nm (EZ) and 470 nm (ZE).16 The diazo moiety may
substitute a two-atom linker separating two aromatic groups in
an existing inhibitor to form an azoster.17,18 Alternatively, a
phenyldiazo group is appended at a position such that E/Z
photoisomerization modulates inhibition.19 The latter design
has been used in all photoswitchable ion channel inhibitors
reported to date,20 including the four cases of TRP ion channel
modulators which were used for optical control of nociceptor
TRPV121 and sensory neuron receptors TRPC2/3/6.22,23 In all of
these cases the phenyldiazo group replaced or extended a
lipophilic group present in the parent inhibitor, and the Z-diazo
isomer was more inhibitory than the E-diazo isomer (Figure
S1).
1
N
N
IC50 = 0.32 ± 0.12 µM
X
9b
9c
(X = CH)
(X = N)
N
N
N
N
N
2
N
X
N
IC50 = 1.70 µM
9d
9e
(X = N)
(X = CH)
Figure 1. Chemical structures of TRPV6 inhibitors 1-2 and
investigated analog photoswitches 9a-e.
Phenyldiazo analogs 9a-b were prepared by first reacting N-
Boc-protected 4-amino or 4-nitro-aryl-piperazines (3a-b) with
nitrosobenzene to obtain the corresponding diazo intermediates
(4a-b), followed by Boc removal to the free piperazines 5a-b
and reductive alkylation of the piperazine with 4-phenyl-
cyclohexanone 6. Analog 9c was similarly obtained from a
To design our TRPV6 photoswitches, we followed the
phenyldiazo appendage approach starting from inhibitors 1 and
2. Structure-activity relationship studies had shown that only
minimal perturbations were allowed on the cis-4-phenyl-
cyclohexyl group, while a range of different aromatic groups
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ACS Medicinal Chemistry Letters
diazo intermediate 5c obtained by converting 3-aminopyridine
(3c) to the corresponding phenyldiazonium (4c) and coupling
with 1-phenylpiperazine (7). On the other hand we obtained 9d-
e by alkylation of (4-phenylcyclohexyl)piperazine 824,25 with 4-
hydroxymethyl or 4-bromomethyl-aryldiazoaryl intermediates
5d-e obtained separately (Scheme 1).26 In all cases the TRPV6
inhibitory cis-1,4-cyclohexyl stereoisomer with the diazo group
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in the dark, E-configuration, was isolated by column
chromatography. All diazo compounds were finally
precipitated as di-hydrochloride salts. The structure of our
photoswitches was confirmed by X-ray crystallography in the
case of E-9a, E-9c, and E-9e, which showed that the phenyl
substituent was axial and the piperazine equatorial relative to
the cyclohexane ring.
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Scheme 1. Synthesis of TRPV6 photoswitches 9a-e,a and X-ray crystal structure of E-9a, E-9c, and E-9e.
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NH2
N
N
N
N
a
c
N
N
N
N
N
N
N
N
Boc
RN
9a
E-
3a
9a
4a
5a
(R = Boc)
(R = H)
b
NO2
N
N
N
N
c
d
N
N
N
N
N
Boc
RN
4b
5b
(R = Boc)
(R = H)
3b
9b
b
Ph
N
HN
N
N
N
N
N
7
c
N
N
N
R
N
HN
N
3c
5c
(R = NH2)
(R = N2)
9c
9c
E-
e
4c
NH2
N
N
N
f-i
j
N
O
N
N
N
N
HO
N
O
3d
5d
9d
N
N
NH2
k-l
m
N
N
N
N
Br
3e
5e
9e
9e
E-
aReagent and conditions: (a) Nitrosobenzene, NaOH, benzene, H2O, reflux, 10 min (45 %); (b) HCl, H2O, reflux, o.n. (84 %); (c) (i) 4-
phenyl-cyclohexanone (6), Et3N, NaBH(OAc)3, DCE, r.t., 48 h; (ii) HCl, MeOH (34-40 %, over 2-steps); (d) (i) H2, Pd/C, AcOEt, r.t., 48 h;
(ii) Nitrosobenzene, AcOH, r.t., 24 h (61 %, over 2-steps); (e) NaNO2, HCl, H2O, 0 °C to r.t., 17 h (65 %); (f) LiAlH4, THF, Ar, 0 °C to r.t.,
24 h (76 %); (g) TBDMSCl, DIPEA, DMAP, CH2Cl2, DMF, 0 °C to r.t., 8 h (53 %); (h) Nitrosobenzene, NaOH, Toluene, H2O, 50 °C, 17 h
(55 %); (i) TBAF, THF, r.t., 2 h (90 %); (j) (i) MsCl, Et3N, THF, 0 °C to r.t., 2 h; (ii) (4-phenylcyclohexyl)piperazine (8), K2CO3, NaI, DMF,
60 °C, 2 h; (iii); HCl, MeOH (20 %, over 3-steps); (k) Nitrosobenzene, AcOH, r.t., 24 h (93 %); (l) NBS, BPO, CCl4, reflux, 24 h (99 %);
(m) (i) (4-phenylcyclohexyl)piperazine (8), K2CO3, DMF, r.t., 24 h; (ii) HCl, MeOH (46 %, over 2-steps).
We analyzed the photoswitchable behavior of 9a-e using
RP-UHPLC and UV-Vis spectrometry. The 500 µM stock
solutions in DMSO maintained in the dark for at least 48h
contained almost pure E-isomers (Table 1). These E-isomers
UV-Vis (λmax(Z)) between 400 and 500 nm corresponding to the
Z-isomer.27,28 In the case of 9a, irradiation at  = 365 nm did not
result in a new λmax(Z), probably due to overlap of both E- and Z-
λmax. The photostationary state of 9a, 9d and 9e was reached
within 5 minutes and featured the less stable Z-isomer (Z/E
ratios of 1:7 to 3:1), which returned to E-isomer in the dark
within minutes to days upon thermal isomerization (Figure 2B
and 2C). We did not observe any switching effect for 9b-c
(Figure S3), probably due to fast thermal isomerization.29,30
displayed a characteristic maximum absorbance (λmax(E)
)
between 300 and 400 nm (Figure 2). In the case of 9d and 9e,
UV irradiation at  = 365 nm resulted in a decrease of this
absorbance concomitantly with the appearance of a new peak at
RP-UPLC (Figures S4-S6) and a new maximum absorbance at
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Figure 2. (A) Individual UV-Vis spectra of compounds 9a, 9d and 9e (DMSO, 500 µM), in the dark-adapted (black), UV-adapted (magenta,
365 nm), blue-adapted (blue, 470 nm) states. The DMSO solutions were irradiated for 5 min in a self-built chamber equipped with LED
lamps31 (90 mW). (B) The light-magenta lines represent the UV-Vis spectra of the thermal relaxation of the UV-adapted state (dark-magenta
line) in direction to the dark-adapted state (black line) over time. (C) The absorption at λmax(E) was plotted as function of time (blue cross),
with exponential fit (black line).
Table 1. Photoisomerization properties of compounds 9a-e.
control for E-9a (30 %), E-9d (70%) and E-9e (13%) (Figure 3,
black bars).
c
Entry Dark-adapteda UV-adaptedb
T1/2
8.2 min
-
To access the Z-isomers, we irradiated the 10 mM DMSO
stock solutions of each individual compound at  = 365 nm for
5 min. Then, the solutions containing Z-isomers were
immediately diluted with assay buffer to the same concentration
of screening used before (10 µM). Switching did not affect
channel blockage of 9a but increased the inhibitory activity of
compounds 9d and 9e (Figure 3, magenta bars).
9a
9b
9c
9d
9e
100:0
93:7
88:12
93:7
100:0
100:0
97:3
100:0
50:50
25:75
-
3.7 h
3.9 d
aE/Z ratio for the dark state. bE/Z ratio after photoisomerization at
λ = 365 nm for 5 min. Ratios determined by RP-UHPLC.bHalf-life
for thermal ZE isomerization in the dark, determined by UV-Vis
spectroscopy.
To evaluate TRPV6 inhibition by our diazo analogs, we
measured their effect on channel activity by means of cadmium
(Cd2+) uptake into HEK293 cells stably overexpressing human
TRPV6 (HEK-hTRPV6) as described previously.15 In this
assay, a solution of Cd2+ is applied extracellularly, and its
transport through TRPV6 leads to complexation and
fluorescence of an intracellular dye. Screening of the dark state
revealed partial inhibition of Cd2+ transport compared to vehicle
Figure 3. Inhibition of Cd2+ (50 µM) influx into HEK293-hTRPV6
cells. Data was normalized to the maximum entry in the vehicle
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group (buffer/ DMSO). Compounds 1-2 (positive controls) 9a, and
differences in the two assay formats. Upon irradiation of E-9e
at 365 nm, the currents were further reduced, which was
reversible upon excitation with 470 nm. Subsequent irradiation
via alternating wavelength (365/470 nm) showed the
repeatability and photostability of this process (Figure 5B). The
current-voltage-relationship of TRPV6 currents before and after
the application of 9e revealed positive reversal potentials,
indicating unchanged Ca2+ selectivity (Figure 5C).
1
2
3
4
5
6
7
8
9d-e were tested at 10 µM. Diazo derivatives 9a-e showed limited
solubility and could not be measured above 10 µM. **P < 0.01;
***P < 0.001. Data shown are mean+SEM (n = 3 or 6).
Unfortunately dose-response measurements for the dark and
photoisomerized states of 9d had comparable IC50 values (IC50
~ 0.6 µM, Figure S8). On the other hand, we observed a striking
switching effect with 9e, which upon UV irradiation turned
from a weak E-isomer (< 15 % inhibition at 10 µM) to a potent
Z-isomer (IC50 = 1.7 ± 0.4 µM, Figure 4A) matching the
inhibitory potency of its template 2 (IC50 = 1.7 µM).15
Furthermore, irradiation of 9e with UV light at alternate 365 nm
and 470 nm led to alterations of the UV-Vis spectrum indicating
fully reversible isomerization between E-9e and Z-9e,
suggesting that this inhibitor would be well suited for reversible
photoswitching of TRPV6 (Figure 4B).
A
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B
C
Figure 5. Electrophysiological characterization of the
photoswitchable TRPV6 inhibitor 9e. (A) Averaged time course of
whole-cell currents of YFP-TRPV6 transfected HEK293 cells. The
diagram illustrates the whole-cell inward current density at -74 mV
during a 200 ms voltage ramp from -90 to +90 mV (applied every
5 s, HP = 50 mV), recorded by patch clamp technique, across time.
Black arrows indicate time points of 9e photoswitching by
continuous irradiaton at
λ = 365 nm (Polychrome IV
Figure 4. TRPV6 inhibition and photoswitching of 9e. (A)
Inhibition curve of Cd2+ influx into HEK293-hTRPV6 cells by Z-
9e. Data shown are mean ± SEM (n = 6/concentration) from 4
independent experiments. (B) UV absorbance at 327 nm indicating
photoswitching between E-9e and Z-9e upon irradiation
alternatively at 365 nm and 470 nm, measured at 500 µM in
DMSO.
monochromator, 150 W Xe lamp, first arrow: turn-on) and 470 nm.
Data shown are mean - SEM (n = 4, DMSO or n = 10, 9e) from at
least 2 independent experiments. I-VI illustrate the data points used
for graphs (B) and (C). (B) Bar chart (mean+SEM) of current
density of YFP-TRPV6 expressing HEK293 cells before and after
(I, white; II, grey) addition of 10 μM of 9e and after the exposure
with 365 nm (III, V, magenta) and 470 nm (IV, VI, blue). **P <
0.0005. (C) Representative current-voltage characteristic before the
addition of 9e (I, black), as well as after the exposure with 365 nm
(III, magenta) and 470 nm (IV, blue).
To evaluate photoswitching of the TRPV6 channel with
inhibitor 9e we conducted electrophysiological experiments in
transiently transfected HEK293-hTRPV6 cells (Figure 5). The
experiment started in 10 mM Ca2+ solution. After 120 seconds
the constitutively active TRPV6 currents reached a plateau and
a subsequent 10 mM Ca2+ solution containing 10 μM of E-9e or
an equivalent amount of DMSO as control were applied (Figure
5A, time point I), followed by applying continuous irradiation
at λ = 365 nm and 470 nm. Finally, 10 µM La3+ was added for
full inhibition at the end of the experiment. While currents were
stable over the course of the experiment in the control sample,
addition of E-9e led to a significant decrease of the TRPV6
current densities. The partial inhibition by E-9e in this
experiment contrasts with the very weak inhibition observed in
the FLIPR assay (Figure 3) and probably reflects inherent
In summary, by surveying phenyldiazo analogs of the
previously reported TRPV6 inhibitors 1 and 2, we identified 9e
as the first photoswitchable inhibitor of TRPV6. This inhibitor
switches reversibly between a weak E-isomer in its dark state
and an inhibitory Z-isomer upon UV irradiation at  = 365 nm
which matches the activity of its template inhibitor 2. Rapid
photoswitching of TRPV6 was illustrated by monitoring time
dependent inhibition of TRPV6 by 9e using electrophysiology.
Further studies with 9e should provide new insights into the
physiological and pathological roles of TRPV6.
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AUTHOR INFORMATION
Corresponding Authors
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*(C. R.) E-mail: Christoph.Romanin@jku.at
(M.A.H.) E-mail: matthias.hediger@ibmm.unibe.ch
(J.L.R.) E-mail: jean-louis.reymond@dcb.unibe.ch.
(15)
Author Contributions
MRC designed, synthesized, and purified the compounds. RB and
MRC performed FLIPR assays. SL and CB designed and
performed electrophysiology experiments. MRC and JLR wrote the
manuscript. MAH, CR, and JLR supervised the study. All the
authors discussed the results and commented on the manuscript.
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Funding Sources
Morstein, J.; Hill, R. Z.; Novak, A. J. E.; Feng, S.; Norman, D.
D.; Donthamsetti, P. C.; Frank, J. A.; Harayama, T.; Williams, B.
M.; Parrill, A. L.; Tigyi, G. J.; Riezman, H.; Isacoff, E. Y.;
Bautista, D. M.; Trauner, D. Nat. Chem. Biol. 2019, 15 (6), 623–
631.
This work was supported financially by the Swiss National Science
Foundation, NCCR TransCure.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
MRC thanks the Swiss Excellence Scholarship for Foreign
Students and Scholars (ESKAS – 2017.0670).
ABBREVIATIONS
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TRPV, Transient Receptor Potential Vanilloid; TRPA, Transient
Receptor Potential Ankyrin; TRPC, Transient Receptor Potential
Channel; SAR, Structure-Activity Relationships; RP-UHPLC,
Reversed Phase-Ultra High Performance Liquid Chromatography;
NCF, Nominal Calcium Free.
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N
N
N
N
365 nm
470 nm
N
N
N
N
9e
9e
Z-
E-
IC50 >> 10 µM
IC50 = 1.7 ± 0.4 µM
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T
R
P
V
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T
R
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Ca2+; Cd2+
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