Optical Control of CRAC Channels Using Photoswitchable Azopyrazoles

Article abstract of DOI:10.1021/jacs.0c02949

The Ca2+ release-activated Ca2+ (CRAC) channels control many Ca2+-modulated physiological processes in mammals. Hyperactivating CRAC channels are known to cause several human diseases, including Stormorken syndrome. Here, we show the design of azopyrazole-derived photoswitchable CRAC channel inhibitors (designated piCRACs), which enable optical inhibition of store-operated Ca2+ influx and downstream signaling. Moreover, piCRAC-1 has been applied in vivo to alleviate thrombocytopenia and hemorrhage in a zebrafish model of Stormorken syndrome in a light-dependent manner.

Full text of DOI:10.1021/jacs.0c02949

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Article
Optical control of CRAC channels using photoswitchable azopy-razoles
Xingye Yang, Guolin Ma, Sisi Zheng, Xiaojun Qin, Xiang
Li, Lupei Du, Youjun Wang, Yubin Zhou, and Minyong Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c02949 • Publication Date (Web): 24 Apr 2020
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Optical control of CRAC channels using photoswitchable azopyrazoles
Xingye Yang,†,# Guolin Ma,‡,# Sisi Zheng,§,# Xiaojun Qin,† Xiang Li,† Lupei Du,† Youjun Wang,*,§ Yubin
Zhou,*,‡ Minyong Li*,†,ǁ
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†Department of Medicinal Chemistry, Key Laboratory of Chemical Biology, School of Pharmacy, Shandong University,
Jinan, Shandong 250012, China.
‡Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston,
TX 77030, USA.
§Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal
University, Beijing 100875, China.
ǁHelmholtz International Lab, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong
250100, China.
ABSTRACT: The Ca2+ release-activated Ca2+ (CRAC) channels control many Ca2+-modulated physiological processes in
mammals. Hyperactivating CRAC channels are known to cause several human diseases, including Stormorken syndrome. Here
we show the design of azopyrazole-derived photoswitchable CRAC channels inhibitors (designated piCRACs), which enable
optical inhibition of store-operated Ca2+ influx and downstream signaling. Moreover, piCRAC-1 has been applied in vivo to
alleviate thrombocytopenia and hemorrhage in a zebrafish model of Stormorken syndrome in a light-dependent manner.
delivery of exogenous genes and expression of non-native
INTRODUCTION
proteins. By contrast, photopharmacological approaches29-
Ca2+ release-activated Ca2+ (CRAC) channel comprising
STIM and ORAI proteins is a prototypical example of store-
operated Ca2+ entry (SOCE) and serves as an indispensable
highly-selective Ca2+ entry route in both excitable and non-
excitable cells1-3. SOCE is initiated by Ca2+ store depletion
within the lumen of the endoplasmic reticulum (ER) and
subsequent multimerization of the STIM1 ER-luminal
domain4. Activated STIM1 molecules accumulate at ER-PM
junctions to engage and directly gate ORAI1 channels,
permitting Ca2+ flux into cells5-10. Aberrant STIM-ORAI
signaling has been linked to several human diseases,
including immunodeficiency, autoimmunoinflammatory
32
use photoswitchable molecules to exert precise
spatiotemporal control over the action of bioactive targets,
such as ion channels33-35, receptors36-38, enzymes39 and
nucleic acids40. To more precisely control CRAC channel
activity, we set out to develop a series of photoswitchable
inhibitors that allow for optical inhibition of Ca2+ signaling
with good spatiotemporal resolution, thereby permitting
photopharmacological modulations of diseases associated
with overactivated CRAC channels, such as Stormorken
syndrome. These tools are anticipated to complement the
existing genetically-encoded optogenetic actuators that
allow light-inducible activation of CRAC channels28, 41-44
.
R'
disorders, cardiac hypertrophy, cancer metastasis, and the
F
R
F
N
Stormorken syndrome3,
. Stormorken syndrome is
11
R
N
N
NH
N
UV
N
F
R'
F
N
Blue
R
O
N
N
believed to arise from gain-of-function mutations in either
ORAI1 or STIM1 (e.g., R304W) that lead to constitutive Ca2+
entry12-13. Therefore, CRAC channel has been actively
pursued as a promising therapeutic target. Several small
molecules targeting CRAC channels have been developed,
with some entering clinical trials14-16. Among them, GSKs
series17-19 and Synta 6620-24 are regarded as relatively
specific CRAC channels inhibitors with IC50 values in the
range of 1-4 µM17, 25. The field still lacks switchable CRAC
channel blockers that might cause less systemic side effects.
N
N
F
F
GSKs
OCH3
OCH3
OCH3
N
H3CO
UV
H3CO
O
H3CO
F
N
Blue
N
N
N
N
H
N
Synta 66
N
Scheme 1. Converting CRAC channel inhibitors, GSKs and
Synta 66, into photoswitchable derivatives, piCRACs.
piCRACs undergo reversible trans-to-cis isomerization
upon illumination with light emitting at different
wavelengths. Irradiation at the UV range leads to
conversion from the trans to cis configuration, whereas blue
light stimulation switches the cis isomer back to a trans
conformation.
Optogenetic approaches have been recently employed to
modulate Ca2+ channels with high spatiotemporal
resolution26-28
.
However, optogenetic manipulation is
limited in certain applications because it requires the
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CF3
1
Br
CF3
lithium bis(trimethylsiy)amide
2
3
4
5
6
7
8
9
F
R1
R1
N
N
anhydrous THF, r.t
N
N
N
HN
R2
F
R1
R1
N
NH2
R1
R1
HN
Oxene
N
N
NH2
DCM/H2O, r,t
NO
R1
R2
AcOH, r,t
R2
1-1
6-1
7-1
8-1
9-1
R1
R2
: R1=F, R2=H
: R1=H, R2=H
: R1=F, R2=methyl ester
: R1=F, R2=F
: R1=F, R2=Cl
1-3
6-3
7-3
8-3
9-3
piCRAC-1
piCRAC-6
piCRAC-7
piCRAC-8
piCRAC-9
: R1=F, R2=H
: R1=H, R2=H
: R1=F, R2=H
: R1=H, R2=H
: R1=F, R2=methyl ester
: R1=F, R2=F
: R1=F, R2=Cl
1-2
6-2
7-2
8-2
9-2
: R1=F, R2=H
: R1=H, R2=H
: R1=F, R2=methyl ester
: R1=F, R2=F
: R1=F, R2=methyl ester
: R1=F, R2=F
: R1=F, R2=Cl
: R1=F, R2=Br
: R1=F, R2=Cl
: R1=F, R2=Br
10-1
: R1=F, R2=Br
10-3
piCRAC-10
: R1=F, R2=Br
10-2
10
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R3
O
R3
R5
R5
Br
PBr3
LiAlH4
OH
OH
OH
Br
THF, 0 oC, reflux
DCM, 0 o
C
NaOH, CH3CN, r.t
R4
2-1
R4
R6
R6
2-2
2-3
3-3
: R5=CF3, R6=benzyl ether
: R5=benzyl ether, R6=CF3
2-2
3-2
: R3=CF3, R4=OH
: R3=OH, R4=CF3
: R3=CF3, R4=OH
: R3=OH, R4=CF3
: R5=CF3, R6=benzyl ether
: R5=benzyl ether, R6=CF3
3-1
3-2
R5
R3
lithium bis(trimethylsiy)amide
anhydrous THF, r.t
F
F
F
N
N
N
TiCl4
N
N
N
N
R6
R4
N
DCM, r.t
N
F
HN
F
N
N
2-2
3-2
: R5=CF3, R6=benzyl ether
: R5=benzyl ether, R6=CF3
piCRAC-2
piCRAC-3
: R3=CF3, R4=OH
: R3=OH, R4=CF3
1-3
F
O
F
O
CuI, DMF,120 o
C
OH
PBr3
O
H
O
O
NaBH4, MeOH, r.t
lithium bis(trimethylsiy)amide
F
F
N
N
H
+
N
N
O
OH
Br
DCM, 0
oC
anhydrous THF, r.t
N
F
F
HN
4-1
4-2
4-3
4-4
4-5
piCRAC-4
N
N
1-3
5-4
OCH3
NH2
OCH3
PdCl2(PPh3)2, K2CO3
OCH3
NO2
N
+
H3CO
H3CO
toluene/EtOH/H2O, 80 o
C
N
H3CO
Br (HO)2B
xylene, PTC, NaOH, K2CO3, r.f
N
NO2
N
5-1
5-2
5-3
piCRAC-5
Scheme 2. Synthesis of piCRACs based on the prototypic compounds of well-known CRAC channel inhibitors GSK-5498A,
GSK-7975A, GSK-5503A and Synta 66
alcohol, substitution, and deprotection reaction. PiCRAC-4
was produced by following Ullmann reaction, reduction,
bromination, and substitution. With Suzuki coupling and
diazo-coupling reaction, piCRAC-5 was synthesized.
RESULTS AND DISCUSSION
Design, synthesis and characterizations of photo-
inducible CRAC channel inhibitors (termed as piCRACs).
Pyrazole-derived inhibitors of CRAC channels such as GSK
series17-18 and Synta 6620 have heterocyclic N-aryl
benzamide45 backbone structures. We resorted to
bioisosteric replacement by substituting the amide group of
these compounds with azo-groups to lend themselves
toward azologization for photoconversion45. We envisioned
that the engineered photoswitchable compounds could only
inhibit SOCE under one configuration, and the light-induced
trans-to-cis isomerization of the azobenzene moiety would
enable light-switchable inhibition on SOCE (Scheme 1 and
Figure S1).
We then moved on to characterize the photochemical
properties of piCRACs in vitro. To identify the optimal
activation window for photoconversion, we measured the
absorption spectra of piCRACs after irradiation under
various wavelengths (Figure 1c, Figure S2a-d). The UV-Vis
spectra of piCRAC-1 to -5 were similar to the signature
absorption spectra of azobenzenes with photoconverted
characteristics between trans (E) and cis (Z) states. The best
photoconversions were generally achieved with 365 nm (E
− Z) and 415 nm (Z − E) irradiation (Figure 1c, Figure S2a-
d). These wavelengths were thus used in following
experiments.
Four synthesis routes were developed based on the
prototype compounds from well-known CRAC channel
inhibitors, including GSK-5498A, GSK-7975A, GSK-5503A
and Synta 66 (Schemes 2 and Figure S1). A series of
nitrosaniline analogs were obtained by aniline oxidation.
Azopyrazoles analogs were formed by Mills reaction in
glacial acetic acid followed by substitution to yield piCRAC-
1, -6 to -10. PiCRAC-2 and -3 were prepared by reducing
benzoic acid, protecting phenol, bromo substituted benzyl
By using thapsigargin (TG)-induced store-operated
calcium entry (SOCE) and Ca2+-dependent nuclear entry of
NFAT as two independent readouts, we examined the
biological activities of piCRACs. We firstly assessed
piCRACs’ photo-responsiveness and compared their
abilities to inhi-
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Table 1. Photostationary states (PSS) for piCRAC-1 and piCRAC-6 to -10 at different irradiation wavelengths
1
2
3
4
5
6
7
8
Dark
365 nm
415 nm
455 nm
530 nm
Compound
R1
R2
(area % cis)
3.7 ± 0.1
4.6 ± 0.2
4.7 ± 0.4
0
(area % cis)
49.0 ± 0.3
60.3 ± 0.7
50.4 ± 3.0
80.3 ± 0.2
52.3 ± 0.5
73.1 ± 0.6
76.9 ± 0.3
(area % cis)
15.5 ± 0.1
19.8 ± 0.6
12.9 ± 3.5
18.4 ± 0.1
18.5 ± 1.5
22.8 ± 3.9
19.5 ± 0.1
(area % cis)
22.9 ± 0.1
25.3 ± 0.4
11.0 ± 0.1
28.6 ± 0.7
20.0 ± 1.5
24.2 ± 0.1
24.8 ± 0.1
(area % cis)
33.2 ± 0.1
25.8 ± 0.4
10.7 ± 0.1
47.5 ± 0.2
26.5 ± 0.1
32.8 ± 0.1
33.6 ± 0.1
piCRAC-1
piCRAC-1
piCRAC-6
piCRAC-7
piCRAC-8
piCRAC-9
piCRAC-10
-F
-F
-H
-F
-F
-F
-F
-H
-H
-H
-methyl ester
9
-F
0
10
11
12
13
14
15
-Cl
-Br
0.9 ± 0.5
15.8 ± 3.0
The percentages of photostationary state area at dark, 365 nm (9.8 mW), 415 nm (14.4 mW), 455 nm (9.5 mW), 530 nm (6.8
mW) in acetonitrile containing 0.5% DMSO (50 M) or E3 embryo medium containing 0.1% DMSO (10 M) were
determined by HPLC analysis at 302 nm. Data were shown PSS ± s.e.m..
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-bit CRAC channels under the following conditions: the
initially synthesized compounds without any light
illumination defined as the “dark” status, which was further
exposed to 365 nm or 415 nm light, respectively. Among the
five tested piCRACs (1-5, at 10 μM), piCRAC-1 (Figure 1)
derived from GSK-5498A showed the most potent UV-light-
inducible suppression of TG-induced SOCE (Figure S3a)
and NFAT nuclear translocation (Figure S3b). PiCRAC-2
and piCRAC-5 failed to inhibit CRAC channel regardless of
UV and blue light. Compared with piCRAC-1, piCRAC-3 and
piCRAC-4 showed strong background and poor
isomerization. Thus, piCRAC-1 as the lead compound was
further characterized and optimized.
configuration (> 95 %) in the dark (Table 1), but contained
49.0 % cis-isomer under 365 nm illumination. To obtain a
higher conversion rate, we moved forward to design and
synthesize new GSK-5498A derivatives piCRAC-6 to -10 by
removing steric-effect-inducing groups or adding additional
para-electron-withdrawing groups to piCRAC-146-47
.
Consistent with previous reports46-47, these modifications
significantly increased photoisomerization rates of
piCRAC-6 to -10 (Table 1 and Figure S2e-i). However, in
cellulo functional testing results showed that these new set
of piCRACs had no biological activity as they did not inhibit
SOCE (Figure S4). We thus focused on piCRAC-1 and
thoroughly tested its photochemical and biological
properties.
We further examined the real-time isomerization of
piCRAC-1 by monitoring UV−Vis spectra, HPLC and 1H/19F-
NMR profiles. Prior to 365 nm illumination (at 415 nm or in
the dark), piCRAC-1 primarily adopted
a
trans
configuration, as reflected by a strong absorbance peak at
314 nm in the UV-Vis spectra (Figure 2a, black trace) and a
single peak corresponding to trans-piCRAC-1 in the HPLC
elution profile (Figure 2c, top trace at 0 s). Upon UV
irradiation at 365 nm, the trans-piCRAC-1 was converted
into a cis state, as characterized by the decrease of
absorbance at 314 nm (Figure 2a, violet curves) and a
newly-emerged secondary peak in the HPLC profile (Figure
2c). After 3-5 min UV illumination, 49.0 % of trans-piCRAC-
1 was switched to the cis state based on the HPLC profiles
(Figure 2c and S5c, Table 1). A similar trend was visualized
by using NMR to monitor the resonance signals of the CH2
group (H7) adjacent to the pyrazole ring and F21/F22
within the azobenzene group (Figure 2d and Figure S5d).
As shown in Figure 2b, S5e-f, cis PSS reversed to trans PSS
upon irradiation at 415 nm. Rapid and reversible
photoisomerization was quantified based on HPLC profiles
during illuminations at 365 nm (violet trace) or 415 nm
(blue trace, Figure 2e), with the photoisomerization half-
lives determined to be 22.1 ± 4.2 s at 365 nm and 14.3 ± 4.5
s at 415 nm (Figure 2e). Following exposure to UV light, the
half-life of the cis isomer of piCRAC-1 was determined to be
255.9 days, as measured with the method described by
Priimagi et al48 at 25 °C (Figure S6a-d). Furthermore, no
appreciable photodegradation was observed after repeated
cycles of photoconversions between 365 nm and 415 nm
Figure 1. Design and photochemical properties of piCRAC-
1. a-b, Chemical structures of GSK-5498A (compound A)
and light-inducible isomerization of its photoswitchable
derivative piCRAC-1 (compound 1). PiCRAC-1 undergoes
reversible trans-to-cis isomerization upon illumination with
lights emitting at different wavelengths. Irradiation at 365
nm leads to conversion from the trans to cis configuration,
whereas 415 nm light stimulation switches the cis isomer
back to a trans conformation. c, UV-Vis spectra of piCRAC-1
(50 M) at different irradiation wavelengths in acetonitrile
containing 0.5% DMSO.
We then monitored the relative abundance of the trans
and cis isomers of piCRAC-1 by HPLC (Table 1). The results
showed that all PSS of piCRAC-1 contained a mixture of
both isomers. PiCRAC-1 mostly existed in
a trans
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(Figure S6e-f). These findings establish piCRAC-1 as a
bistable compound with good photochemical characteris-
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Figure 2. Photochemical properties of piCRAC-1. a-b, Time-resolved UV-Vis spectra of piCRAC-1 (in acetonitrile
containing 0.5% DMSO) after illumination at 365 nm and 415 nm. Inset, the changes of absorbance monitored at 314 nm upon
illuminated at 365 nm and 415 nm. c-d, HPLC elution profiles (c) and selected regions of NMR spectra (d) showing the
transition of trans-piCRAC-1 to cis-piCRAC-1 at different UV illumination time points. The representative groups of piCRAC-1
(35.4 mM) in 1H-NMR and 19F-NMR spectra were indicated in MeOD-d4. e, piCRAC-1 underwent multiple rounds of reversible
isomerization with photostability when repeatedly alternating the illumination between 365 nm and 415 nm. The cis and
trans isomers of piCRAC-1 (50 µM) were indicated in acetonitrile containing 0.5% DMSO by the HPLC elution profiles based
on Figure 2c and Figure S5c-e (t1/2 = 22.1 ± 4.2 s at 365 nm and t1/2= 14.3 ± 4.5 s at 415 nm, Error bars are ± s.e.m.).
-tics, thus making it a promising candidate for subsequent
functional studies.
S7a). Meanwhile, the light-activated piCRAC-1 and GSK-
5498A had comparable inhibition rates over SOCE (Figure
S7b, c).
Optical control of CRAC channel activities and
downstream signaling. We next set out to test the light-
dependent inhibitory effects of the piCRAC-1 on CRAC
channel per se and the downstream Ca2+-responsive
transcription factor, the nuclear factor of activated T-cells
(NFAT), in mammalian cells. We first examined its ability to
suppress SOCE in HEK cells stably expressing GEM-GECO, a
genetically-encoded ratiometric Ca2+ indicator49-50. Newly-
synthesized piCRAC-1 had minimal effect on SOCE at
concentrations lower than 50 µM prior to UV illumination
(IC50 = 73.2 µM; Figure 3a). However, after 5 min UV
illumination, piCRAC-1 showed pronounced inhibition on
SOCE, with the IC50 value plummeting down to the sub-
In parallel, we examined the effect of piCRAC-1 on the
CRAC channel current (ICRAC) in HEK cells stably co-
expressing STIM1 and ORAI151. We found that piCRAC-1
suppressed ICRAC by up to 80 % when exposed to UV light
(365 nm) but not under blue light at 415 nm (Figure 3b-c),
indicating that cis-piCRAC-1 is a good CRAC channel
inhibitor. To evaluate the specificity piCRAC-1, we also
examined its effects on three other major types of Ca2+
channels. The results showed that piCRAC-1 did not inhibit
Ca2+ responses mediated by CaV1.2, TRPC3, and IP3Rs
channels at a concentration that could inhibit SOCE by 86%
(10 μM) (Figure S7f-h). Therefore, similar to its prototype
GSK-5498A, cis-piCRAC-1 is a specific inhibitor of CRAC
channels. We next examined the light-induced effects on the
downstream effector NFAT by monitoring its subcellular
micromolar range (IC50
= 0.5 µM; Figure 3a). The
performance of piCRAC-1 at 365 nm seemed to be superior
over its prototype, GSK-5498A (IC50 = 3.10 µM; Figure
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Figure 3. Optical control of CRAC channel and downstream signaling using piCRAC-1. a, Dose-dependent effects of
piCRAC-1 on SOCE in HEK293 GEM-GECO cells before (black curve; IC50: 73.2 µM) and after 365 nm light stimulation (purple
curve; IC50: 0.5 µM). b, Representative time courses of normalized inward CRAC channel currents (ICRAC, pA/pF), measured at
−100ꢀmV in HEK 293 STIM1-ORAI1 stable cells bathed with 10 μM piCRAC-1. Cells were either kept in the dark prior to light
stimulation (dark) and illuminated at 365 nm (violet) or 415 nm (blue), respectively. Whole-cell ICRAC currents were greatly
inhibited after 4 min of UV illumination. (n ≥ 6, -16.1 ± 1.4 pA/pF vs -3.4 ± 1.3 pA/pF) c, I-V relationships at the peak of ICRAC
indicated by arrows in Figure 3b. d, Representative confocal images of HeLa cells stably expressing NFAT1-GFP after
treatment with DMSO (control) or piCRAC-1 (10 μM) in the dark (prior to UV light) or with 365 nm illumination. 1 μM TG
was used to trigger Ca2+ influx with subsequent NFAT nuclear translocation. Scale bar, 10 μm. Three times experiments
repeated, 60 - 100 cells were measured in one image. e, Quantification of the inhibitory activity of 10 μM piCRAC-1 against
TG-induced NFAT nuclear entry. The ratio of nuclear GFP signals over the whole cell signals was used as readout. n=6, with
50 - 100 cells measured for each condition. f, Typical whole-cell currents of HEK293 cells expressing ORAI1-SS-GFP bathed
in DVF solution containing 5 μM piCRAC-1 (left panel), I-V relationships measured at different time points indicated by
arrows (right panel). g, Statistics from cells shown in panel f (***, P<0.0005, Paired Student’s t-test, n=6). h, Typical cellular
images showing spatial control of SOCE by piCRAC-1. Left panel, one imaging field showing UV exposed area (within the white
circle) and non-UV region (the rest of the image). Middle panel, showing the resting cellular GEM-GECO ratios; left panel,
showing cellular GEM-GECO ratios at the peak of SOCE responses. i, Time traces (left) and statistics (right) from cells shown
in panel h. Data were shown as mean ± s.e.m.
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localization
with
confocal
microscopy.
thrombocytopenia, as well as overt hemorrhage in the tail
as revealed by o-dianisidine staining of hemoglobin (Figure
Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase inhibitor
TG was used as a passive store depletion inducer to trigger
SOCE and subsequent nuclear translocation of NFAT. Upon
exposure to UV light, TG-induced NFAT nuclear
accumulation was significantly suppressed (Figure 3d-e
and Figure S8). Although to a less extent, piCRAC-3 and
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4c-d). After ensuring that the protein levels of expressed
STIM1_R304W were similar among all experimental groups
(Figures S10), we treated 3 dpf (day 3 post-fertilization)
zebrafish embryos with 5 μM piCRAC-1 with and without
exposure to UV or blue light. We found that 365 nm-excited
piCRAC-1 markedly rescued thrombocytopenia, as
indicated by significantly enhanced GFP signals emitting
from mature thrombocytes (78.9 ± 6.9 ％; Figure 4a-b). By
contrast, embryos incubated with piCRAC-1 without 365
nm illumination or under 415 nm light stimulation failed to
effectively rescue thrombocytopenia (Figure S9b).
Interestingly, we noticed that dark PSS and 415 nm PSS of
piCRAC-1 rescued thrombocytopenia slightly, probably
owing to incomplete light-induced conversion of cis-
piCRAC-1. Most importantly, piCRAC-1 effectively
ameliorate the abnormal bleeding at the tail exposed to
brief UV light illumination (Figure 4c-d).
piCRAC-4 also exhibited
inhibitory effect on SOCE and nuclear translocation of NFAT
(Figure S8).
a similar photoswitchable
9
We next exploited the use of piCRAC-1 for spatial and
temporal control of SOCE or CRAC channel activity in
HEK293 cells. Ca2+ imaging results showed that the UV light-
induced suppressive effect on SOCE could be partially
reversed after blue light illumination for 15 min (Figure
S7d). In 15 minutes, SOCE responses of piCRAC-1 treated
cells recovered by 57%, an extent significantly larger than
those of GSK-5498A treated cells after washing out the
residual compound (30%) (Figure S7d vs S7e). Thus, the
reversibility of piCRAC-1 seemed to be improved from its
mother compound. Further results from whole-cell patch-
clamping confirmed that the inhibitory effect of piCRAC-1
was at least partially reversible (Figure 3f-g). We further
examined the photoswitchable inhibitory potential of
piCRAC-1 on ICRAC with pulses of UV light. UV irradiation
inhibited ICRAC in HEK293 cells (Figure 3f). Upon the UV
light withdrawal, the amplitudes of ICRAC gradually
increased, indicating partial recovery from cis-piCRAC
inhibition (Figure 3g). This cycle of UV-induced-inhibition
and dark-recovery of ICRAC could be repeated multiple times
(Figure 3f), suggesting that the biological activity of
piCRAC-1 could be controlled temporally with partial
reversibility. To explore spatial control of SOCE with
piCRAC-1, we monitored SOCE responses of HEK293 cells
bathed in piCRAC-1 after exposures to locally-applied UV
flashes. We found that, under the same imaging field, only
cells exposed to locally applied UV light showed marked
reduction in SOCE (Figure 3h-i). Therefore, piCRAC-1
indeed enabled spatial control of SOCE responses. Together,
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Taken together, these results indicated that piCRAC-1
could serve as
a photo-tunable agent to alleviate
pathological conditions associated with hyperactive CRAC
channel activities, such as the Stormorken syndrome.
CONCLUSIONS
In summary, we developed a series of photoswitchable
CRAC channel modulators named as piCRACs through
bioisosteric replacement in heterocyclic N-aryl benzamide
derived SOCE inhibitors. The introduction of azoster moiety
into these compounds enables rapid and reversible
photoconversion between the trans and cis configurations,
thereby allowing piCRACs to exhibit contrasting inhibitory
effects on CRAC channels (with IC50 values varying by ~150-
fold for piCRAC-1 in the dark vs 365 nm). Among them, the
bistable piCRAC-1 effectively inhibits SOCE or ICRAC in a
light-dependent manner, with the affinity and kinetics
comparable to the unmodified prototype GSK-5498A.
PiCRAC-1 permits light-inducible modulation of both CRAC
channels and Ca2+-dependent physiological processes in
cellulo with appropriate spatiotemporal precision. Most
excitingly, we have successfully demonstrated the use
piCRAC-1 as a potential therapeutic agent to optically
intervene in pathological conditions associated with
dysregulated Ca2+ signaling, such as the Stormorken
syndrome arising from over-activating mutations of CRAC
these data firmly established piCRAC-1 as
photoswitchable CRAC channel blocker at the cellular level.
a
Optical control of Stormorken syndrome in zebrafish
model. Moving toward in vivo applications, we further
investigated whether piCRAC-1 could be used as a non-
invasive optical tool to treat CRAC-related channelopathies
in a zebrafish model of Stormorken syndrome13, 52, which
was generated by overexpressing a STIM1 mutant (R304W)
to cause constitutive activation of CRAC channels13. The
resulting zebrafish embryos have been reported to exhibit
Stormorken syndrome-like pathological conditions,
including thrombocytopenia and bleeding13. We injected
STIM1_R304W mRNA into Tg (CD41:EGFP) line with
mature thrombocytes (equivalent of human platelets) and
hematopoietic stem and progenitor cells labeled by GFP53 to
monitor their expression levels in developing embryos
(Figure 4a-b and Figure S9a-b). As anticipated, embryos
expressing STIM1_R304W showed a significant reduction in
GFP signals in the tails that were indicative of
channels13, 52
.
The majority of azologs seem to be more bioactive in their
trans-form45, 54, but cis-active photoswitches are equally
appealing as they are inactive in the dark state and can be
conveniently activated with light. PiCRAC-1 takes action in
its cis-configuration, making it a new addition to the cis-on
photopharmacology toolbox. We anticipate that this
chemical biology toolkit may not only facilitate the study of
structure-function relations of Ca2+ channels but also has
the potential to interrogate a myriad of Ca2+-regulated
signaling events and human disorders associated with
imbalanced Ca2+ homeostasis1-3, 11
.
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Figure 4. Optical control of piCRAC-1 attenuate Stormorken syndrome-like pathological conditions in zebrafish
embryos. a, Whole-body lateral views of Tg (CD41:EGFP) embryos expressing wild type (WT) STIM1 or the activating mutant
R304W in the absence or presence of 5 µM piCRAC-1. The embryos were either shielded from UV light prior to
photostimulation (dark) or exposed to 365 nm light stimulation for 5 min. Boxed areas with overt changes in thrombocyte
progenitors were enlarged and shown on the right. Scale bar, 200 µm. b, Quantification of the GFP signals representing
thrombocyte progenitors in Figure 4a. Three independent experiments were performed, with 25-78 embryos per experiment.
Data were shown as mean ± s.e.m. (**P < 0.01, ****P < 0.0001, two-tailed unpaired Student’s t-test) c-d, Representative images
showing o-dianisidine staining of embryos expressing WT, STIM1_WT or STIM1_R304W (c). The embryos were bathed in the
presence of 10 µM piCRAC-1, either kept in the dark or exposed to UV light (365 nm; 5 min). Quantification of the degrees of
hemorrhage was shown on the bar graph (d). Four independent experiments were performed, with 25-75 embryos per
experiment. Data were shown as mean ± s.e.m. (**P < 0.01, two-tailed unpaired Student’s t-test).
by TLC on glass coated with silica gel 60F254 (0.2-mm
thickness) and visualized by UV irradiation at 254 nm.
EXPERIMENTAL SECTION
Chemical synthesis. The compounds were chemically
Column chromatography was performed using silica gel
(SiO2, 200–300 mesh, Haiyang Chem). The preparation
methods were described in Synthesis.
synthesized by following the general procedures55-56
,
including modifications (synthetic routes shown in
Supporting Information). All reagents and solvents were
purchased from commercial sources (Meryer, Aladdin, J&K
Chemical, Energy Chemical, and Heowns Inc.), and were
used without further purification unless otherwise noted.
Dry tetrahydrofuran (THF) was distilled from
Na/benzophenone before use. All reactions were monitored
Photochemical characteristical. UV–Vis spectra were
recorded using TU-1901 and TU-1905 (PERSEE) with 10-
mm light path cuvettes. All piCRAC compounds were
dissolved at a concentration of 50 µM in acetonitrile
containing 0.5 % DMSO. PiCRAC-1 was dissolved at a
concentration of 10 µM in E3 embryo medium (5 mM NaCl,
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0.17 mM KCl, 10 mM HEPES, 0.33 mM MgSO4, 0.33 mM mM Cs-aspartate, 8 mM MgCl2, 10 mM EGTA, and 10 mM Cs-
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3
4
5
6
7
8
CaCl2) containing 0.1 % DMSO. The cuvettes were directly
irradiated by a fiber-coupled LED (Thorlabs). An initial
spectrum was obtained for each newly-synthesized
compounds (defined as dark PSS). Illumination at 365 nm
was used to initiate the π–π* transition for trans-to-cis
isomerization57. 415 nm, 455 nm, 530 nm LED were further
used to illuminate the sample during UV-Vis spectra
acquisition. Time-resolved UV-Vis spectra of compounds
were recorded at the same conditions by monitoring the
absorbance at a fixed wavelength at different time points.
HEPES (pH 7.2), with the osmotic pressure set at 320 ± 3
mOSM/L. The standard extracellular solution contained the
following (mM): 115 NaCl, 10 CaCl2, 10 TEA, 4.5 KCl, and 5
HEPES, and 10 Glucose (pH = 7.4, adjusted with NaOH;
osmotic pressure = 310 ± 3 mOSM/L). The osmotic pressure
of the solution was measured by
a freezing point
osmometer (OM807, Loser). UV or blue light was remained
on during the entire recording. To examine the reversibility
of piCRAC-1, divalent free (DVF) solution (mM) were used:
150 NaCl, 10 EDTA, 10 TEA, and 5 HEPES, and 10 Glucose
(pH = 7.4, adjusted with NaOH; osmotic pressure ~ 307
mOSM/L)61. The compounds were added to the standard
extracellular solution. After stabilization of whole cell
current, cycles of UV (365 nm) illumination was applied
with various intervals. The HEKA Fitmaster and Matlab
2014b software packages were used for offline data
analysis. Data were presented from at least six individual
cells for each condition.
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The 1H, 19F, and 13C NMR spectra were respectively
obtained on a Bruker Avance AVIII-400 spectrometer at 400
(1H), 376 (19F) and 101 MHz (13C). CDCl3, MeOD-d4, DMSO-
d6 (CIL, Energy Chemical) were used as the solvent. δH, δF,
and δC were expressed in parts per million (p.p.m., δ scale).
1
The H and 19F NMR spectra of 35.4 mM of piCRAC-1 in
MeOD-d4 was acquired by illuminating the sample at 365
nm or 415 nm at the indicated time points. The changes in
spectra were measured at each time point. NMR spectra
were displayed below. HR-ESI-MS was performed by using
an Agilent 6510 Q-TOF mass spectrometer. ESI-MS spectra
Fluorescence Imaging. HeLa cells stably expressing
NFAT1(1-460)-GFP were seeded in a black glass-bottom
96-well microplate with 3000 cells/well and were cultured
in 40 μL Dulbecco’s modified eagle’s medium (DMEM)
supplemented with 10 % FBS in 5 % CO2 at 37 ᵒC. 6 h after
culture, the old media were replaced by fresh media
containing 10 μM compound (diluted from 5 mM stock
dissolved in DMSO). Each compound was added
individually to six wells, which was subdivided into two
groups: UV and Dark. The dark group was shielded from
light stimulation by sealing the wells with aluminum foil.
The UV group was illuminated by UV light at 365 nm for 10
min. 1 μM TG was subsequently added to both groups, with
the mixture incubated for 20 min in the dark or lit
conditions, respectively. Next, the plate was washed with
PBS twice, and the cells were fixed with 4 % PFA for 15 min
at room temperature, then treated with 0.1 % Triton X-100
for 10 min. The cells were subsequently stained with DAPI
(1 mg/mL) for 2 min and followed by washing with PBS
twice prior to imaging.
were obtained by using
instrument.
a Thermo Fisher CQ Fleet
Cell culture. HEK 293 or HEK 293 cells stably expressing
GEM-GECO (Addgene; #32442) or ORAI1/STIM1 were used
as described previously 51, 58-59. HEK cells were cultured in
DMEM (HyClone) supplemented with 10 % FBS (Gemini
Bio-Products), 5 % penicillin and streptomycin (Thermo
Scientific) at 37 °C with 5% CO258. The GEM-GECO stable cell
line was made as previously described58-59, and was
maintained in the presence of 2 μg/mL puromycin (Gibco).
Intracellular Ca2+ imaging. Cytosolic Ca2+ was
conducted using a ZEISS Oberserver-Z1 imaging system
controlled with Zen2 software58-59. All filters or filter sets
were purchased from Semrock (BrightLine). Ca2+ signals
indicated by GEM-GECO fluorescence were acquired using a
customized GEM filter set purchased from Semrock.
Fluorescence signals were acquired with a rate of one frame
per two seconds by setting the excitation at 387 ± 12 nm,
and the emission at 549 ± 21 nm (FGreen) and 465 ± 32 nm
(FBlue). The cytosolic Ca2+ level was presented as the ratio of
FBlue/FGreen51. The imaging solution consisted of 107 mM
NaCl, 7.2 mM KCl, 1.2 mM MgCl2, 11.5 mM glucose, and 20
mM HEPES-NaOH at pH 7.2. A UV light was used as the
external source for photostimulation27. For assays to
demonstrate the spatial control of piCRAC-1 on SOCE,
localized UV light was applied to store-depleted cells in the
center of the view-field by setting the aperture for
excitation light to minimum and keeping UV light on for 2
min. Representative traces from at least three independent
experiments were shown as mean ± s.e.m. as we did
Fluorescence imaging was performed on a Nikon Eclipse
Ti-E microscope (Nikon) equipped with an A1R-A1 confocal
module with LU-N4 laser sources (argon-ion: 405 and
488ꢀnm), and a CFI (chrome-free infinity) plan Apochromat
VC series objective lenses (40 ×). Each well was captured for
four fields with a built-in automated sampling module. The
confocal images were analyzed by the NIS-Elements AR
microscope imaging software (Nikon, NIS-element AR
version 4.5). The cytosolic and nuclear intensities of
NFAT1-GFP signals were statistically analyzed by a semi-
automatic image analysis tool of the NIS-element AR
software. The collected data were further plotted by the
GraphPad Prism software.
previously51, 58, 60
.
Zebrafish experiments. Adults zebrafish was mated
according to standard procedures, and zebrafish embryos
were maintained in E3 embryo medium (5 mM NaCl, 0.17
mM KCl, 10 mM HEPES, 0.33 mM MgSO4, 0.33 mM CaCl2, 60
mg/L penicillin G sodium salt and 100 mg/L streptomycin
sulfate; pH 7.8). To generate capped human mRNA, the
Electrophysiological
measurements.
Whole-cell
recordings were performed using HEK STIM1-ORAI1 dual-
expression stable cells as described previously51. Briefly,
currents were recorded using an EPC-10 (HEKA Elektronik)
at room temperature. The membrane potential was held at
0 mV, and 50 ms voltage ramps ranging from -100 to 100
mV were delivered every 2 s. A 10 mV junction potential
compensation was applied to correct the liquid junction
potential of the pipette solution relative to the extracellular
solution. The intracellular or pipette solution contained 135
cDNAs
encoding
STIM1_WT
(NM_003156)
and
STIM1_R304W were first inserted into a pCS2+ vector by
using the Gateway cloning technology, followed by
linearization with EcoRI digestion. Capped mRNAs were
prepared by using the mMESSAGE mMACHINE SP6
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Transcription Kit (Invitrogen). Capped mRNAs were
dissolved in PBS and microinjected into fertilized eggs at
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AUTHOR INFORMATION
Corresponding Author
100 pg/embryo. Embryos were maintained in 0.2 mM 1-
phenyl-2-thio-urea (Aladdin) to prevent pigment
formation. The Tg (CD41:EGFP) line53 was obtained from
the China Zebrafish Resource Center. The developing
embryos were kept at least 3 dpf. Embryos were then
treated with DMSO (control), piCRAC-1 (in trans- or cis-
configurations) and GSK-5498A at 5 µM for 2.5 h.
*yubinzhou@tamu.edu,
*wyoujun@bnu.edu.cn
*mli@sdu.edu.cn.
Author Contributions
To quantify the number of cells with high levels of GFP
signals, Tg(CD41:EGFP) embryos were anesthetized with
0.016 % tricaine (Bide Pharm) and mounted on glass
culture dish coated with 3 % agarose (BioFr). Images were
acquired using an OLYMPUS SZX16 microscope equipped
with an AxioCam MRm CCD camera and a light source for
EGFP excitation (488 nm). Images were processed by using
the Image J software (NIH). The data were plotted with the
GraphPad Prism software.
# These authors contributed equally.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by grants from the National
Natural Science Foundation of China (81673393, 91954205,
81874308, and 31671492), the Taishan Scholar Program at
Shandong Province, the Qilu Scholar Program at Shandong
University, the Shandong Natural Science Foundation
(ZR2018ZC0233), the Key Research and Development
Project of Shandong Province (2017CXGC1401), the Welch
Foundation (BE-1913-20190330), and the American
Cancer Society (RSG-16-215-01-TBE)
For spontaneous bleeding, embryos stained with 0.6
mg/mL o-dianisidine (J&K Chemical), 0.01M NaOAc (pH
4.6), 0.65% H2O2, and 40% (vol/vol) EtOH in the dark for 15
min. Stained embryos were washed with 1% PBST for 3
times. Freshly prepared 4% paraformaldehyde was used to
fixed at room temperature for 30min and then scored for
bleeding phenotype.
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collected. The total protein content in the supernatant was
determined by the bicinchoninic acid assay. Next, an equal
volume of loading buffer was added, with the mixture boiled
for 4 min for denaturation. The samples were resolved on a
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All animal studies were approved by the Ethics
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ASSOCIATED CONTENT
Supporting Information
The detailed synthetic procedures, spectra, and
additional data were included.
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Ca2+
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trans-piCRAC
cis-piCRAC
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