Abnormally
activated Ca2+ channels are related to many human diseases,
including Stormorken Syndrome, which makes Ca2+ release-activated Ca2+
(CRAC) channel a very promising therapeutic target. Several small molecule therapeutics
targeting CRAC channels have been developed, including the GSKs series
and Synta 66. Those compounds have relatively high specificity. Meanwhile,
a controllable system that can be activated with a switch can probably serve as
a convenient tool for further related research.
Photoswitchable
chemistry has been applied to a lot of bioactive targets such as ion channels,
receptors enzymes and nucleic acids. Recently, the Li group developed CRAC
channel inhibitors that can be turned ‘on’ and ‘off’ by UV-light exposure.
Scheme 1. Converting
CRAC channel inhibitors, GSKs, into photoswitchable derivatives, piCRACs.
Starting
from the well-established GSK-based CRAC inhibitor (Scheme 1), the
authors developed a series of nitrosaniline analogs (piCRACs) according to the
rule of bioisosteric replacement, substituting amide groups with photoisomerizable
azo-groups.
Before
jumping into in vivo studies, they first tested the photochemical
properties of piCRACs in vitro.
They
measured the absorption spectra of piCRACs and determined that the best
photoconversions were achieved with excitations at 365 nm for E-Z conversion
and 415 nm for Z-E conversion, respectively (Fig. 1b, 1c). To
test biological activities of piCRACs, the authors used SOCE and NFAT as
two independent readouts to determine the responsiveness of photoconversion and
the ability of inhibiting CRAC channels. Among those, piCRAC-1 showed the most potential. They also attempted to optimize
piCRAC-1 to increase the photoconversion rate, however at the price of
losing the biological activity. Therefore, all the following in vitro
and in vivo tests was performed with piCRAC-1.
Figure
1. a) Chemical structures of GSK-5498A; b) Chemical structure of piCRAC-1;
c) UV-Vis spectra of piCRAC-1 (50 mM)
at different irradiation wavelengths in acetonitrile containing 0.5% DMSO.
PiCRAC-1 prefers a low-energy
trans configuration primarily. It can achieve 49 % of conversion to the cis
state upon 3-5 min UV (365 nm) illumination. The cis configuration is
very stable with a half-life of 255.9 days at room temperature. For the
inhibition of SOCE activity, IC50 of piCRAC-1 plummeted down dramatically from 73.2 µM to 0.5 µM after 5
min UV illumination (Fig 2. Left), which had better potency compared with
the lead compound GSK-5498A (3.1 µM). Moreover, the activity of
SOCE could be restored after blue light treatment for 15 min, demonstrating the
temporally controlling capability of PiCRAC-1.
Figure
2. Left: 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). Right: 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.
The
suppressive effect on the nuclear accumulation of downstream effector NFAT is
also notable by examining the subcellular localization of NFAT (Fig 2.
Right). The specificity of piCRAC-1
is also comparable to its prototype GSK-5498A, showing no inhibition
over other Ca2+ channels at relatively high concentrations.
Furthermore,
this compound is also tested in a live zebrafish model. Zebrafish embryos were
treated with piCRAC-1 with and
without UV exposure, and it is found that piCRAC-1
markedly rescued the related pathological conditions associated with CRAC
channel aberrant activities.
Overall,
this paper demonstrated that azo-containing piCRAC-1 enables rapid and
reversible activation of SOCE and can be a useful research tool to tune the
activities of ion channels.
Reference:
1.
J. Am. Chem. Soc. 2020 Apr 24. doi:
10.1021/jacs.0c02949.
Woo! You guys made wise use of social distancing time...
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