Photoswitchable azopyrazoles Flipping the Switches of Ion Channels

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.