Activatable Photosensitizer for Targeted Ablation of lacZ-Positive Cells with Single-Cell Resolution

Abstract

To achieve highly selective ablation of lacZ-positive cells in a biological milieu in vivo, we developed an activatable photosensitizer, SPiDER-killer-βGal, targeted to β-galactosidase encoded by the lacZ reporter gene. Hydrolysis of SPiDER-killer-βGal by β-galactosidase simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles, so that the phototoxic products generated by light irradiation are trapped inside the lacZ-positive cells. The combination of SPiDER-killer-βGal and light irradiation specifically killed lacZ-positive cells in coculture with cells without lacZ expression. Furthermore, β-galactosidase-expressing cells in the posterior region of cultured Drosophila wing discs and in pupal notum of live Drosophila pupae were selectively killed with single-cell resolution. This photosensitizer should be useful for specific ablation of targeted cells in living organisms, for example, to investigate cellular functions in complex networks.

Fluorescence imaging of drug target proteins using chemical probes

“…The same strategy is applicable to target ß -galactosidase based on a rhodol scaffold [ [33] , [34] , [35] ]. Furthermore, the selenium-substituted rhodol and rhodamine are active as potent photosensitizers for enzyme-activated cell ablation [ 36 , 37 ]. The ring-switching mechanism favors high-contrast imaging with a low background because the ‘ring-closed’ form principally emits no fluorescence.…”

“…The future work on enzymatically activatable probes is likely to focus on improving the imaging resolution by enhanced enzyme specificity, increased reaction kinetics, and limited diffusion upon activation (e.g., cell-trappable probes [ 37 , 65 , 66 ]). For targeted probes, selective visualization of intracellular proteins remains challenging.…”

A self-immobilizing near-infrared fluorogenic probe for sensitive imaging of extracellular enzyme activity in vivo

“…Incorporating a fluorescent probe and the chemistry of QM, Withers and co-workers developed a fluorogenic and self-immobilizing probe to image the β-glucuronidase activity in an Arabidopsis thaliana plant line. 10 While this approach has been further explored in the fluorescent probes of various enzymes and promising results have been reported in cell-based imaging, 11 visualization of enzymatic transformations in live animals with these reagents has not been realized, mainly due to short absorption and emission wavelengths. The key characteristics of ideal fluorescent probes for in vivo imaging include a high fluorescence turn-on ratio, brightness, and bio-stability, as well as absorption and emission wavelengths in the NIR region and high retention of activated fluorophore.…”

BSJ 2019 “Single-cell PRESTO” session

“…Among a variety of their synthesized probes, she mainly introduced two new molecules. These molecules target lacZ positive cells and make the cells fluorescent (Ito et al 2018) or able to be ablated (Chiba et al 2019) with single-cell resolution. She demonstrated that their probes are applicable from in vitro cell cultures to in vivo models.…”