Directionally Modified Fluorophores for Super-Resolution Imaging of Target Enzymes: A Case Study with Carboxylesterases

In the need for improving the labeling
quality of super-resolution
imaging, multifarious fluorescent labeling strategies have sprang
up. Among them, a small molecule inhibitor-probe (SMI-probe) shows
its advancement in fine mapping due to its smaller size and its specific
binding to a specific site. Herein, we report a novel protocol of
mechanism-guided directional modification of fluorophores into fluorescent
inhibitors for enzyme targeting, which could half the size of the
SMI-probe. To confirm the feasibility of the strategy, carboxylesterase
(hCE) inhibitors are designed and developed. Among the constructed
molecule candidates, NIC-4 inhibited both isoforms of hCE1 and hCE2,
with IC50 values of 4.56 and 4.11 μM. The CE-targeting
specificity of NIC-4 was confirmed by colocalizing with an immunofluorescent
probe in fixed-cell confocal imaging. Moreover, NIC-4 was used in
live-cell super-resolution microscopy, which indicates dotlike structures
instead of the larger staining with the immunofluorescent probe. Moreover,
it enables the real-time tracking of dynamic flow of carboxylesterases
in live cells.

为提升超分辨率成像（super-resolution imaging）的标记质量，各类荧光标记策略应运而生。其中，小分子抑制剂探针（small molecule inhibitor-probe，SMI-probe）凭借体积更小、可特异性结合特定位点的优势，在精细定位领域展现出卓越性能。在此，我们报道一种基于机制导向的定向修饰策略，将荧光团改造为靶向酶的荧光抑制剂，可将SMI-probe的体积缩减一半。为验证该策略的可行性，我们设计并开发了针对羧酸酯酶（carboxylesterase，hCE）的抑制剂。在所构建的分子候选物中，NIC-4可同时抑制hCE1与hCE2两种亚型，其半抑制浓度（IC50）分别为4.56 μM与4.11 μM。通过与免疫荧光探针在固定细胞共聚焦成像（fixed-cell confocal imaging）中共定位，验证了NIC-4的羧酸酯酶靶向特异性。此外，将NIC-4应用于活细胞超分辨率显微镜成像后，观察到其呈现点状结构，而非免疫荧光探针所产生的弥散性较强的染色信号。同时，该探针可实现活细胞内羧酸酯酶动态流动的实时追踪。