Bidirectional Regulation of Intracellular Enzyme Activity Using Light-Driven Nano-Inhibitors

Photochemical regulation provides precise control over enzyme activities with high spatiotemporal resolution. A promising approach involves anchoring "photoswitches" at enzyme active sites to modulate substrate recognition. However, current methods often require genetic mutations and irreversible enzyme modifications for the site-specific anchoring of "photoswitches", potentially compromising the enzyme activities. Herein, we present a pioneering reversible nano-inhibitor based on molecular imprinting technique for bidirectional regulation of intracellular enzyme activity. The nano-inhibitor employs a molecularly imprinted polymer nanoparticle as its body and azobenzene-modified inhibitors ("photoswitches") as the arms. By using a target enzyme as the molecular template, the nano-inhibitor acquires oriented binding sites on its surface, resulting in a high affinity for the target enzyme and non-covalently firm anchoring of the azobenzene-modified inhibitor to the enzyme active site. Harnessing the reversible isomerization of azobenzene units upon exposure to ultraviolet and visible light, the nano-inhibitor achieves bidirectional enzyme activity regulation by precisely docking and undocking inhibitor at the active site. Notably, this innovative approach enables the facile in situ regulation of intracellular endogenous enzymes, such as carbonic anhydrase. Our results represent a practical and versatile tool for precise enzyme activity regulation in complex intracellular environments.