Rational design of an environment-sensitive fluorescent probe for monitoring butyrylcholinesterase activity and its application in Alzheimer's disease

Abstract

In the progression of Alzheimer's disease (AD), the levels of butyrylcholinesterase (BChE) increase significantly, making it a promising biomarker and therapeutic target. To investigate the functional role of BChE in AD pathogenesis, it is essential to develop diagnostic tools capable of visualizing its activity in a structure–function context. Herein, we designed and synthesized a series of fluorescent probes by conjugating environment-sensitive fluorophores to potent BChE inhibitors via rationally designed amine-functionalized linkers. Among these, the optimal probe LY36 integrates a naphthalimide-based fluorophore as the responsive unit and a selective BChE inhibitor pharmacophore as the recognition and therapeutic moiety. LY36 exhibits high selectivity for BChE over acetylcholinesterase (AChE), demonstrating a distinct “turn-on” fluorescence response upon binding to BChE while effectively inhibiting BChE enzymatic activity. Molecular dynamics simulations confirmed a highly stable and specific binding mode within the BChE active site. Furthermore, LY36 displays excellent in situ imaging performance in both cellular and animal models, and successfully visualizes elevated BChE levels. These findings suggest that LY36 holds promise as a simple and effective visualization tool for AD diagnosis, progression monitoring, and therapeutic evaluation.