A fixation-compatible fluorescent probe for sensitive and specific imaging of exogenous and endogenous esterase activity

Enzymatic ester hydrolysis is a fundamental biochemical reaction in living organisms that plays a pivotal role in cell biology as well as chemical biology and pharmacology/pharmacokinetics during drug development. This makes esterases attractive targets for activating functional molecules such as prodrugs and fluorescent probes. However, most of the currently available fluorescent probes for esterases are optimized for live-cell applications and fail to retain their signals after chemical fixation. Here, we report Mcyp-FL-FM, a fixation-compatible fluorescent probe, to measure esterase activity. Mcyp-FL-FM incorporates an ester moiety that is resistant to spontaneous hydrolysis and is designed to covalently bind to intracellular proteins via quinone-methide chemistry following enzymatic activation. Upon hydrolysis by exogenously expressed porcine liver esterase (PLE), Mcyp-FL-FM exhibited a marked increase in fluorescence intensity and successfully labeled coexisting proteins, enabling the visualization of reporter esterases, including both exogenously expressed marker esterases and protein-protein interaction (PPI) split esterases, in both live and fixed cells. Furthermore, Mcyp-FL-FM was identified as a fluorogenic substrate for endogenous carboxylesterases (CESs), resulting in durable fluorescent labeling in CES-overexpressing cancer cells. These findings highlight Mcyp-FL-FM is a versatile chemical tool for both reporter-based cell labeling and post-fixation visualization of endogenous esterase activity.