Fluorescein diacetate (FDA) should not be used to study human carboxylesterase 2 (CES2) in complex biological systems...

The utility of fluorogenic chemical tools for investigating processes in biologically complex systems depends on validating their specificity in the same system. This is particularly crucial for tools that report on enzymatic activity, especially when enzymes with similar activity are also present which is often true for serine hydrolases. One such serine hydrolase is human carboxylesterase 2 (CES2). CES2 is a key xenobiotic metabolism enzyme responsible for the hydrolysis of drugs belonging to multiple classes. Due to its importance in drug metabolism and potential as a cancer biomarker, there is a need for reliable and accessible tools to study CES2. Fluorescein DiAcetate (FDA) is a commercially available fluorogenic compound that has been employed to study CES2 activity in various systems. Studies carried out in vitro have shown FDA prefers hydrolysis by CES2 over closely related CES1. However, the specificity of FDA for CES2 over CES1 and other hydrolases in complex biological systems has not been rigorously evaluated. This study aimed to characterize FDA as a tool for studying CES2 in complex biological systems using computational, biochemical, and live cell imaging. Our results indicate that FDA is unsuitable for studying CES2 in systems with multiple hydrolases, like HepG2 cells, but may be used when CES2 is the predominant hydrolase, such as in overexpression models. We advise caution when using FDA to study CES2 and emphasize that rigorous validation is necessary to ensure that chemical tools used in complex biological systems specifically report on the intended molecular target in each experimental context.