Fluorescein Diacetate (FDA) Should Not Be Used to Study Human Carboxylesterase 2 (CES2) in Complex Biological Systems Without Validation

Human Carboxylesterase 2 (CES2) is a key xenobiotic metabolism enzyme responsible for the hydrolysis of various drugs. Due to its importance in drug metabolism and its potential as a cancer biomarker, there is a need for reliable and accessible tools to study CES2 activity. Fluorescein Diacetate (FDA) is a commercially available fluorogenic compound that has been employed to study CES2 activity in various systems, including purified enzymes, microsomes, and tissues. 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. In this study, we aimed to complete the characterization of FDA as a chemical tool for studying CES2 in complex biological systems and determine if FDA could be used to study CES2 activity in live cells by combining computational, biochemical, and live cell imaging experiments. Our results indicate that FDA is not suitable for studying CES2 in biological systems where other hydrolases are present like the HepG2 cell line. 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.