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.

中文翻译：

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未经验证，不应用于复杂生物系统中的人羧酸酯酶 2 （CES2） 研究荧光素二乙酸酯酶 （FDA）


人羧酸酯酶 2 （CES2） 是一种关键的外源性代谢酶，负责各种药物的水解。由于其在药物代谢中的重要性及其作为癌症生物标志物的潜力，因此需要可靠且可访问的工具来研究 CES2 活性。荧光素二乙酸酯 （FDA） 是一种市售的荧光化合物，已用于研究各种系统中的 CES2 活性，包括纯化的酶、微粒体和组织。体外研究表明，FDA 更喜欢 CES2 水解而不是密切相关的 CES1。然而，FDA 对复杂生物系统中 CES2 相对于 CES1 和其他水解酶的特异性尚未得到严格评估。在这项研究中，我们旨在完成 FDA 作为在复杂生物系统中研究 CES2 的化学工具的表征，并确定 FDA 是否可用于通过结合计算、生化和活细胞成像实验来研究活细胞中的 CES2 活性。我们的结果表明，FDA 不适合在存在其他水解酶（如 HepG2 细胞系）的生物系统中研究 CES2。我们建议在使用 FDA 研究 CES2 时谨慎行事，并强调必须进行严格的验证，以确保复杂生物系统中使用的化学工具在每个实验环境中专门报告预期的分子靶标。