Development and Characterization of Fluorescent Chemical Tools to Study Human Carboxylesterase 2 (CES2).

The two major human carboxylesterases (CESs), CES1 and CES2 play a key role in the hydrolysis of xenobiotic esters including aspirin, methylphenidate (Ritalin), and the illicit narcotics cocaine and heroin. CES-mediated metabolism of ester containing drugs can result in their activation or deactivation and enhance clearance. Despite the established role of CESs in drug metabolism, few techniques exist to study their activity in live cells. Fluorescent chemical tools have been developed to be able to expand the methods used to study CES activity, however, many existing fluorescent chemical tools are not sufficiently characterized to be able to confidently study CES activity in live cells. This is particularly true for CES2 where substrate specificity can overlap with other cellular esterases. To address this issue, we report the design and synthesis of new fluorescent chemical tools to study CES2. We subsequently evaluated their specificity for CES2 over CES1 in vitro and in live cells using small molecule inhibitors and shRNA. Overall, our studies resulted in a full characterization of fluorescent chemical tools for studying human CESs and will lead to improved methods for studying the role of CESs in human health and drug metabolism.