Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes

Area of Science:

• Biochemistry
• Pharmacology
• Chemoproteomics

Background:

• Understanding prodrug activation mechanisms is essential for accurate preclinical testing and predicting drug interactions.
• Functional annotation of prodrug-activating enzymes is challenging, hindering drug development.
• Serine hydrolases are a key enzyme family involved in prodrug activation.

Purpose of the Study:

• To develop and apply a modified activity-based protein profiling (ABPP) approach to identify the enzyme responsible for activating the oseltamivir prodrug.
• To investigate the role of carboxylesterase 1 (CES1) in oseltamivir activation.
• To establish methods for analyzing prodrug activation mechanisms in different species.

Main Methods:

• Utilized a substrate-competitive activity-based protein profiling (ABPP) strategy.
• Employed reporter-linked fluorophosphonates for covalent labeling of serine hydrolases.
• Used specific carbamate inhibitors (WWL50 and WWL79) to probe enzyme activity.

Main Results:

• Identified carboxylesterase 1 (CES1) as the primary enzyme activating oseltamivir in human intestinal and liver cell homogenates.
• Demonstrated that oseltamivir competes with fluorophosphonate labeling of CES1, reducing inactivation rates.
• Confirmed CES1 as the main oseltamivir-activating enzyme in human cells using selective inhibitors.

Conclusions:

• A substrate-competitive ABPP approach is effective for identifying prodrug-activating enzymes.
• CES1 is the principal enzyme responsible for activating the oseltamivir prodrug in humans.
• This methodology aids in ester prodrug design and preclinical development by elucidating activation kinetics.