Mammalian Esterase Activity: Implications for Peptide Prodrugs.

Area of Science:

• Biochemistry and Chemical Biology
• Enzymology
• Drug Delivery

Background:

• Esterification of peptides and proteins offers a traceless, bioreversible modification to enhance clinical utility.
• Quantifying esterase-catalyzed hydrolysis rates for these esterified biologics is a significant challenge.
• Existing methods lack the sensitivity and continuous monitoring needed for precise kinetic analysis.

Purpose of the Study:

• To develop a continuous Förster resonance energy transfer (FRET) assay for quantifying esterase activity.
• To establish a kinetic model for the release of esterified groups from biologics.
• To facilitate the rational design of effective peptide prodrugs.

Main Methods:

• Development of a continuous FRET assay utilizing a peptidic substrate and the protease Glu-C.
• Validation of the assay with pig liver esterase (PLE) and human carboxylesterases using simple and self-immolative esters.
• Extension of the Michaelis-Menten model to a substrate → intermediate → product (SIP) model incorporating first-order intermediate decay.

Main Results:

• The FRET assay successfully detected esterase activity, with simple esters showing no cleavage.
• The SIP model allowed evaluation of rate constants for PLE-catalyzed ester cleavage (kcat/KM = 1.63 × 10^3 M^-1 s^-1) and quinone methide elimination (kI = 0.00325 s^-1).
• Esterase activity was also detectable in human intestinal S9 fraction, demonstrating in vivo relevance.

Conclusions:

• The developed FRET assay and SIP model provide a robust method for assessing esterase activity and release kinetics of esterified biologics.
• This approach enhances the understanding of peptide prodrug behavior.
• It facilitates the rational design and optimization of novel peptide-based therapeutics.