Enzyme kinetics model for the coronavirus main protease including dimerization and ligand binding

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• The coronavirus main protease (MPro) is crucial for viral replication and a key target for SARS-CoV-2 antiviral therapies.
• Some MPro enzymatic activities show biphasic concentration-response curves (CRCs), with activation at low and inhibition at high ligand concentrations.
• This biphasic behavior is hypothesized to result from ligand-induced dimerization, but lacks quantitative kinetic modeling.

Purpose of the Study:

• To develop and validate a quantitative kinetic model that integrates dimerization and ligand binding for MPro.
• To elucidate the mechanisms underlying the biphasic CRCs of MPro enzymatic activity.
• To characterize the interactions of inhibitors and substrates with MPro dimers.

Main Methods:

• Development of a novel kinetic model incorporating dimerization and ligand binding.
• Global fitting of the model to diverse biochemical and biophysical data using Bayesian regression.
• Analysis of the effects of a reversible covalent inhibitor (GC376) and a fluorescent peptide substrate on MPro activity.

Main Results:

• The developed kinetic model successfully explains the biphasic CRCs of MPro.
• The inhibitor GC376 strongly induces MPro dimerization and exhibits positive cooperativity in binding to the dimer.
• The fluorescent peptide substrate has a minimal impact on dimerization but binds to the dimer with negative cooperativity.

Conclusions:

• The biphasic concentration-response curve of MPro is explained by differential effects of inhibitors and substrates on dimerization and catalytic turnover.
• Inhibitors like GC376 accelerate turnover in the opposite catalytic site of the dimer, contributing to the observed biphasic behavior.
• This quantitative model provides a framework for understanding MPro kinetics and guiding antiviral drug design.