Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MPro(WT)) was assessed using a predominantly monomeric mutant (MPro(M)). Rates of MPro(WT) and MPro(M) catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MPro(WT), to MPro(M) leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites. The binding of a drug targeting the active site of a predominantly monomeric SARS-CoV-2 main protease (MPro(M)) favors an equilibrium shift to MPro(M) dimer formation with two equivalent active sites. These results suggest targeting the monomeric active site and/or the dimer interface to interfere with the conformational rearrangements to active dimer formation as an alternative drug design strategy against MPro.

Automated summary

This study assesses the impact of dimer formation on the catalytic activity of SARS-CoV-2 main protease. The results show that the addition of an inhibitor can increase the catalytic activity, but too high concentrations can decrease the activity. These findings reveal the correlation between dimer formation and enzymatic activity, providing a strategy for designing new drugs.