Comparative analysis of production and kinetic assay conditions and parameters for SARS-CoV-2 main protease (Mpro): a systematic review

The main protease (Mpro) of SARS-CoV-2 plays a crucial role in the viral replication cycle and is a key target for COVID-19 antiviral drugs. In this review, we conducted an extensive bibliographic search on the main challenges and experimental limitations reported in the production and biochemical characterization of the Mpro. A large variability in the kinetic parameters was revealed, which compromises comparisons across various studies and the evaluation of inhibitor efficacy. The studies indicate that these discrepancies are mainly associated with experimental limitations, such as low substrate solubility, optical irregularity due to the inner-filter effect, and variations in the oligomeric state of Mpro. This review summarizes and discusses the different strategies for Mpro expression in Escherichia coli and purification, with emphasis on obtaining an N-terminal tag-free enzyme, essential for homodimerization into a highly active form for structural and functional characterization. The main FRET-based substrates used in enzymatic assays were compiled and analyzed, as well as alternative methodologies for assessing Mpro enzymatic activity. Furthermore, the effects of assay conditions, such as buffer composition, pH, and the presence of reducing agents were addressed. The intrinsic structural features of Mpro that impact its catalytic function are discussed, including functional asymmetry in substrate binding and the positive allosteric cooperation between monomers. Overall, this review provides a critical and integrated perspective on the main experimental limitations in the production and characterization of Mpro, applicable to other proteases, contributing to the standardization of approaches and advances in the development of more effective inhibitors.