Comparative analysis of production and kinetic assay conditions and parameters for SARS-CoV-2 main protease (Mpro): a systematic review.
- 1University of Brasilia, Institute of Biological Sciences, Biophysics Laboratory, Brasília-DF, 70910-900, Brazil.
- 2University of Brasilia, Institute of Biological Sciences, Biophysics Laboratory, Brasília-DF, 70910-900, Brazil. [email protected].
- 3University of Brasilia, Institute of Biological Sciences, Biophysics Laboratory, Brasília-DF, 70910-900, Brazil. [email protected].
Abstract
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.
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BibTeX
@article{schwartz2026comparative,
title = {{Comparative analysis of production and kinetic assay conditions and parameters for SARS-CoV-2 main protease (Mpro): a systematic review.}},
author = {Matheus Ferroni Schwartz and Aisel Valle Garay and Amanda Araújo Souza and Napoleão Fonseca Valadares and Sonia Maria de Freitas},
journal = {Molecular biology reports},
year = {2026},
month = oct,
doi = {10.1007/s11033-026-12966-6},
url = {https://europepmc.org/article/MED/42961523},
note = {CC BY},
}
RIS
TY - JOUR
TI - Comparative analysis of production and kinetic assay conditions and parameters for SARS-CoV-2 main protease (Mpro): a systematic review.
AU - Matheus Ferroni Schwartz
AU - Aisel Valle Garay
AU - Amanda Araújo Souza
AU - Napoleão Fonseca Valadares
AU - Sonia Maria de Freitas
PY - 2026
DA - 2026/10/30
JO - Molecular biology reports
DO - 10.1007/s11033-026-12966-6
UR - https://europepmc.org/article/MED/42961523
AB - 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.
SN - 0301-4851
SN - 1573-4978
ER -