Determination of SARS-CoV-2 Main Protease (M^pro) Activity Based on Electrooxidation of Tyrosine Residue of a Model Peptide

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Tatiana A. Filippova^1,2, Rami A. Masamrekh^1,2, Tatiana E. Farafonova¹, Yulia Yu. Khudoklinova², Victoria V. Shumyantseva^1,2, Sergei A. Moshkovskii^2,3, Alexey V. Kuzikov^1,2,a*

¹Institute of Biomedical Chemistry, 119121 Moscow, Russia

²Pirogov Russian National Research Medical University, 117513 Moscow, Russia

³Max Planck Institute for Interdisciplinary Research, Göttingen, 37077, Germany

^* To whom correspondence should be addressed.

Received: October 23, 2024; Revised: December 6, 2024; Accepted: December 10, 2024

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The proposed approach for determining catalytic activity of the SARS-CoV-2 main protease (M^pro) is based on registration of the peak area of electrochemical oxidation of tyrosine residue in the model peptide substrate CGGGAVLQSGY immobilized on the surface of a graphite screen-printed electrode (SPE) modified with gold nanoparticles (AuNP). The AuNP were obtained by electrosynthesis. Steady state kinetic parameters of M^pro in the reaction with the model peptide were determined: catalytic constant (k_cat) was (3.1 ± 0.1) × 10⁻³ s⁻¹; Michaelis constant (K_M) was (358 ± 32) × 10⁻⁹ M; catalytic efficiency (k_cat/K_M) was 8659 s⁻¹/M. The limit of detection (LOD) determined for M^pro using the proposed electrochemical system was 44 nM. The proposed approach is a promising tool to search for new M^pro inhibitors as drugs for treatment of coronavirus infections.

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KEY WORDS: M^pro protease, tyrosine electrooxidation, screen-printed electrodes, gold nanoparticles, model peptide

DOI: 10.1134/S0006297924603836

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