Determination of SARS-CoV-2 main protease (Mpro) activity based on electrooxidation of the tyrosine residue of a model peptide

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Abstract

The proposed approach for determining the catalytic activity of SARS-CoV-2 main protease (Mpro) is based on the registration of the peak area of the electrochemical oxidation of the tyrosine residue of 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. The steady state kinetic parameters of Mpro towards the model peptide were determined: catalytic constant (kcat) was (3.1 ± 0.1)·10–3 s–1; Michaelis constant (KM) was (358 ± 32)·10–9 M; catalytic efficiency (kcat/KM) was 8659 s–1/M. The limit of detection (LOD) determined for Mpro using the proposed electrochemical system was 44 nM. The proposed approach is a promising tool to search for new Mpro inhibitors as drugs for the treatment of coronavirus infections.

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About the authors

T. A. Filippova

Institute of Biomedical Chemistry; N. I. Pirogov Russian National Research Medical University

Email: alexeykuzikov@gmail.com
Russian Federation, 119121 Moscow; 117513 Moscow

R. A. Masamrekh

Institute of Biomedical Chemistry; N. I. Pirogov Russian National Research Medical University

Email: alexeykuzikov@gmail.com
Russian Federation, 119121 Moscow; 117513 Moscow

T. E. Farafonova

Institute of Biomedical Chemistry

Email: alexeykuzikov@gmail.com
Russian Federation, 119121 Moscow

Yu. Yu. Khudoklinova

N. I. Pirogov Russian National Research Medical University

Email: alexeykuzikov@gmail.com
Russian Federation, 117513 Moscow

V. V. Shumyantseva

Institute of Biomedical Chemistry; N. I. Pirogov Russian National Research Medical University

Email: alexeykuzikov@gmail.com
Russian Federation, 119121 Moscow; 117513 Moscow

S. A. Moshkovskii

N. I. Pirogov Russian National Research Medical University; Max Planck Institute for Multidisciplinary Sciences

Email: alexeykuzikov@gmail.com
Russian Federation, 117513 Moscow; 37077 Göttingen, Germany

A. V. Kuzikov

Institute of Biomedical Chemistry; N. I. Pirogov Russian National Research Medical University

Author for correspondence.
Email: alexeykuzikov@gmail.com
Russian Federation, 119121 Moscow; 117513 Moscow

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Supplementary files

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1. JATS XML
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2. Scheme 1. Proposed mechanism of electrochemical oxidation of tyrosine

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3. Scheme 2. Fragment of a peptide cleaved by Mpro, where P1 is a glutamine residue

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4. Fig. 1. Modification of PGE AuNPs obtained by electrosynthesis and immobilization of the model peptide CGGGAVLQSGY due to the formation of a chemical bond between the mercapto group of cysteine ​​and AuNPs. The arrow shows the cleavage site of the peptide under the action of Mpro

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5. Fig. 2. Cyclic voltammograms of PGE/AuNPs after incubation with different concentrations of the model peptide CGGGAVLQSGY (0–6 mM) for 2 h at 4 °C. Measurements were performed in 60 μl of 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaCl. Scan rate was 50 mV/s. The arrow shows the scanning direction.

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6. Fig. 3. Dependence of the surface concentration (G0) of the electroactive peptide CGGGAVLQSGY on its concentration in the solution applied to PGE/AuNPs with subsequent incubation for 2 h at 4 °C. The average values ​​from at least three replicate experiments are presented ± standard deviations.

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7. Fig. 4. Cyclic voltammograms of PGE/AuNPs with immobilized model peptide CGGGAVLQSGY after incubation with different concentrations of Mpro (0–1500 nM) for 1200 s at 37 °C. Measurements were performed in 60 μl of 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaCl. Scan rate was 50 mV/s. The arrow shows the scanning direction.

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8. Fig. 5. Dependences of the proportion of undigested peptide CGGGAVLQSGY (%) on the time of its incubation (t) with different concentrations of Mpro (0–1500 nM). The average values ​​from at least three replicates of the experiments are presented ± standard deviations.

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9. Fig. 6. Dependence of the proportion of undigested peptide (%) on the logarithm of Mpro concentrations. The average values ​​from at least three replicate experiments are presented ± standard deviations.

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10. Fig. 7. Dependences of the proportion of the cleaved model peptide CGGGAVLQSGY under the action of Mpro (θ) on the incubation time (t) with different enzyme concentrations (50–1500 nM). The average values ​​from at least three replicate experiments are presented ± standard deviations.

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11. Fig. 8. Dependence of the effective constant (keff) on the concentration of Mpro applied to the surface of PGE/AuNPs with the immobilized peptide CGGGAVLQSGY. The average values ​​from at least three replicates of the experiments are presented ± standard deviations.

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