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bioRxiv · 10.64898/2026.03.11.711159

Discovery of Semicarbazone and Thiosemicarbazone Analogs as Competitive SARS-CoV-2 Virus Main Protease (Mpro) Inhibitors

Barreto, L. V.·Lourenco, E. M. G.·da Silva, E. B.·de Godoy, M. O.·Martins, L. C.·Laureano de Souza, M.·Almeida, R. G.·Cunha, V. L. S.·Pires, M. C.·Lavorato, S. N.·de Souza, T. B.·Bretas, A. C. O.·Ottoni, F. M.·Junior, E. N. S.·Oliva, G.·Alves, R. J.·de Oliveira, R. B.·Guido, R. V. C.·Ferreira, R. S.
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Abstract

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 M. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 M and 70 M. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.

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Barreto, L. V., Lourenco, E. M. G., da Silva, E. B., de Godoy, M. O., Martins, L. C., Laureano de Souza, M., Almeida, R. G., Cunha, V. L. S., Pires, M. C., Lavorato, S. N., de Souza, T. B., Bretas, A. C. O., Ottoni, F. M., Junior, E. N. S., Oliva, G., Alves, R. J., de Oliveira, R. B., Guido, R. V. C., Ferreira, R. S.. 2026-03-12. Discovery of Semicarbazone and Thiosemicarbazone Analogs as Competitive SARS-CoV-2 Virus Main Protease (Mpro) Inhibitors. https://doi.org/10.64898/2026.03.11.711159

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