Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis

Abstract

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases M^pro and PL^pro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC₅₀ values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC₅₀ against PL^pro at approximately 10-fold higher micromolar concentrations. Although originally developed as PL^pro inhibitors, the comparison between IC₅₀ and EC₅₀ of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

GRAPHICAL ABSTRACT

Keywords:

• SARS-CoV-2 viral cysteine proteases Mpro and PLpro
• bis(benzylidene)cyclohexanone inhibitors
• in vitro antiviral effect
• enzyme inhibition assays

Acknowledgements

The financial support provided by the Slovak Research and Development Agency (APVV) and the Research Grant Agency of the Ministry of Education and the Slovak Academy of Sciences (VEGA), grants PP-COVID-20–0010 (main source), APVV-19–0376, VEGA-02/0026/22, VEGA 1/0223/20 and APVV-21–0108, are gratefully acknowledged.

For providing access to the SARS-CoV-2 strain Slovakia/SK-BMC5/2020, we thank the EVA GLOBAL consortium (funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 871029, https://www.european-virus-archive.com/virus/sars-cov-2-strain-slovakiask-bmc52020-fd).

Notes

1 Interferon-stimulated gene 15 (ISG15) is a secreted protein that covalently links to lysine residues on newly synthesized proteins. The effects of protein ISGylation involve activation and inhibition of antiviral immunity.

2 LUMO - lowest unoccupied molecular orbital.

3 More negative value of $\mathrm{\Delta }{\mathrm{E}}_{\mathrm{\text{int}}}$ means stronger binding of the ligand to PL^pro.