The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Open access for this article was enabled by the participation of The Francis Crick Institute in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with JISC. https://creativecommons.org/licenses/by/4.0/

Cite as

Lim, C., Tan, K., Wu, M., Ulferts, R., Armstrong, L., Ozono, E., Drury, L., Milligan, J., Zeisner, T., Zeng, J., Weissmann, F., Canal, B., Bineva-Todd, G., Howell, M., O'Reilly, N., Beale, R., Kulathu, Y., Labib, K. & Diffley, J. 2021, 'Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease', Biochemical Journal, 478(13), pp. 2517-2531. https://doi.org/10.1042/BCJ20210244

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Last updated: 16 June 2022
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