Key result
Previously reported SARS-CoV-2 PLpro inhibitors fail to bind or inhibit the protease, invalidating their specificity.
Why the study?
Several compounds were recently identified as SARS-CoV-2 PL pro inhibitors via high-throughput screening, but required validation using target-specific enzymatic, binding, and cell-based assays.
Previously reported SARS-CoV-2 PLpro inhibitors were invalidated using orthogonal assays, demonstrating they are not specific inhibitors.
Prior PLpro inhibitors should not advance as leads; leaves open validated compounds for SARS-CoV-2 antiviral research.
SARS-CoV-2 encodes two viral cysteine proteases, the main protease (M pro ) and the papain-like protease (PL pro ), both of which are validated antiviral drug targets. The PL pro is involved in the cleavage of viral polyproteins as well as immune modulation through removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PL pro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PL pro inhibitors through high-throughput screening. In this study, we aim to validate/invalidate the reported PL pro inhibitors using a combination of PL pro target specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and the cell based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of the PL pro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular inhibition of PL pro as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for specific and potent SARS-CoV-2 PL pro inhibitors.
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Ma et al. (2021) studied SARS-CoV-2. Putative SARS-CoV-2 PLpro inhibitors (e.g., YM155, cryptotanshinone, tanshinone I, 6-thioguanine) vs. GRL0617 (positive control) was evaluated on Enzymatic inhibition (IC50), thermal shift binding, and cellular inhibition (FlipGFP). Previously reported SARS-CoV-2 PLpro inhibitors demonstrated weak enzymatic inhibition, failed to bind or destabilized the protease, and lacked cellular inhibition, invalidating them as specific inhibitors.
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