Validation and Invalidation of SARS-CoV-2 Papain-like Protease Inhibitors

[Image: see text] 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. PL(pro) is involved in the cleavage of viral polyproteins as well as immune modulation by 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 screenings. 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 cell-based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of 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 PL(pro) inhibition as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for potent and specific SARS-CoV-2 PL(pro) inhibitors.