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

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.