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.