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. 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.