Invalidation of dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) as SARS-CoV-2 main protease inhibitors and the discovery of PGG as a papain-like protease inhibitor

The COVID-19 pandemic spurred a broad interest in antiviral drug discovery. The SARS-CoV-2 main protease (M^pro) and papain-like protease (PL^pro) are attractive antiviral drug targets given their vital roles in viral replication and modulation of host immune response. Structurally disparate compounds were reported as M^pro and PL^pro inhibitors from either drug repurposing or rational design. Two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 M^pro inhibitors. With our continuous interest in studying the mechanism of inhibition and resistance of M^pro inhibitors, we report herein our independent validation/invalidation of these two natural products. Our FRET-based enzymatic assay showed that neither dieckol nor PGG inhibited SARS-CoV-2 M^pro (IC₅₀ > 20 µM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 PL^pro with an IC₅₀ of 3.90 µM. The binding of PGG to PL^pro was further confirmed in the thermal shift assay. However, PGG was cytotoxic in 293T-ACE2 cells (CC₅₀ = 7.7 µM), so its intracellular PL^pro inhibitory activity could not be quantified by the cell-based Flip-GFP PL^pro assay. In addition, we also invalidated ebselen, disulfiram, carmofur, PX12, and tideglusib as SARS-CoV-2 PL^pro inhibitors using the Flip-GFP assay. Overall, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PL^pro inhibitor might worth further pursuing. [Figure not available: see fulltext.]