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) (IC(50) > 20 µM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 PL(pro) with an IC(50) 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 (CC(50) = 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: see text]