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 main protease (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 papain-like protease (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.