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

Abstract

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. Graphical abstract

Disulfiram: Mechanisms, Applications, and Challenges

“…These proteases and many others contain catalytic cysteine residues that are crucial for enzymatic function [ 121 , 122 ]. Because disulfiram is known to bind and covalently modify these residues, researchers have investigated its ability to inhibit coronavirus activity [ 20 , 121 , 123 , 124 , 125 , 126 , 127 ]. In 2018, one in vitro study found that disulfiram inhibited proteases in the MERS-CoV and SARS-CoV-1 coronavirus strains [ 20 ].…”

“…The evidence is mixed as to whether disulfiram inhibits papain-like protease (PL pro ), an essential enzyme involved in the generation of the replicase complex. One 2020 study found that disulfiram inhibits SARS-CoV-2 by binding to cysteine in PL pro [ 126 ], whereas a 2022 study found that disulfiram had no inhibitory activity against PL pro [ 127 ].…”

A Patent Review on SARS Coronavirus Papain‐Like Protease (PLpro) Inhibitors

“…Hence, we believe an investigation into its ability to inhibit human cysteine proteases is warranted as this will likely result in adverse side‐effects. Subsequently, disulfiram was found to be inactive (IC 50 >10 μM) in a cellular SARS‐CoV‐2 PL pro inhibition assay by Rutgers University and The University of Arizona researchers in a 2022 academic paper [88] . Based on this, we opine disulfiram lacks sufficient potency for further development as an antiviral for treating COVID‐19.…”

“…Hence, further investigation into its ability to inhibit human cysteine proteases is warranted as this may cause adverse side‐effects. Interestingly, tideglusib was later reported to be inactive (IC 50 >60 μM) in a cell‐based SARS‐CoV‐2 PL pro inhibition assay conducted by Rutgers University and The University of Arizona researchers in a 2022 journal publication [88] …”

COVID-19 therapeutics: Small-molecule drug development targeting SARS-CoV-2 main protease

“…The target selectivity and cellular target engagement of purported M pro inhibitors were addressed by several recent investigations using a variety of secondary assays. 61 , 111 , 112 The compounds can be tested for their ability to inhibit unrelated cysteine proteases, such as the viral EV-A71 2A pro and EV-D68 2A pro , the host cathepsins B, L, and K, caspases, and calpains I, II, and III, among others, to determine their ability to specifically target M pro . Most of the time, substances that block numerous cysteine proteases are promiscuous substances that might act by causing protein aggregation, redox cycling, or alkylating the catalytic cysteine residue Cys145.…”