Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

Abstract

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

Propolis, Bee Honey, and Their Components Protect against Coronavirus Disease 2019 (COVID-19): A Review of In Silico, In Vitro, and Clinical Studies

“…Efforts to control the pandemic of COVID-19 are directed toward inhibiting viral endocytosis and replication, preventing and repairing viral-induced tissue damage, and boosting host immunity. Molecular docking studies suggest that some flavonoids may play dual roles in targeting SARS-CoV-2, e.g., inhibiting TMPRSS2 [ 5 ], ACE-II [ 24 ], S protein, and NSPs [ 25 , 34 , 47 , 51 , 80 , 81 ] ( Table 1 ). Therefore, these flavonoids may lower the chances of viral entry into host cells and decrease viral load and inflammatory reaction following infection.…”

“…Rutin exhibited the highest inhibitory potential (−59.9 kcal/mol) relative to GRL-0617 (−36.3 kcal/mol), which was confirmed by time-dependent docking involving 100 μs molecular dynamics simulation trajectory of the PLpro as well as in vitro investigations (detailed below) [ 34 ]. Docking rutin in the naphthalene-inhibitor binding site of PLpro caused displacement of ubiquitin in a conformation that involves dysfunctional DUB activity [ 80 ].…”

“…A derivative of podophyllotoxin compounds known as sulabiroins A inhibited Mpro at an affinity of −7.6 kcal/mol through a hydrophobic interaction with the catalytic site His41 [ 42 ]. Altogether, flavonoids may inhibit NSPs of SARS-CoV-2 and interrupt viral replication [ 25 , 34 , 42 , 47 , 51 , 80 , 81 , 86 ].…”

“…Two studies [ 34 , 80 ] compared PLpro inhibitory activity of rutin (3.1–200 μM) with that of GRL-0167 (100 μM) and hypericin (50 and 100 μM) in vitro using the proprietary papain-like protease (SARS-CoV-2 Assay Kit) for enzymatic inhibition assay. The percentage of inhibition of PLpro protease activity was 94% by GRL-0167, 97% and 87% by hypericin (50 and 100 μM, respectively), and 94% by rutin (100 μM) [ 34 ].…”

“…The percentage of inhibition of PLpro protease activity was 94% by GRL-0167, 97% and 87% by hypericin (50 and 100 μM, respectively), and 94% by rutin (100 μM) [ 34 ]. GRL-0167, hypericin, and rutin, in order, inhibited DUB activity in another study [ 80 ].…”