Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

Abstract

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

The SARS‐CoV‐2 main protease (Mpro): Structure, function, and emerging therapies for COVID‐19

“…In the meantime, tanshinone I and tanshinone IIA were identified as SARS-CoV-2 PL pro inhibitors. [223][224][225] Jin et al 16 found that shikonin exhibits potent inhibition of SARS-CoV-2 3CL pro activity with an IC 50 of 15.75 μM. Moreover, shikonin presented a noncovalent binding configuration with multiple interactions at the S1-S4 subsites of the binding pocket and occupied the space of one water molecule of 3CL pro .…”

“…These results substantiate the use of tanshinone derivatives as antiviral agents. In the meantime, tanshinone I and tanshinone IIA were identified as SARS‐CoV‐2 PL pro inhibitors 223–225 . Jin et al 16 .…”

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

“…Neither of these studies performed additional rescue or validation experiments testing the MSI2-dependence of the Ro phenotype. Another study also found that Ro decreased SARS-CoV-2 nucleocapsid production, however in this case the putative target is the viral papain-like protease 18 . We found that Ro inhibits steroidogenesis doses lower than those that affect cell viability (Figure 1A-D).…”

Drugs targeting the viral enzymes of SARS-CoV-2

“…2). GRL-0617 was one of the first small molecules discovered to act as an inhibitor of PLpro [18][19][20]. The co-crystal structure of SARS-CoV-2 PLpro C111S in complex with GRL-0617 showed that GRL-0617 binds to the catalytic triad in the ubiquitin-specific protease (USP) domain of PLpro [21].…”