Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

Abstract

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.

Fig. 1. Structure of PLpro from SARS-CoV-2 and comparison with human USP12.

A PLpro model showing secondary structure and “thumb-palm-fingers” architecture, with domains and subdomains labeled and active site residues Cys111/His272/Asp286 represented as spheres, zinc ion is in blue. B Model of human USP12 (PDB id: 5K1B) showing similar fold and domain architecture with active site residues Cys48/His317/Asp333 represented as spheres and zinc ion in blue.

Fig. 2. Biochemical activity assays for compounds 1–7.

Activity assays were performed using substrate shown in Supplementary Fig. 3. Error bars represent the standard error of the mean for n = 3 replicates.

Fig. 3. Virus inhibition in whole cell assay.

A Virus replication activity assays for compounds 1, 4, 5, 6, and 7. Data are mean % percent spike positive cells relative to DMSO treated cells +/− SEM of three biological replicates. B whole cell assay for compound 4. Percent Spike positive cells, n = 100. Scale bar is 100 μM.

Fig. 4. Ligands binding to SARS-CoV-2 PLpro.

A Compound 1 binding to PLpro. B Compound 2 binding to PLpro. C Compound 3 binding to PLpro. D Model of compound 4 (yellow sticks) binding to PLpro. Ligands are shown as green sticks and PLpro is in magenta. Dashed lines show hydrogen bonds, water molecules are shown as blue spheres. In A–C the 2F_o − mF_c electron density maps are shown as a grey mesh, contoured at 1.2 σ. E Compound 2 (green sticks) binds to a groove on the surface of PLpro protein (surface of palm subdomain is in white and thumb subdomain is in light blue) with the active site catalytic triad surface is shown in red in the end of a slender tunnel. Peptide LRGG from ubiquitin structure in complex with SARS PLpro (PDB id: 4MOW) is shown in yellow and peptide positions corresponding P1–P4 sites are marked in white.