Crystal structure of SARS-CoV-2 papain-like protease

Abstract

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC 50 of 2.2 ± 0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.

An in-silico study on selected organosulfur compounds as potential drugs for SARS-CoV-2 infection via binding multiple drug targets

“…Papain-like protease or PLpro of SARS-CoV-2 shares 83% sequence similarity with SARS-CoV but distant from MERS-CoV PLpro [33] . PLpro is a multifunctional cysteine protease that process viral polyproteins to a functional replicase complex leading to viral spread [34] . PLpro also involved in deubiquitination, de-ISGylation which obstruct the important signaling pathways causing viral invasion of the innate immune response by the expression of type I interferon [35] .…”

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

“…GRL-0617 was the control and this has previously been found to potently inhibit the SARS-CoV and SARS-CoV-2 PL pro in a non-covalent manner (Ratia et al, 2008 ; Freitas et al, 2020 ; Shin et al, 2020 ). The GRL-0617 inhibitor occupies the S3 and S4 pockets of the SARS-CoV and SARS-CoV-2 PL pro (Ratia et al, 2008 ; Gao et al, in press ). Based on the molecular docking results from the current study, GRL-0617 was predominantly surrounded by hydrophobic residues in the SARS-CoV and SARS-CoV-2 crystal structures ( Figure 1 and Supplementary Figures 3 – 6 ) (Ratia et al, 2008 ).…”

“…This ligand was predicted to form inter-atomic contacts with the protein residues and this included D164 in the 6wuu SARS-CoV-2 structure, as well as Q269 in the 7jrn SARS-CoV-2 structure. In the crystal structure determined by Gao et al, GRL-0617, which was the co-crystallized ligand, was found to form hydrogen bonds with these critical residues (Gao et al, in press ). In saying this, D164 and Q269 were found to surround GRL-0617 in the 6xaa, 6w9c, and 6wx4 structures of the SARS-CoV-2 PL pro .…”

“…In terms of SARS-CoV-2, the deubiquitinating and deISGylating activities of the PL pro enzyme have been described (Bosken et al, 2020 ; Klemm et al, 2020 ; Rut et al, 2020 ; Shin et al, 2020 ). GRL-0617 is a naphthalene-based inhibitor that has been found to interfere with the protease, deubiquitinase and deISGylating activities of the SARS-CoV and SARS-CoV-2 PL pro enzymes (Ratia et al, 2008 ; Freitas et al, 2020 ; Gao et al, in press ). The SARS-CoV-2 PL pro has consequently been identified as an attractive drug target and in this study, the deubiquitinase activity of this viral protein was of interest (McClain and Vabret, 2020 ).…”

Drug discovery and development targeting the life cycle of SARS-CoV-2

“…It was reported that GRL-0617 could also inhibit the activity of the SARS-CoV-2 PLpro with an IC 50 value of 2.4 µM and the replication of SARS-CoV-2 in cells with an EC 50 value of 27.6 µM. The crystal structure of the SARS-CoV-2 PLpro in complex with GRL-0617 revealed that GRL-0617 bound to an allosteric site adjacent to the active site through an induced-fit mechanism, where the flexible blocking loop 2, which contains Y268, adopts a closed conformation in order to interact well with the inhibitor 76 , 77 , 78 . The amide bond of GRL-0617 formed hydrogen bonds with the side chain of D164 and the main chain of Q269.…”