Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease

Abstract

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC₅₀ value against Jurkat (9.67 μM) and MOTL-4 cells (11.8 μM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.

Keywords: COVID-19; Deubiquitination; Leukemia; SARS-CoV-2 papain-like protease (PLpro); Ubiquitin-specific protease 2 (USP2).

Graphical abstract

Fig. 1

Correlation between the AutoDock Vina docking score and IC₅₀ for 20 known human-USP2 binders. (A) Plot of AD Vina docking score against pIC₅₀. (B) Plot of MMGBSA value (total binding free energy calculated after 5 ns MD simulations) against pIC₅₀.

Fig. 2

Chemical structure of selected compounds from in silico screen. CHEMBL3392809, potent USP2 inhibitor is included as reference.

Fig. 3

Dose-response curve for Z93 and cisplatin against Jurkat and MOLT-4 cell lines.

Fig. 4

Molecular modelling of human USP2 and SARS-CoV-2 papain-like protease (PLpro). (A) Overall structure of the USP12 comprises of finger (green), palm (purple) and thumb (sea green) domains. The catalytic centre (Cys box) is shown in orange between the palm and thumb domains. (B) Surface representation of the structure of the human USP2. (C) The SARS-CoV-2 PLpro monomer (PDB: 6W9C) consists of four domains: beginning from N- to the C-terminus, the extended UBL, the thumb, palm and fingers domain. The active site is circled red. (D) An overlay of SARS-CoV-2 PLpro (salmon) with USP2 (cornflower blue) displaying the conserved USP fold. The catalytic triad residues are magnified.

Fig. 5

(A) The energy plot of top hits between corresponding AD Vina docking score and MMGBSA total binding free energy calculated after a production run of 20 ns MD simulations). The selected compounds are highlighted red. Root-mean-square-deviation of USP2 (B) and SARS-CoV-2 PLpro (C) backbone are estimated over a period of 20 ns with bound Z93 ligand (red). (D) RMSD plot of binding site residues of corresponding proteases. The MD simulated binding modes of Z93 (sticks representation) inside the active site of USP2 (E) and SARS-CoV-2 PLpro (F).