Fig 2: Inhibitory activity of GRL0617 against SARS-CoV-2 PLpro.a The inhibitory activity of GRL0617 and compound 6 against PLpro was measured using the peptide RLRGG-AMC as a substrate. IC50 was presented as mean ± SEM, n = 3 independent experiments. b In-cell deISGylating (left) and deubiquitinating (right) activities of PLpro, HEK293T cells were transfected for 24 h with plasmids encoding GFP-PLpro, ISG15, and E1(Ube1L)/E2(UbcH8)/E3(HECR5) enzymes, alone or in combination. Cells were treated for an additional 24 h with indicated concentrations of GRL0617. Cell lysates were subjected to immunoblotting with anti-ubiquitin, anti-ISG15, and anti-GFP antibodies. GAPDH served as a loading control. A representative from three independent experiments is shown. c HEK293T cells were treated with or without 500 U/mL interferon β (IFN-β) for 48 h. The cell extracts were incubated with purified recombinant PLpro (100 nM) and indicated concentrations of GRL0617 for 60 min at 37 °C, followed by immunoblotting analysis with anti-ISG15. A representative from three independent experiments is shown. d Antiviral activity of GRL0617 on SARS-CoV-2 and the cytotoxicity of GRL0617 on Vero E6 cells. Vero E6 cells were infected with SARS-CoV-2 using a multiplicity-of-infection (MOI) of 0.01. The quantification of absolute viral RNA copies (per mL) in the supernatant at 48 h post-infection was determined by qRT-PCR analysis. The cytotoxicity of GRL0617 on Vero E6 cells was measured using CCK8. All data are shown as mean ± SEM, n = 3 independent experiments. e EC50 was presented as mean ± SEM, n = 3 independent experiments, the cytopathic effect (CPE) analysis revealed an EC50 of 21 ± 2 μM.

Fig 3: NMR studies show that GRL0617 blocks the binding of ISG15 to SARS-CoV-2 PLpro.a 1H,15N-HSQC spectrum of 15N-ISG15. b HSQC spectrum of 15N-ISG15 (0.1 mM) and 0.15 mM PLpro. Peak broadening and peak intensity loss indicate binding of ISG15 to PLpro. c HSQC spectrum of 15N-ISG15 (0.1 mM) in the mixture of 0.15 mM PLpro and 0.25 mM GRL0617. Recovery of peak intensity suggests that GRL0617 binds to PLpro and displaces ISG15. d SARS-CoV-2 PLproC111S/GRL0617 structure in cartoon model. e ISG15 in the complex structure of human ISG15 C-UBL-PA/SARS-CoV-2 PLpro (PDB 6XA9 [10.2210/pdb6xa9/pdb]) was superimposed on (d) showing steric clash of GRL0617 with the C-terminal tail of ISG15. f Ub in the complex structure of UbPA/SARS-CoV-2 PLpro (PDB 6XAA [10.2210/pdb6xaa/pdb]) was superimposed on (d), showing steric clash of GRL0617 with the C-terminal tail of Ub.

Fig 4: The C-terminal tail of ISG15 is dominant for binding SARS-CoV-2 PLpro.a Superposition of the 1H,15N-HSQC spectra of 15N-ISG15-FL (0.1 mM) and the mixture of 15N-ISG15 (0.1 mM)/0.15 mM SARS-CoV-2 PLpro. Massive peak broadening and peak intensity loss indicate binding of ISG15-FL to PLpro. b Superposition of the 1H,15N-HSQC spectra of 15N-ISG15-ΔC6 (0.1 mM) and the mixture of 15N-ISG15-ΔC6 (0.1 mM)/SARS-CoV-2 PLpro (0.15 mM). Negligible peak perturbation indicates minimum binding of ISG15-ΔC6 to PLpro. c ITC measurement for the binding of SARS-CoV-2 with ISG15-FL (black) and ISG15-ΔC6 (blue), respectively. d Structural analysis of the complex structure of ISG15/SARS-CoV-2 PLpro (PDB 6XA9 [10.2210/pdb6xa9/pdb]) shows that the sidechains (black dashed lines) of D164 and E167 of SARS-CoV-2 PLpro are involved in the binding with the C-terminal tail of ISG15, other interactions are involved with the backbone (red dashed lines). e DUB cleavage assay using Ub-AMC, ISG15-AMC, or peptide-AMC shows that D164A and E167A mutants have impaired enzyme activity compared with wild-type SARS-CoV-2 PLpro. Data are presented as mean ± SEM, n = 3 independent experiments.

Fig 5: GRL0617 occupies the same binding pocket in the USP domain as other USP7 and USP14 inhibitors.a Binding pocket of GRL0617 (orange) in SARS-CoV-2 PLproC111S mutant (PDB 7CJM [10.2210/pdb7cjm/pdb]), the S111 was labeled in place of C111. b Binding pocket of FT671 (PDB 5NGE [10.2210/pdb5gne/pdb], pink), XL188 (PDB 5VS6 [10.2210/pdb5vs6/pdb], cyan), and ALM2 (PDB 5N9R [10.2210/pdb5n9r/pdb], purple) in USP7 with active Cys223 label. c Binding pocket of IU1 (yellow) in USP14 (PDB 6IIK [10.2210/pdb6iik/pdb]) with active Cys114 label; the BL1 and BL2 loops were labeled in (a, b, c). d Superposition of GRL0617, USP7, and USP14 in the ISG15 C-terminus binding pocket in SARS-CoV-2 PLpro.