The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

Abstract

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC₅₀ of 2.1 μM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLpro^C111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro.

Fig. 1. 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. 2. Structural and mechanistic analysis of SARS-CoV-2 PLpro^C111S in complex with GRL0617, and in comparison with SARS-CoV and MERS PLpro.

a Surface and cartoon structures of SARS-CoV-2 PLpro in complex with GRL0617 (orange sticks) showing the N-terminal UBL domain (magenta) and C-terminal USP domain (marine). b The binding pocket of GRL0617 in PLpro. The PLpro residues involved in GRL0617 binding are shown as marine sticks. The 2Fo-Fc omit map (contour level = 1.6 σ, shown as purple mesh). c Schematic diagram of SARS-CoV-2 PLpro^C111S/GRL0617 interactions shown in (b). d Comparison of GRL0617-bound (marine) PLpro^C111S and unbound (cyan) PLpro^C111S (PDB ID: 6WRH [10.2210/pdb6wrh/pdb]) structures. Y268 and Q269 on the BL2 loop shifted toward GRL0617 upon binding. GRL0617 shown as orange sticks; the catalytic triad residues (S111 in place of C111, H272, and D286) are shown in yellow; Y268 and Q269 are shown in marine in bound state, and in cyan in unbound state. e Comparisons of the binding sites of SARS-CoV PLpro/GRL0617 (slate sticks, PDB ID: 3E9S [10.2210/pdb3e9s/pdb]), SARS-CoV-2 PLpro^C111S/GRL0617 (marine sticks), and MERS-CoV PLpro (deep teal sticks, PDB ID: 4RNA [10.2210/pdb4rna/pdb]).

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 PLpro^C111S/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. The extensive hydrogen-bond and electrostatic interaction network between ISG15 C-terminus and SARS-CoV-2 PLpro.

a The complex structure of human ISG15 C-UBL domain and SARS-CoV-2 PLpro (PDB 6XA9 [https://doi.org/10.2210/pdb6xa9/pdb]). b Close-up view for interactions of the C-terminus of ISG15 (green) with PLpro (marine), sidechain interactions in black dashed lines, and backbone interactions in red dashed lines. c Surface model showing the superposition of the C-terminus of ISG15 on apo-PLpro (PDB 6W9C [10.2210/pdb6w9c/pdb]). d Surface model showing the C-terminus of ISG15 in PLpro (PDB 6XA9 [10.2210/pdb6xa9/pdb]). e Cartoon model showing the shift of BL2 loop from the apo-state (orange) to bound state (marine), a distance of 4.5 Å was labeled for the shifting of sidechain η-OH of Y268 in PLpro.

Fig. 6. 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 PLpro^C111S 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.