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.