Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease

Abstract

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Fig. 1. Crystal structures of SARS-CoV-2 PLpro complexes with the three natural compounds.

PLpro domains are depicted in a right-handed architecture, ubiquitin-fold like (blue), thumb (green), palm (salmon pink), and fingers (light orange). Catalytic active site residues Cys 111, His 272, and Asp 286 are represented as sticks and a zinc ion in the fingers domain is shown as a gray sphere. The flexible blocking loop (BL2 loop) that changes conformation in the context of substrate binding is shown in blue. YRL (green spheres), HBA (yellow spheres) and HE9 (pink spheres) compounds bind at the allosteric site that is located about 30 Å apart to the active site. S2 helix involved in the interaction of the ISG15 molecule is indicated. The inset shows an enlarged view of the two compounds HBA and YRL in the binding site.

Fig. 2. Interaction of the ISG15 molecule to PLpro is disrupted by the binding of the three natural compounds.

a Superposition of the crystal structures of SARS-CoV-2 PLpro-C111S in complex with mouse-ISG15 (PDB code 6YVA, ISG15 molecule in blue) with SARS-CoV-2 PLpro+HE9 (PDB code 7OFU, in gray surface representation). The three compounds YRL, HBA, and HE9 are depicted as spheres. b Close-up view of the ISG15 binding site. ISG15 molecule is shown as a cartoon representation (blue) with the interacting residues Ser 22, Met 23, and Glu 27 in sticks. The bound inhibitor compounds (spheres) clearly prevent the binding of the ISG15 molecule to the S2 binding site of PLpro.

Fig. 3. Inhibition of PLpro by the three natural compounds in deISGylation assay with ISG15-Rh substrate.

a IC₅₀ determination was performed with ISG15-Rhodamine as the substrate at a concentration of 250 nM. A gradient concentration of all three compounds YRL, HBA, HE9, and the inhibitor GRL-0617 as a control in the range from 2 to 50 µM was used in the reaction mixture. IC₅₀ values were calculated by fitting the data to a sigmoidal dose-response-inhibition function and are presented in the log scale for interpolation. Individual data points represent the mean of normalized relative fluorescence unit per min ±SD from triplicates. b Summary of the inhibition profiles for the three natural compounds YRL, HBA, HE9, and the control compound GRL-0617 (TTT) obtained from enzyme activity assays and cell line antiviral assays.

Fig. 4. Effect of the natural compounds on SARS-CoV-2 loading in Vero cells.

a The viral titer and cell viability were quantified by qRT-PCR (●) and CellTiter-Glo luminescence method (■), respectively. IC₅₀- and R-squared values for viral titers are shown. IC₅₀-values were calculated by fitting the data to the sigmoidal function as previously described. Compounds concentrations are presented in log scale for interpolation. HE9 was diluted to a stock concentration of 100 mM in DMSO, while YRL was diluted in sterile water to a 50 mM stock concentration. All compounds were stored at −20 °C. Individual data points represent means ± SD from four independent replicates in two biological experiments. Values were plotted in a line graph with error bars displaying standard deviation. b Cell viability in the presence of the three compounds was determined by CellTiter-Glo luminescence method. Individual data points from three independent replicates in three biological experiments. c CPE inhibition was determined by CellTiter-Glo luminescence method. IC₅₀- and R-squared values are shown. IC₅₀-values were calculated by fitting the data to the sigmoidal function. Individual data points represent means ± SD from three independent replicates in one biological experiment. Values were plotted in a line graph with error bars displaying standard deviation.