The inhibitory effects of PGG and EGCG against the SARS-CoV-2 3C-like protease

Abstract

The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has led to a global health crisis. Given that the 3 chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition halts viral replication and thus constrains virus spread. Therefore, developing an effective SARS-CoV-2 3CLpro inhibitor to treat COVID-19 is imperative. A fluorescence resonance energy transfer (FRET)-based method was used to assess the proteolytic activity of SARS-CoV-2 3CLpro using intramolecularly quenched fluorogenic peptide substrates corresponding to the cleavage sequence of SARS-CoV-2 3CLpro. Molecular modeling with GEMDOCK was used to simulate the molecular interactions between drugs and the binding pocket of SARS-CoV-2 3CLpro. This study revealed that the V_max of SARS-CoV-2 3CLpro was about 2-fold higher than that of SARS-CoV 3CLpro. Interestingly, the proteolytic activity of SARS-CoV-2 3CLpro is slightly more efficient than that of SARS-CoV 3CLpro. Meanwhile, natural compounds PGG and EGCG showed remarkable inhibitory activity against SARS-CoV-2 3CLpro than against SARS-CoV 3CLpro. In molecular docking, PGG and EGCG strongly interacted with the substrate binding pocket of SARS-CoV-2 3CLpro, forming hydrogen bonds with multiple residues, including the catalytic residues C145 and H41. The activities of PGG and EGCG against SARS-CoV-2 3CLpro demonstrate their inhibition of viral protease activity and highlight their therapeutic potentials for treating SARS-CoV-2 infection.

Keywords: 3CL protease (3CLpro); COVID-19; EGCG; PGG; SARS-CoV-2.

Graphical abstract

Fig. 1

The proteolytic activity of recombinant SARS-CoV-2 3CLpro. (A) Coomassie blue staining and western blot analysis of His-tagged 3CLpro. (B) Edans quantification. The RFU was plotted with respect to each free Edans concentration. (C) 3CLpro catalyzed substrate proteolytic rate at different IQF peptide substrate concentrations. The velocities of SARS-CoV 3CLpro and SARS-CoV-2 3CLpro were determined. (D) Kinetic parameters of SARS-CoV 3CLpro and SARS-CoV-2 3CLpro. From the fitted curve shown in (B), the V_max (black) and K_m (gray) of SARS-CoV-2 3CLpro were characterized, with SARS-CoV 3CLpro shown alongside. (E) Cleavage of IQF peptide substrates by 3CLpro over time. (F) Yield of IQF substrate cleavage by 3CLpro at the 3-hr time point. Data (N = 3) are expressed as the mean ± SEM (∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Screening for candidates of 3CLpro inhibitors. The inhibitory activities of natural compounds against SARS-CoV-2 3CLpro were compared in parallel with SARS-CoV 3CLpro. Changes in the 3CLpro activity were normalized by the corresponding control. The dotted line indicates 50% of 3CLpro activity. Drugs vs. control in the SARS-CoV group: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; Drugs vs. control in the SARS-CoV-2 group: #, p < 0.05; ##, p < 0.01; ###, p < 0.001, using one-way ANOVA post hoc Dunnett’s multiple comparisons tests. Each drug in SARS-CoV vs. in SARS-CoV-2: $, p < 0.05; $$, p < 0.01, using Student’s t-tests.

Fig. 3

Inhibitory effects of PGG and EGCG on SARS-CoV-2 3CLpro. (A–D) Dose-response curves of PGG and EGCG against SARS-CoV-2 3CLpro and SARS-CoV 3CLpro. Changes in the relative SARS-CoV 3CLpro activity (B and D) and the relative SARS-CoV-2 3CLpro activity (C and E) are shown. Data (N = 3) are expressed as the mean ± SEM.

Fig. 4

Molecular modeling of PGG and EGCG in the binding pocket of SARS-CoV-2 3CLpro. (A) Ribbon diagram of SARS-CoV-2 3CLpro with a vacant substrate binding site. S1, S1′, S2 and S4 subsites are labeled (black), along with the catalytic residues (red), H41 and C145. (B–C) PGG (B) and EGCG (C) docking in SARS-CoV-2 3CLpro. Left: PGG (yellow) and EGCG (purple) are depicted with balls and sticks, while the 3D binding pocket is shown in shaded white. The locations of the catalytic residues and subsites are labeled in red and black, respectively. Middle: The main residues (cyan) of SARS-CoV-2 3CLpro around PGG (yellow) or EGCG (purple) in the binding pocket are labeled, including the catalytic residues. Right: Amino acids engaged in the binding of PGG or EGCG are arrayed around the chemical structure, with the involved subsites shaded in beige. Hydrogen bonds are shown as gray dashed lines, while the Van der Waals forces have been omitted from the diagram for simplicity. (D) Interaction energy (kcal/mol) between PGG or EGCG and the residues of SARS-CoV-2 3CLpro. The crucial interacting residues of SARS-CoV-2 3CLpro in the binding models of PGG and EGCG are shown at the top, with catalytic residues highlighted in red. The involved types of forces, hydrogen (H) and/or Van der Waals (V), were indicated above each interaction energy. The strength of the interaction is correlated positively with the brightness of the color; no interaction is shown in black. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)