Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay

Figures

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  Figure 1. The 384-well high-throughput screening assay for SARS-CoV-2 PLpro. The signal to base ratio (S/B) is 11.2, and the calculated Z′ factor is 0.688.

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  Figure 2. HTS and hit validation of SARS-CoV-2 PLpro inhibitors. (A) IC50 values of the screening hits in the FRET-based enzymatic assay, the red line indicates the IC50 = 10 μM. (B) Differential scanning fluorimetry assay of the screening hits in stabilizing the SARS-CoV-2 PLpro. (C) Chemical structures of GRL0617, Jun9-13-7, and Jun9-13-9. (D) Inhibitory activity of Jun9-13-7 and Jun9-13-9 against SARS-CoV-2 PLpro using Ub-AMC and ISG-AMC substrates. (E) Native MS binding assay of Jun9-13-9 and Jun9-13-7 to SARS-CoV-2 PLpro.

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  Figure 3. SAR of SARS-CoV-2 PLpro inhibitors. (A) Analogues of GRL0617 purchased from Enamine. (B) Synthetic compounds designed based on the SAR results. Potent compounds with IC50 values less than 1 μM are highlighted in blue.

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  Figure 4. Development of cell-based FlipGFP assay for the quantification of the cellular activity of SARS-CoV-2 PLPro inhibitors. (A) Design principle for the cell-based FlipGFP assay. (B) Sequence of the flipped GFP β10–11 and cleavage site. (C) FlipGFP-PLPro assay development. 293T cells that were not transfected (Ø), or transfected with FlipGFP-Mpro and SARS-CoV-2 Mpro plasmids, or FlipGFP-Mpro and SARS-CoV-2 PLpro plasmids, or FlipGFP-TEV and SARS-CoV-2 PLpro plasmids, or FlipGFP-PLpro and SARS-CoV-2 Mpro plasmids, or FlipGFP-PLpro plasmid alone, or FlipGFP-PLpro and SARS-CoV-2 PLpro plasmids (details are described in Materials and Methods). Representative images of FlipGFP-PLpro assay with the positive control GRL0617 (D) and the negative control GC376 (E). (F) Dose–response inhibition PLpro in the FlipGFP-PLpro assay by nine compounds selected from the FRET-based enzymatic assay.

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  Figure 5. Antiviral activity of PLpro inhibitors against SARS-CoV-2 in Vero E6 and Caco-2 hACE2 cells. (A) Antiviral activity of PLpro inhibitors against SARS-CoV-2 in Vero E6 cells. (B) Antiviral activity of PLpro inhibitors against SARS-CoV-2 in Caco2-hACE2 cells. Antiviral assay results for compounds Jun9-75-5 and Jun9-87-2 are shown in Figure S4.

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  Figure 6. Correlation of the FlipGFP-PLpro, FRET assay results with the antiviral assay results. (A) Correlation of the results between FlipGFP-PLpro assay and the antiviral assay in Vero E6 cells. (B) Correlation of the results between FlipGFP-PLpro assay and the antiviral assay in Caco2-hACE2 cells. (C) Correlation of the results between FRET assay and the antiviral assay in Vero E6 cells. (D) Correlation of the results between FRET assay and the antiviral assay in Caco2-hACE2 cells.

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  Figure 7. Complex structure of SARS-CoV-2 PLpro with GRL0617. The protein and ligand of the SARS-CoV-2 complex are colored in light green and dark green, respectively. C111* indicates the catalytic cysteine. (A) Binding mode of GRL0617 with an unbiased Fo – Fc map, shown in gray, contoured at 2σ. Hydrogen bonds are shown as red dashed lines. (B) Superimposition with apo SARS-CoV-2 PLpro (salmon, PDB ID 6WZU). Significant rearrangement is observed in the loop comprising residues Asn267-Cys270 upon GRL0617 binding. These movements are indicated with arrows. (C) Superimposition with the terminal five residues of the ubiquitin-like protein ISG15 substrate (orange) from the complex with SARS-COV-2 PLpro (PDB ID 6XA9, showing only the ligand). The atoms of the Leu side chain at the P4 position are labeled. (D) Superimposition with the catalytic mutant C/S111 in complex with GRL0617 (PDB ID 7JIR, light blue and dark blue). OAc indicates an acetate molecule in the C/S111 mutant active site that may have caused the structural differences between the WT and mutant complexes.

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  Figure 8. Molecular dynamics simulations of SARS-CoV-2 PLpro with GRL0617, Jun9-53-2, Jun9-72-2, and Jun9-75-4. In (A), (D), (G), and (J) are shown representative frames from 100 ns MD simulations of the complexes between the GRL0617, Jun9-53-2, Jun9-72-2, and Jun9-75-4 inside SARS-CoV-2 PLPro (protein = light blue ribbon and sticks; the ligand’s carbons are shown in green; nitrogen and oxygen are shown in blue and red, respectively; hydrogen bonding interactions are shown with red dashes). In (B), (E), (H), and (K) are shown stabilizing interactions inside the binding area of PLpro with inhibitors; hydrogen bonding interactions are depicted in blue, π–π stacking in yellow, hydrophobic interactions in brown, and water bridges in green. The binding interactions are considered important when the frequency bar is ≥0.2. In (C), (F), (I), and (L) are shown the RMSD plots of Cα carbons of the protein (blue diagram) and of the ligand (red diagram).