Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay

Figures

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  Figure 1. Development of the FlipGFP-3CLpro assay. (A) Sequence of the 3CLpro-FlipGFP construct. T2A, a short peptide sequence derived from viral origins, possesses a unique self-cleavage property. K5, a coiled-coil forming peptide, pairs with its complementary partner peptide E5. They dimerize and induce the alignment of strands into a parallel conformation. (B) Schematic representation of the FlipGFP assay principle. Cleavage of the linker in the β10–11 fragment restores GFP fluorescence. (C) Fluorescence images of HEK293T cells expressing FlipGFP and mutant 3CLpro (C139A) or wild-type 3CLpro. (D) Quantification of fluorescence intensity using a high-content screening system. (E) Dose–response curve of GC376 inhibition of 3CLpro, determined using the FlipGFP assay. Error bars represent mean ± SD (n = 3). Statistical significance was assessed using an unpaired Welch’s t-test (two-sided): *** p ≤ 0.001.

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  Figure 2. Virtual screening of potential 3CLpro protease inhibitors. (A) Workflow for identifying 3CLpro inhibitors. Structure of 3CLpro (PDB ID: 8U1V). The catalytic triad (His30, Glu54, Cys139) is highlighted within the dashed box. (B) Line chart of Max Affinity scores from high-throughput screening, and high-accuracy screening (C). (D) Pairwise plot of Max Affinity scores from HTS and HAS. (E) Line chart of Max Affinity scores from refined screening. (F) Ranking of 17 candidate compounds based on Max Affinity scores.

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  Figure 3. Structure and inhibition activity of hit compounds in the FlipGFP Assay. IC50 curves for (A) Gedatolisib, (B) EGFR-IN-8, (C) Akt inhibitor VIII, (D) Bavdegalutamide, (E) Lifirafenib, (F) TAM-IN-2, (G) GSK1904529A, and (H) IKK 16.

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  Figure 4. Structural interactions of hit compounds with 3CLpro. Three-dimensional visualization of Norovirus 3CLpro in complex with (A) Gedatolisib, (B) EGFR-IN-8, (C) Akt inhibitor VIII, (D) Bavdegalutamide, (E) Lifirafenib, (F) TAM-IN-2, (G) GSK1904529A, and (H) IKK 16, highlighting key interacting residues.

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  Figure 5. Molecular dynamics simulation of hit compound–3CLpro complexes. (A) RMSD plots showing stable trajectories for all eight complexes over 100 ns. (B) Rg plots indicating compactness of the complexes. (C) RMSF plots highlighting regions of flexibility, particularly residues 165–180 and 345–355. (D) SASA plots demonstrating stability of the complexes during simulation.