Discovery of non-covalent rhinovirus 3Cpro inhibitors by molecular docking, in vitro assays, molecular dynamics simulations and DFT analyses

Figures

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  FIGURE 1. The X-ray structure of Rupintrivir covalently bound to HRV-14 3C protein (PDB ID: 1CQQ). The overall structure of HRV-14 3Cpro is shown as a blue cartoon with a transparent surface, and Rupintrivir was displayed in stick representation (cyan). The inset on the right highlights the covalent interaction between Rupintrivir and the catalytic residue CYS-147, demonstrating the irreversible covalent bonding mechanism.

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  FIGURE 2. Chemical structures of some known HRV-14 3Cpro inhibitors.

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  FIGURE 3. Multi-step computational and experimental workflow for identifying HRV-14 3Cpro inhibitors. The process includes: molecular docking and MM/GBSA analysis, ADMET prediction, in vitro enzymatic assays, molecular dynamics simulations, and DFT and IRI calculations.

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  FIGURE 4. ADMET properties and toxicity profiles of the 44 top compounds. The left panel summaries pharmacokinetic properties, including gastrointestinal absorption (ABS), water solubility, blood–brain barrier (BBB) permeability, P-glycoprotein (P-gp) substrate status, and toxicity class. The right panel displays predicted toxicity profiles, including hepatotoxicity, neurotoxicity, respiratory toxicity, carcinogenicity, immunotoxicity, and mutagenicity. Data were classified as “active” or “inactive” based on computational predictions.

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  FIGURE 5. The concentration response curves of S21, S33, S34, and Rupintrivir are measured through enzyme inhibition experiments. The data in the figure is represented as mean ± standard error (mean ± SEM).

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  FIGURE 6. Structural stability and flexibility analysis of HRV-14 3Cpro in the apo form (pink) and in complex with S33 (green) and S43 (blue) over 200 ns of MD simulation. (a) The RMSD for apo-3Cpro, S33 and S43 along with their respective distributions. (b) The SASA for apo-3Cpro, S33 and S43 along with their respective distributions. (c) The SASA for apo-3Cpro, S33 and S43 along with their respective distributions. (d) The RMSF for apo-3Cpro, S33 and S43 along with their respective distributions.

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  FIGURE 7. PCA of HRV-14 3Cpro in apo form and in complex with S33 and S43. (a) Representation of the 2D projections of apo-3Cpro, S33 and S43 complexes conformational changes during the simulation. (b) Projection of the first principal component onto the protein structure to visualize the motion direction of each residue.

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  FIGURE 8. FEL and DCCM analyses of apo-3Cpro, S33, and S43 systems. (a) Free energy landscapes for apo-3Cpro, S33 and S43 systems, projected onto RMSD and radius of gyration (Rg) to visualize conformational stability and metastable states. (b) DCCM of the Cα atoms of apo-3Cpro, S33 and S43 systems, showing correlated (red) and anti-correlated (blue) motions between residue pairs during the simulation.

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  FIGURE 9. Protein ligand interactions in S33 and S43. (a) Three dimensional and two-dimensional interaction diagrams between S33 and ligand. (b) Three dimensional and two-dimensional interaction diagrams of S43 with ligands.

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  FIGURE 10. Energy contribution of residues near the binding site for each complex and total individual energy terms for each complex. The upper panel presents total energy components, including ΔEvdW, ΔEele, ΔEgb, ΔEnp, ΔH, (–TΔS), and ΔG. The lower panels display the residue-level energy contributions near the binding site for each compound.

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  FIGURE 11. Frontier molecular orbital and IRI of compounds S33 and S43. (a) HOMO-LUMO energy plot and electron density surface map of compounds S33 and S43. (b) IRI analyses and IRI vs. sign (λ2) ρ scatter plot of c compounds S33 and S43 at the M06-2X/def2-TZVP level.