Computational and experimental studies of salvianolic acid A targets 3C protease to inhibit enterovirus 71 infection

FIGURE 1. Flowchart of the work. This work consists of three parts: drug screening, inhibition mechanism, and interaction mode.

FIGURE 2. SA is a novel inhibitor of EV71 3Cpro. (A) EV71 3Cpro inhibitor binding pocket. (B) EV71 3Cpro b-factor. (C) Virtual Screening flowchart. (D) Inhibitory effect of candidates (10 μM) on EV71 infection. (E) SA and 3Cpro docking conformation. 3Cpro is shown in cartoon. SA is shown as yellow sticks. NK-1.8K in the crystal structure is shown as white sticks. (F) Fluorescence resonance energy transfer curves of 3Cpro hydrolysis substrates. (G) IC50 of SA inhibition of 3Cpro hydrolysis activity. All the data are means ± SD (n = 3).

FIGURE 3. Antiviral activity of SA on different cell lines. (A–C) Inhibition of EV71 luciferase reporter virus infection of RD cells by SA (5 μM), NK-1.8k (2 μM), and GPP3 (1 μM) at various addition times (0 hpi indicates the time supplied inhibitors and virus simultaneously). (D–F) Quantification of EC50 on RD, HEK-293T, and Vero cell lines. (G–I) Cytotoxicity of SA on RD, HEK-293T, and Vero cell lines. All the data are means ± SD (n = 3).

FIGURE 4. Structural basis of 3Cpro activity and construction of simulation system. (A,D,G) The charge distribution in the initial state of different simulated systems. (B) ESP-mapped molecular VDW surface of E71, H40, C147, and SA. (C) Two protonated structural formulae of histidine in the uncharged state. (E,H) The root means square deviation (RMSD) of the SA heavy atoms. (F,I) The final state of receptors and ligands of the two Holo systems.

FIGURE 5. Structural basis of 3Cpro inhibition by SA. (A,B) Free energy landscape of 3Cpro conformations in Apo and Holo systems. (C) Overlap of the 3Cpro stable state conformations in the Apo and Holo systems. (D) Time courses of the H-bond forming between the SA and E71. (E) H-bond occupancy between the SA and E71. (F) H-bond between SA and E71. (G) ESP-mapped molecular VDW surface of SA and E71. The stick represents SA. The sphere model represents residues. (H) Representative structure of the final structure of Apo and Holo systems. (I) Dihedral angle of H40. (J) The absolute value of dihedral of H40 in Apo and Holo systems at 300 ns (n = 3; **p < 0.01, Apo vs. Holo).

FIGURE 6. SA and 3Cpro binding mechanism. (A) The binding free energy decomposition of SA and 3Cpro. (B) The binding free energy value mapping diagram (Where red indicates the regions contributing to ligand binding). (C) Schematic diagram of ligand reaction coordinates. (D) Free energy landscape of the SA binding mode. (E) and (F) representations of the binding mode of the SA and 3Cpro in their lowest binding energy conformation.

FIGURE 7. SA and 3Cpro dissociation mechanism. (A) The initial state conformation of steered molecular dynamics (SMD). (B) Schematic diagram of SMD reaction coordinates. (C) PMF profiles along the reactions coordinate for SA (n = 30). (D) Representative structures in the dissociation process of the SA from three pockets during SMD simulations. Dissociation path (DP). (E) Illustrations of the molecular mechanism of SA binding to 3Cpro and inhibition of its activity.