Design, synthesis and activity evaluation of dual-target inhibitors against papain-like and main proteases of porcine epidemic diarrhea virus

Figures

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  Fig. 1. A novel dual-target inhibitor structure against Mpro and PLpro was obtained by drug merge.

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  Fig. 2. Analysis of the binding pocket of Mpro and PLpro. (a) Residues and pockets at the PEDV Mpro catalytic site (PDB ID: 6 W81). (b) Identification of potential ligand binding sites I−Ⅲ (PDB: 7MC9).

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  Scheme 1. Synthesis of compounds. Reagents and conditions: (a) CH3COOH, Br2, 50℃; (b) R-COCl, Et3N, DCM, rt, overnight; (c) BBr3, DCM, −78℃ to rt, overnight.

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  Fig. 3. Antiviral activity of compounds against the in Vero-E6 cells. (a) The inhibition ratio of e2, e9, f2-f6, f9, f12-f13, f15, f20-f21 and f23-f24 on the expression of PEDV N gene in Vero-E6 cells. (b) The IFA analysis of the inhibitory effects of f2 on the expression of N protein in PEDV infected Vero-E6 cells.Kinetic analysis of f2 inhibited of (c) Mpro and (d) PLpro activity. c (Mpro) = 100 nM, c (f2) = 0, 1.25, 5, and 10 μM. c (PLpro) = 2 μM, c (f2) = 0, 1.25, 2.5, and 5 μM.

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  Fig. 4. Molecular docking study of f2. (a) Best predicted binding mode of f2 at Mpro binding site. (b) Best predicted binding mode of f2 at PLpro binding site.

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  Fig. 5. Antiviral and anti-inflammatory activities of f2 in IPEC-J2 cells. (a) The inhibition ratio of f2 on the expression of PEDV N gene in IPEC-J2 cells. (b) Cytotoxicity of f2 on IPEC-J2 cells. (c) The IFA analysis of the inhibitory effects of f2 on the expression of N protein in PEDV infected IPEC-J2 cells. (d-f) Total RNA expression levels were extracted from cell lysates, and (d) IL-1β, (e) TNF-α and (f) IL-6 in IPEC-J2 cells were determined by RT-qPCR analysis. Data were expressed as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.01 compared with the PEDV group.

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  Fig. 6. The effect of f2 on the signaling pathway related to inflammatory factors. (a) The protein expression levels of TLR2, p-PI3K, p-Akt, and p65 NF-κB in IPEC-J2 cells were detected using Western blotting, (b-e) and intuitive statistical results were obtained by quantification. Data were expressed as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.01 compared with the PEDV group.

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  Fig. 7. Stability, safety and anti-colitis activity of f2in vivo. (a) Stability of f2 in simulated gastric juice and simulated in testinal juice. (b) Body weight were measured every other day in f2 colitis mice. (c) Length of colon were measured in f2 colitis mice. Effect of compound f2 on the contents of TNF-α (d), IL-1β (e) and IL-6 (f). (g) The different types of lesions in the colon tissue were observed by optical microscope at magnification of 200 × . (h) Immunohistochemistry of p-p65 in colon tissue sections were observed under an optical microscope at a magnification of 200 × . ###p < 0.001, compared with normal control group; Data were expressed as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.01 compared with the PEDV group.

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  Fig. 8. Safety evaluation of f2in vivo. (A) Body weights of mice treated with single vehicle (0.5 % CMC-Na) and f2 (200, 400, and 800 mg/kg) for 7 days (n = 6, male and female). (B) Daily food intake of mice treated with vehicle (0.5 % CMC-Na) and f2 (200, 400, and 800 mg/kg) for 7 days (n = 6, male and female). (C) Histological analysis of heart, liver, kidney, lung and spleen tissues of Vehicle (0.5 % CMC-Na) and f2 (200, 400, and 800 mg/kg) treated mice by H＆E staining. The pictures were captured at 200 × magnification. Data are presented as means ± SD (n = 5).

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  Fig. 9. For the PLpro (blue), PLpro-f2 (Yellow), Mpro (purple) and Mpro-f2 (green) systems during another 80 ns GaMD simulation, (a) RMSD plots, (b) Rg plots and (c) SASA plots. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)