Virtual Screening and Activity Verification of Novel Inhibitors of ApIV 3C Protease†

doi:10.7503/cjcu20170392

Abstract

Abstract:

Antheraea pernyi iflavirus(ApIV) is an important cause of antheraea pernyi vomiting disease(AVD) which is seriously harming the production of Antheraea pernyi(A. pernyi) industry in China. ApIV is a single-stranded, positive-sense RNA virus, belong to the picornavirida family. Due to the significant role of 3C protease in virus replication, it is considered to be an attractive drug target for developing antiviral therapeutic agents. Although great efforts have been made to develop 3C protease inhibitors, no effective anti-viral therapy for the prevention or treatment of diseases caused by ApIV infection is available. In this study, an effective inhibitor of ApIV 3C protease, 3',4',5,7-tetrahydroxyisoflavone(Orobol), was identified through virtual screening using molecular docking against natural product library. And the binding mode of Orobol with ApIV 3C protease was stable through molecular docking and molecular dynamics simulation(MD). The biological data displayed that the inhibition ratio of virus replication was stronger with the increase of the concentration of Orobol. And when the concentration of Orobol was 10 μg/mL, the inhibition ratio reached 80.5%. In vivo assay also demonstrated that Orobol could inhibit viral replication in spodoptera frugiperda cell(Sf9) cells. Those results proved that Orobol was a novel inhibitor for the disease of antheraea pernyi caused by ApIV infections.

Key words: Antheraea pernyi, Antheraea pernyi iflavirus, 3C Protease, Inhibitor, Virtual screening

CLC Number:

O626.32

TrendMD:

SHI Yanli, LIU Yubo, WU Sijin, LIU Yajun, ZHANG Jianing, LI Wenli. Virtual Screening and Activity Verification of Novel Inhibitors of ApIV 3C Protease^†[J]. Chem. J. Chinese Universities, 2018, 39(4): 701.

Figures/Tables 7

Fig.1 Homology modeling and model validation of ApIV 3C protease(A) Multiple alignment of query sequence and templates(PDB: 2XYA and 4GHT) protein sequence. Navy blue represents conserved residues; (B) Homology model(3D structure) of ApIV 3C protease after molecular dynamics(MD) simulation. The active pocket is constituted by green amino acid residues; (C) the romachandram plot of ApIV 3C protease model; (D) the profile 3D model of ApIV 3C protease.

Fig.2 Virtual screening and molecular docking(A) Binding energy distribution of the docking results about liginds; (B) binding mode of fifteen hit compounds in the ApIV 3C protease model by docking studies using AutoDock.

Table 1 Summary of virtual screening results

No.	Compd.	Biological activity	Dock-Score		Interaction residues
No.	Compd.	Biological activity	AutoDock	LibDock	HBond	π-π Bond
1	Orobol	Hypotensivel, enzyme inhibitor	-6.4	114.345	Gly167, Ser164	Tyr163
2	Tanshinlactone	Anti-tumor	-6.1	99.7829	-	Tyr163
3	Maackiain	Antifungal	-5.6	81.7844	Cys123	Cys123
4	(-)-Epiafzelechin	Antioxidant, enzyme inhibitor, anti-inflammatory	-5.4	83.1081	-	Val166
5	2-α,3-α,23-Trihyd-roxy- olean-12-en-28-oic acid	Anti-inflammatory	-5.1	78.322	Cys123	Tyr163
6	(+)-Conocarpan	Analgesic, antifungal	-5.3	80.6224	-	Glu112, Tyr163
7	Sakuranetin	Phytoalexin	-5.3	80.6224	-	Tyr163
8	Dehydro-α-lapachone	Antivascular, antifungal	-5.3	84.7844	-	-
9	Ilicic acid	Anti-inflammatory	-5.2	82.089	-	-
10	Naringenin	Anti-tumor, anti-cancer, antiulcer, antiatherogenic	-5.2	75.367	-	Tyr163
11	Luteolin	Anti-inflammatory, antispasmodic, antitussive, antioxidant, antiallergic	-5.2	78.959	Cys123	Tyr163
12	Carabrone	Antifungal	-5.1	72.633	-	-
13	Dehydrocostus lactone	Antibacterial, antimicrobial	-5.1	73.830	-	-
14	Apigenin	Anti-tumor, anti-cancer, anti-inflammatory	-5.1	70.493	-	Tyr163
15	1-Hydroxyrutaecarpine	Antiplatelet, aggregation	-5.1	75.472	-	Tyr163

Fig.3 Analysis of binding mode between orobol and ApIV 3C protease(A) Binding mode of orobol with ApIV 3C protease by molecular docking studies using AutoDock. In the solid surface, the pocket(green) is labeled. Local amplify docking interaction structure of orobol along with hydrogen bonds; (B) the root mean square fluctuation(RMSF) of ApIV 3C protease model over 20 ns molecular dynamics(MD) simulation. The red dot represents the amino acid residue in the active site procket.

Fig.4 Assays of antiviral activity and cell cytotoxicity in vitro(A) q-PCR analysis of the effect of 15 compounds at 1 μg/mL concentration for ApIV replication. a. Control, b. 1, c. 2, d. 3, e. 4, f. 5, g. 6, h. 7, i. 8, j. 9, k. 10, l. 11, m. 12, n. 13, o. 14, p. 15; (B) q-PCR analysis of the effect of Orobol for ApIV replication. a. negative control, b. positive control, c(orobal)/(μg·mL-1): c. 0.1, d. 1, e. 10; (C) the analysis of cell cytotoxicity for Orobol. a. blank; b. 10 μg/mL orobol; c. ApIV; d. ApIV+10 μg/mL orobol.

Fig.5 q-PCR analysis of the effect of orobol for ApIV replicationa. negative control; b. positive control; c(orobal)/(μg·mL-1): c. 0.1; d. 1; e. 10; F. 100.

Fig.6 Color change of epicraniam of the antheraea pernyi pupae treated with ApIV and different dose orobol

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