Identifying HSV-1 Inhibitors from Natural Compounds via Virtual Screening Targeting Surface Glycoprotein D

Abstract

Herpes simplex virus (HSV) infections are a worldwide health problem in need of new effective treatments. Of particular interest is the identification of antiviral agents that act via different mechanisms compared to current drugs, as these could interact synergistically with first-line antiherpetic agents to accelerate the resolution of HSV-1-associated lesions. For this study, we applied a structure-based molecular docking approach targeting the nectin-1 and herpesvirus entry mediator (HVEM) binding interfaces of the viral glycoprotein D (gD). More than 527,000 natural compounds were virtually screened using Autodock Vina and then filtered for favorable ADMET profiles. Eight top hits were evaluated experimentally in African green monkey kidney cell line (VERO) cells, which yielded two compounds with potential antiherpetic activity. One active compound (1-(1-benzofuran-2-yl)-2-[(5Z)-2H,6H,7H,8H-[1,3] dioxolo[4,5-g]isoquinoline-5-ylidene]ethenone) showed weak but significant antiviral activity. Although less potent than antiherpetic agents, such as acyclovir, it acted at the viral inactivation stage in a dose-dependent manner, suggesting a novel mode of action. These results highlight the feasibility of in silico approaches for identifying new antiviral compounds, which may be further optimized by medicinal chemistry approaches.

Keywords: glycoprotein D; herpes simplex virus type 1; molecular docking; natural compounds; virtual screening.

Figure 1

Schematic diagram of the molecular docking workflow for identifying natural glycoprotein D inhibitors with antiviral activity against HSV-1.

Figure 2

Structure of the eight natural compounds selected as potential glycoprotein D inhibitors by molecular docking. All eight compounds were subjected to in vitro validation. Listed active compounds (ACs) are: #7,1-(1-benzofuran-2-yl)-2-[(5Z)-2H,6H,7H,8H-[1,3]dioxolo[4,5-g]5soquinoline-5-ylidene]ethenone; #10,13-[3-(4-methylpiperazin-1-yl)-3-oxopropyl]-8,13-dihydroindolo[2′,3′:3,4]pyrido[2,1-b]quinazolin-5(7H)-one; #12,(2S,5Ar,6Ar,9S,9Ar)-2,5a-dimethyl-9-((4-(5soquino-2-yl)piperazin-1-yl)methyl)octahydro-2H-oxireno[2′,3′:4,4a]naphtho[2,3-b]furan-8(9Bh)-one; #16,(1Ar,2S,5Ar,6Ar,9S,9Ar,9Bs)-2,5a-dimethyl-9-((4-phenylpiperazin-1-yl)methyl)octahydro-2H-oxireno[2′,3′:4,4a]naphtho[2,3-b]furan-8(9Bh)-one; #17, (1Ar,2S,5Ar,6Ar,9S,9Ar,9Bs)-9-((4-(5-chloro-2-methylphenyl)piperazin-1-yl)methyl)-2,5a-dimethyloctahydro-2H-oxireno[2′,3′:4,4a]naphtho[2,3-b]furan-8(9Bh)-one,#27,5-(7-Hydroxy-1H-benzofuro[3,2-b]pyrazolo[4,3-e]5soquino-4-yl)-1H-pyrrolo[3,2,1-ij]5soquinol-4(2H)-one; #28, N-((S)-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)-2-(3-oxoisoindolin-1-yl)acetamide, #29, Arcyriaflavin A. Their molecular structures were illustrated using ChemDraw 18.2 (PerkinElmer).

Figure 3

Binding interaction diagrams of the eight compounds selected for in vitro validation with the (herpes virus entry mediator) HVEM (A) and nectin-1 (B) binding interfaces of HSV-1 glycoprotein D as predicted by molecular docking. Red lines represent hydrophobic contacts, and broken green lines represent hydrogen bonds, with distances in angstroms.

Figure 4

Antiviral screening assays for three selected active compounds. The antiviral activity of active compounds (ACs) was investigated at three different stages. The antiviral activity of ACs #7, #27, and #28 was tested at 10 µg/mL. (A) Schematic diagram of the viral inactivation assay performed by preincubating Herpes Simplex Virus type 1 (HSV-1) with ACs for 1 h prior to addition to cells is. (B) Results of the viral inactivation assay. Viral attachment and entry inhibition of ACs were investigated by simultaneously adding the virus and ACs to cells, as shown in (C); results are shown in (D). The post-entry antiviral effect of ACs was tested by adding ACs after infection (E); results are plotted in (F). Cells infected with the virus and treated with dimethyl sulfoxide (DMSO) at 0.1% v/v were included as solvent controls. This experiment was performed four times with triplicates. Means of the four experiments are plotted with standard error of the mean. Friedman test, followed by Dunn’s test, was used to determine statistical significance (* p < 0.05, ** p < 0.01).

Figure 5

Quantification of the antiviral activity of AC #7. The antiviral activity of AC#7 was investigated by plaque reduction assay in African green monkey kidney cell line (VERO). (A) AC#7 demonstrated significantly high antiviral activity at a concentration of 25 µM compared to the dimethyl sulfoxide (DMSO) control (0.08% v/v). (B) AC#7 exhibited antiviral activity in a dose-dependent manner compared with the DMSO controls. The DMSO controls were tested at concentrations of 1.33%, 0.67%, 0.33%, 0.16%, 0.08%, 0.04%, and 0.02% (v/v), which are the concentrations at which AC#7 was dissolved in Dulbecco’s Modified Eagle Medium (DMEM) media. The plaque reduction assay was performed three times with duplicates. Means of the three experiments are plotted with standard error of the mean. One-sample t-test was applied to compare the antiviral activity of the DMSO control and AC#7 (* p < 0.05).

Figure 6

Toxicity of AC#7 in VERO cells at 800 µM. The cytotoxicity of AC#7 was tested by incubating Vero cells with AC#7 (800 µM) for 2 h. AC#7 was then removed, and cells were incubated in fresh media for a further 46 h before cell viability was evaluated using water-soluble tetrazolium 1. Cells without any treatment were included as cell-only control and used to calculate cell viability. Cells treated with dimethyl sulfoxide (DMSO) (2.7% v/v), corresponding to the solvent concentration in the tested samples, were used as the solvent control. The wells treated with 1% (v/v) Triton X-100 were used as a negative control. The cytotoxicity assay was performed three times with duplicates. Means of the three experiments are plotted with standard error of the mean. Wilcoxon test was applied to compare the cytotoxicity of DMSO control and AC#7 (ns indicates not significant, p > 0.05).