Inhibition of herpes simplex virus-1 infection by MBZM-N-IBT: in silico and in vitro studies

Abstract

Introduction: The emergence of drug resistance and cross-resistance to existing drugs has warranted the development of new antivirals for Herpes simplex viruses (HSV). Hence, we have designed this study to evaluate the anti-viral activity of 1-[(2-methyl benzimidazole-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT), against HSV-1.

Method: Molecular docking was performed to assess the affinity of MBZM-N-IBT for HSV-1 targets. This was validated by plaque assay, estimation of RNA and protein levels as well as time of addition experiments in vitro.

Result: Molecular docking analysis suggested the inhibitory capacity of MBZM-N-IBT against HSV-1. This was supported by the abrogation of the HSV-1 infectious viral particle formation with the IC₅₀ value of 3.619 µM. Viral mRNA levels were also reduced by 72% and 84% for UL9 and gC respectively. MBZM-N-IBT also reduced the protein synthesis for gC and ICP8 significantly. While mRNA of ICP8 was not significantly affected, its protein synthesis was reduced by 47%. The time of addition experiment revealed the capacity of MBZM-N-IBT to inhibit HSV-1 at early as well as late stages of infection in the Vero cells. Similar effect of MBZM-N-IBT was also noticed in the Raw 264.7 and BHK 21 cells after HSV-1 infection. Supported by the in silico data, this can be attributed to possible interference with multiple HSV targets including the ICP8, ICP27, UL42, UL25, UL15 and gB proteins.

Conclusion: These results along with the lack of acute oral toxicity and significant anti-inflammatory effects suggest its suitability for further evaluation as a non-nucleoside inhibitor of HSV.

Keywords: Herpes simplex virus-1; ICP8; MBZM-N-IBT; UL9; gC.

Fig. 1

Inhibition of HSV-1 infection by MBZM-N-IBT. Vero cells were infected by HSV-1 with MOI 0.001. DMSO, 44.4 µM of Acyclovir and 100 µM, 150 µM and 200 µM of MBZM-N-IBT were added to the Vero cells. DMSO and Acyclovir were used as negative and positive controls respectively. a Morphological changes were observed under a microscope at 20X resolution b Vero cells were seeded onto cover-slips and infected with HSV-1 at MOI 1 whereas cells without HSV-1 infection were considered as Mock.. After infection, cells were treated with DMSO, 100 µM and 200 µM of MBZM-N-IBT The cells were fixed with 4% paraformaldehyde after 18 hpi and probed with ICP8 (II, V, VIII, XI) followed by staining with secondary antibody, anti-mouse Alexa Fluor 488(red) respectively. Nuclei were counterstained with DAPI (blue). c Bar diagrams showing the percentage of HSV-1 ICP8 positive cells in infected and drug treated samples. Data represented as mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparisons test p ≤ 0.05 was considered statistically significant

Fig. 2

Dose dependent inhibition of HSV-1 using different concentrations of MBZM-N-IBT: Vero cells were infected with HSV-1 and MBZM-N-IBT was added with different concentrations (2.5 µM, 5 µM, 10.0 µM, 25.0 µM, 50.0 µM, 100.0 µM, 150.0 µM and 200.0 µM, 250 µM). DMSO was used as a negative control. The supernatant was collected at 24hpi from the infected and drug treated Vero cells and virus titer was determined by plaque assay. Bar diagram represents the log (10) of PFU/mL of the virus after treatment with different concentrations of MBZM-N-IBT. Data represents as mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparison test

Fig. 3

Effect of MBZM-N-IBT in HSV-1 mRNA and DNA levels: a Vero cells were infected by HSV-1 with MOI 1 and treated with DMSO (negative control), 44.4 µM Acyclovir (positive control) and MBZM-N-IBT (100 µM, 150 µM, 200 µM,). Total RNA was extracted from the HSV-1 infected cells at 24 hpi, cDNA was synthsized and HSV-1 UL9, ICP8, gC, gD genes were amplified using respective primers by RT-PCR (a) Bar diagrams (b–e) depicts the relative band intensities of viral mRNA expression pattern in infected and drug treated samples of UL9, ICP8, gC and gD genes respectively. Data represent the mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparison tests.. p ≤ 0.05 was considered statistically significant. GAPDH was used as a loading control and the band intensities were normalized based on GAPDH by the ImageJ software. f Total RNA was isolated from the HSV-1 infected cells at 24 hpi, cDNA was synthesized and the expression levels of HSV-1 UL9 and gC mRNA, were detected using respective primers by qRT-PCR. Bar diagrams showing the relative fold change reduction in viral mRNA expression pattern in infected and drug treated samples. Data represent the mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparisons test. p ≤ 0.05 was considered statistically significant. GAPDH was used as endogenous control. g Vero cells were infected by HSV-1 with MOI 1 and treated with DMSO (negative control) and MBZM-N-IBT (50, 100, 200 µM). Cell supernatants were collected at 24hpi and HSV-1 DNA was isolated by the Qiagen All prep DNA/RNA Mini kit according to the manufacturer’s protocol followed by qPCR of HSV-1 gC gene. Bar diagram represents the viral copy number in percentage which was calculated based on standard curve. Data represent the mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparisons test

Fig. 4

Effect of MBZM-N-IBT in HSV-1 protein levels: Vero cells were infected with HSV-1 at MOI 1 and subsequently treated with DMSO (negative control), Acyclovir (positive control) and different concentration of MBZM-N-IBT (100 µM, 150 µM and 200 μM) and finally harvested at 24 hpi for ICP8 and 15 hpi for gC. The cells were lysed and subjected to Western blot analysis. Western blots were probed with ICP8 (a) and gC (b) antibodies. GAPDH was used as a loading control and the band intensities were normalized based on GAPDH by the ImageJ software. c, d Bar diagrams showing the relative band intensities of ICP8 and gC. p ≤ 0.05 was considered statistically significant. e Flowcytometric dot plot analysis showing percent positive cells for ICP8 at 15 hpi against SSC-H off different samples. f Graphical representation showing percent positive cells for ICP8 with varying concentrations of MBZM-N-IBT. g Mean fluorescence intensity (MFI) analysis also suggests the ICP8 signal intensity in the presence of MBZM-N-IBT in a dose dependent manner as compared to DMSO control. The comparison between the groups with only one parameter was performed by one way ANOVA (nonparametric) with the Bonferroni post-hoc test. p < 0.05 was considered as the statistically significant difference between the groups. (ns, non-significant; ** p ≤ 0.01; *** p ≤ 0.001)

Fig. 5

Inhibition pattern of HSV-1 infection by addition of MBZM-N-IBT at different time points: Vero cells were infected by HSV-1 with MOI 0.001, 2, 5 and 200 µM MBZM-N-IBT was added to different dishes at every 4 h intervals up to 20 hpi. The supernatants, as well as cells were collected from all the experimental samples at 20 hpi and plaque assay was performed to assess the number of infectious particles of HSV-1. The bar diagram represents the virus titer in PFU/mL. Data represent the mean ± SEM from three independent experiments using the one way Anova, Dunnett’s multiple comparisons test. p-value less than 0.001 was considered to be statistically significant in the test