Inhibition of Chikungunya Virus Replication by 1-[(2-Methylbenzimidazol-1-yl) Methyl]-2-Oxo-Indolin-3-ylidene] Amino] Thiourea(MBZM-N-IBT)

Abstract

Chikungunya virus (CHIKV) infection is one of the most challenging human Arboviral infections with global significance and without any specific antiviral. In this investigation, 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) was synthesised as a molecular hybrid of 2-methyl benzimidazole and isatin-β-thiosemicarbazone and its anti-CHIKV property was evaluated. The release of infectious virus particles was calculated by plaque assay, expression profile of viral RNA was estimated by RT-PCR and viral protein profiles were assessed by Western blot and FACS analyses. The safety index of MBZM-N-IBT was found to be >21. The CHIKV infectious viral particle formation was abrogated around 76.02% by MBZM-N-IBT during infection in mammalian system and the viral RNA synthesis was reduced by 65.53% and 23.71% for nsP2 and E1 respectively. Surprisingly, the viral protein levels were reduced by 97% for both nsP2 and E2. In the time-of-addition experiment it abrogated viral infection at early as well as late phase of viral life cycle, which indicates about multiple mechanisms for its anti-CHIKV action. In silico analysis justified development of MBZM-N-IBT with good affinities for potential target proteins of CHIKV and related virus. With predictions of good drug-likeness property, it shows potential of a drug candidate which needs further experimental validation.

Figure 1. Synthesis of MBZM-N-IBT.

(a) Isatin, paraformaldehyde, and 2-methyl benzimidazole were dissolved in ethanol and refluxed for 16h to yield the intermediate compound (1); (b) Compound (1) and thiosemicarbazide were dissolved in warm ethanol and refluxed for 4h following addition of few drops of glacial acetic acid to yield MBZM-N-IBT (2).

Figure 2. Synthesis of MIBT.

N-methyl isatin and thiosemicarbazide were dissolved in warm ethanol and refluxed for 4h following addition of few drops of glacial acetic acid to yield MIBT (1′).

Figure 3. Structure and mass spectrum of (a) MBZM-N-IBT and (b) MIBT.

Mass spectrum was recorded with ESI technique in micrOTOF-QII mass spectrometer.

Figure 4. Inhibition of CHIKV infection by MBZM-N-IBT.

Vero cells were infected with Chikungunya virus prototype strain S 27 (MOI 0.001). DMSO and different doses of the drugs [MBZM-N-IBT (50 μM), 100 μM and 200 μM)] were added to the experimental samples. DMSO was used as negative control and recent reported drugs Ribavirin (4.1 μM) and MIBT (187.8 μM) were used as positive control. The cells and supernatants were harvested at different time post infection based on the assay. (a) Morphological changes were observed under microscope at 15 hpi and pictures were taken with 20X magnification. (b) The supernatants as well as cells were collected from all the experimental samples at 15 hpi and plaque assay was performed to assess the number of infectious particle of CHIKV. (c) MBZM-N-IBT was added in DMEM complete media and kept in CO₂ incubator at 37 °C for 15 hrs. The whole contents was added into the CHIKV infected Vero cells and virus titer was determined by plaque assay as described earlier. The bar diagram represents the virus titer in log (10) scale for all the experimental samples from three independent experiments. The statistical analysis of the experimental data was presented as mean ± SD of three independent experiments (n ≥ 3).P - value less than 0.001 was considered to be statistically significant in the tests (*p < 0.05; **p ≤ 0.01, ***p ≤ 0.0001).

Figure 5. Effect of MBZM-N-IBT in CHIKV RNA and protein level.

(a) Chikungunya virus (S 27) infected with MOI 0.001 and treated with DMSO (negative control), 4.1 μM Ribavirin (positive control), 187.8 μM MIBT and (50, 100, 200 μM) MBZM-N-IBT. Whole cell RNA was isolated from the CHIKV infected samples at 15hpi and CHIKV nsP2 and E1 genes were amplified using respective primers by RT PCR. (b) Bar diagrams showing the relative band intensities in viral RNA expression pattern in infected and drug treated samples as obtained through PRISM Software. Data represented as mean ± SEM from three independent experiments. p ≤ 0.05 was considered statistically significant. (c) As mention above infected Vero cells lysates were separated in 10% SDS PAGE and viral protein expression pattern were assessed by Western blot using antibodies against CHIKV nsP2 and E2 proteins. In both, GAPDH was used as a loading control. (d) Bar diagrams showing the relative band intensities in viral protein expression pattern in infected and drug treated samples as obtained through PRISM Software. Data represented as mean ± SEM from three independent experiments. p ≤ 0.05 was considered statistically significant.

Figure 6. Flow cytometric analysis depicting inhibition of CHIKV nsP2 and E2 protein expression after treatment with MBZM-N-IBT.

Dot plot analysis showing percent positive cells for nsP2 (a) and E2 (b) against SSC-H towards mock + DMSO, S 27 + DMSO, S 27 + MBZM-N-IBT (50, 100 and 200 μM). Graphical representation showing percent positive cells for nsP2 (c) and E2 (d) of S 27 + DMSO, S 27 + MBZM-N-IBT (50, 100 and 200 μM). Student’s t test was performed to calculate p values. p < 0.05 was considered statistically significant between groups (*p ≤ 0.05; **p ≤ 0.01).

Figure 7. Inhibition pattern of CHIKV infection by addition of MBZM-N-IBT at different time points.

Vero cells were infected by Chikungunya virus prototype strain S 27 with MOI 0.001 and 200 μM MBZM-N-IBT was added to each sample at every 2 hrs interval up to 14 hpi. Ribavirin (4.1 μM) was used as a control. The supernatants as well as cells were collected from all the experimental samples at 15 hpi and plaque assay was performed to assess the number of infectious particles of CHIKV. The bar diagram represents the virus titre in PFU/ML scale for all the experimental samples from three independent experiments. Open bar and close bar indicate the DMSO only and with MBZM-N-IBT respectively. The statistical analysis of the experimental data was presented as mean ± standard deviation of three independent experiments. P – value less than 0.001 was considered to be statistically significant in the test.

Figure 8. MBZM-N-IBT shows strong binding affinities for CHIKV proteins.

The best fit complex of MBZM-N-IBT with, (a) mature envelope glycoprotein complex (spontaneous cleavage) of CHIKV (3N41) and (b) nsP2 protease of CHIKV (3TRK). MBZM-N-IBT was screened against the target protein in the AutoDock Vina open-source program for molecular docking and the best fit complex was visualised in the PyMOL viewer.