Inhibition of Hepatitis E Virus Replication by Novel Inhibitor Targeting Methyltransferase

Abstract

Hepatitis E Virus (HEV) is a quasi-enveloped virus having a single-stranded, positive-sense RNA genome (~7.2 kb), flanked with a 5' methylated cap and a 3' polyadenylated tail. The HEV open reading frame 1 (ORF1) encodes a 186-kDa polyprotein speculated to get processed and produce Methyltransferase (MTase), one of the four essential replication enzymes. In this study, we report the identification of the MTase inhibitor, which may potentially deplete its enzymatic activity, thus causing the cessation of viral replication. Using in silico screening through docking, we identified ten putative compounds, which were tested for their anti-MTase activity. This resulted in the identification of 3-(4-Hydroxyphenyl)propionic acid (HPPA), with an IC₅₀ value of 0.932 ± 0.15 μM, which could be perceived as an effective HEV inhibitor. Furthermore, the compound was tested for inhibition of HEV replication in the HEV culture system. The viral RNA copies were markedly decreased from ~3.2 × 10⁶ in untreated cells to ~4.3 × 10^2.8 copies in 800 μM HPPA treated cells. Therefore, we propose HPPA as a potential drug-like inhibitor against HEV-MTase, which would need further validation through in vivo analysis using animal models and the administration of Pharmacokinetic and Pharmacodynamic (PK/PD) studies.

Keywords: 3-(4-Hydroxyphenyl) propionic acid; HEV replication; Hepatitis E Virus; Methyltransferase; antiviral.

Figure 1

A flow chart depicting protocol for Virtual Screening against HEV-MTase.

Figure 2

Computational docking of m⁷ GTP and SAM on HEV-MTase model. (A) 3D structure model of HEV-MTase protein (region 34–353) after MD simulations; Putative MTase region 60 to 240 amino acid is highlighted in light cyan, and 34–59 to 241–353 amino acid regions are in dark cyan. The Surface view of the ligand-binding site in the HEV-MTase model is highlighted in dark pink. (B) For clarity, only the first rank pocket in the structure of HEV-MTase is shown here. Docked poses of m⁷ GTP and SAM are displayed in the binding site of putative HEV-MTase protein. The active site residues are shown as balls and stick with a black colour code. (C) Docked poses of m⁷ GTP and SAM (grey carbon, blue nitrogen, red oxygen, yellow sulfur, and phosphate in pink) are displayed in the binding site of the putative HEV-MTase region. The docked poses show distance (4.71Å) as yellow dotted lines between a carbon atom of methyl group donor and N7 methyl group acceptor in the m⁷GTP and SAM binding site of HEV-MTase protein. An invariant histidine 65 residues (H32), a DXXR Motif residues Asp114, Val115, Gln116 and Arg117 (Asp81, Arg84), and the invariant tyrosine 229 residue (Tyr196) in the HEV-MTase complex with m⁷GTP and SAM are represented in red colour circles. (D,E) The 2D interaction diagrams. The docked poses display interactions in the m⁷GTP and SAM binding site of the HEV-MTase protein. In the charts, residues highlighted with a red circle are common in the HEV, CHIKV and SFV MTase proteins. Conserved residues His32, Asp81, Arg84, and Tyr196 were previously mutated in the SFV MTase site-directed mutagenesis study. Sidechains of interacting residues of the HEV-MTase protein are shown as sticks in pink colour. (F) Amino acid Sequence alignment of HEV-MTase (23 to 207 correspond to (Putative HEV-MTase 56 to240 region) and CHIKV-MTase domain 24 to 259 region. Conserved residues of HEV-MTase and CHIKV-MTase are highlighted in blue and red colours, respectively.

Figure 3

2D structure of the best ten compounds identified through the refined model structure of the HEV-MTase docking study.

Figure 4

The 2D interaction diagram. The docked poses display interactions in the SAM and m⁷GTP binding site of the predicted model of HEV-MTase protein. In the diagrams, residues highlighted in the red circle are common SAM and m⁷GTP binding residues in the HEV-MTase complexes. Sidechains of interacting residues of the HEV-MTase protein are shown as sticks in pink colour.

Figure 5

Cell-free inhibition assay and IC₅₀ determination of HPPA: (A) The graph represents the % of HEV-MTase activity in different compounds concerning compound-free control. The no compound control was taken as 100% MTase activity, and no enzyme control was taken as 0% activity of HEV-MTase. The inhibition assay was performed in triplicates, and the error bar indicates the standard deviation. The statistical analysis was performed using a student t-test, and a p-value < 0.005 was considered statistically significant. * p < 0.0001; n.s p > 0.005. (B) The graph represents the mean value of triplicate measurements for IC₅₀ determination. GraphPad Prism was determined by fitting the curve using log(inhibitor) vs. normalized response. The error bar indicates the standard deviation. The determined IC₅₀ for HPPA was 0.932 ± 0.15 μM.

Figure 6

MicroScale Thermophoresis (MST). Dose-response curve to study the binding affinity between HEV-MTase and HPPA was plotted. The graph’s X-axis represents the concentration of ligand (HPPA), and the Y-axis represents the % fluorescence change. The calculated K_d for HEV-MTase and HPPA was approximately 2 μM. The graph describes the mean value of three independent experiments, and the error bar indicates the standard deviation.

Figure 7

Molecular dynamics simulations of HPPA with HEV-MTase: RMSD of the Cα backbone of apo-MTase and its complexes with respect to time (100 ns), RMSD of compounds over 100 ns, RMSF of Ca atoms of MTase complexes over the 100 ns. Stability of MTase from HEV-MTase: The number of hydrogen bond interactions between MTase and compounds during simulation are shown. The radius of gyration (Rg) of MTase and in complex with compounds over the simulation. Total solvent accessible area (SASA) with respect to time is shown. The apo-MTase (black) and its complex with inhibitor HPPA (red) are represented.

Figure 8

Cell Viability and inhibition of HEV Replicon. The dose-dependent curve represents % inhibition of viral replication in HEV replicon-based assay and cytotoxicity profile of HPPA. The dose-dependent graph was generated by quantifying luminescence, and cytotoxicity was examined using an MTT assay. The blue line in the graph represents the % inhibition, and the orange line represents the cell viability in the presence of HPPA. The calculated CC₅₀ and IC₅₀ of the compound were found to be more than 1 mM and 77.12 ± 18.7 μM, respectively.

Figure 9

Inhibition of HEV Replication. (A) The RNA copy number of HEV was quantified using qPCR. HPPA reduced RNA levels from 3.2 × 10⁶ copies to 4.3 × 10^2.8 copies per μg of total cellular RNA. The graph represents the log₁₀ RNA copy number per μg of total cellular RNA. (B) The ORF2 level was checked using western blot in mock and treated cells, and a decrease in ORF2 expression was seen compared to the untreated group. (C) The expression of ORF2 was also seen using HEV-ORF2 specific antibody detected through Alexa fluor 488 secondary antibody. The decrease in fluorescence was seen when compared to the untreated panel. The fluorescence was quantified using Image J, and the fluorescence level was decreased from 100% in untreated cells to ~25% in an 800 μM treated sample.