Methotrexate, an anti-inflammatory drug, inhibits Hepatitis E viral replication

Figures & data

Figure 1. In silico analysis of HEV helicase. (A) Represents the ribbon structure representation of The HEV helicase modelled structure. (B) Represents the P-loop containing the Helicase domain (85–232 amino acids residues) coloured in red (as predicted by the InterPro web server); the ball-shaped structure represents the GTP-bound binding pocket of helicase. The binding pocket residues are highlighted in red.

Table 1. Conserved motifs of SF1 helicases in TMV and HEV Helicase.

Motif	Consensus	TMV helicase	HEV helicase	Function
I	GxPGTGKT	GVPGCGKT	GVPGSGKS	NTP binding site
Ia	xxTNxAx	VPGRQAA	VPTRELR	Nucleic acid binding site
to	YDExxQ	IDEGLM	IDEAPSL	NTP binding site
III	GDxxQLxx	GDTQQIPYINR	GDPNQIPAIDF	Coordination between NTP and nucleic acid binding site
IIIa	xxYRx	TTLRC	VTHRCPADV	NTP binding site
V	xxTVHx	VHTVHE	VTVHEAQGATY	Nucleic acid binding
Va	FQGxE	VQGET	AQGATY	Coordination between NTP and nucleic acid binding site
Vb	VRxxDxx	IIARDSP	IQSSAAH	Nucleic acid binding
VI	VAxTRARx	VSLSRHTK	VALTRHTE	NTP binding site

The first column represents the conserved motif name present in all SF1 helicases. The second column represents the conserved amino acid residues in respective motifs (x is any amino acid). In contrast, the third column represents the motifs in TMV helicase (highlighted in yellow). The fourth column represents the motif sequence present in HEV helicase, the conserved residues are highlighted in yellow, and the fifth column represents the function of conserved motifs.

Figure 2. Protein–ligand interaction diagram- Figure- represents the interaction diagram of (A) GTP and (B) methotrexate with key amino acid residues at the binding pocket of HEV helicase. (A) Depicts hydrogen bonding and hydrophobic interactions between Helicase and GTP. (B) Depicts hydrogen bonding and hydrophobic interactions between Helicase and Methotrexate. The hydrogen bonds are represented with green dashed lines, and the interacting residues of HEV helicase are labelled.

Table 2. The table represents the ZINC ID, common name, and binding affinity of the ligands selected after the virtual screening of the FDA-approved library from the ZINC database. Rocaglamide and silvestrol were used as docking control.

ZINC ID	Ligand	Binding affinity
ZINC000001529323	Methotrexate	−8.6
ZINC000001530788	Cromolyn	−8.6
ZINC000003917708	Daunorubicin	−8.5
ZINC000002005305	5-Methyltetrahydrofolate	−8.3
ZINC000003833821	Prdl	−7.6
ZINC000005664046	Rocaglamide	−7.3
ZINC000095099283	Silvestrol	−7.3

Figure 3. MD simulations analysis trajectory plot of HEV helicase-methotrexate bound complex. Figure 3 represents the RMSD of the helicase-methotrexate complex during 200 ns MD simulation.

Figure 4. Protein–ligand contacts. The above figure represents protein-ligand contacts of the helicase-methotrexate complex during 200 ns MD simulation. The hydrogen bonds between helicase and methotrexate have been depicted in green, and water–water bridges have been depicted in blue colour; very few ionic interactions were observed between helicase and methotrexate, as illustrated in the figure, and the ionic interactions are displayed in red colour. Hydrophobic interactions are depicted in lavender colour.

Figure 5. 2D depiction of protein-ligand contacts. The figure above represents a 2D depiction of the percentage of protein-ligand contacts between helicase and methotrexate complex. Arg24, Arg128, and Arg48 showed the maximum interaction rate during the 200 ns MD simulation. Asp70 and Asp103 also showed significant interaction with the methotrexate.

Figure 6. Ni-NTA (IMAC) protein purification of HEV helicase. Figure 6(A) represents Coomassie Brilliant Blue stained 12% SDS-PAGE. Lane 1 contains the pre-stained protein marker; Lanes 2 and 3 have the 0.5% NLS solubilised protein fraction sample; lane 4 contains flow-through; lanes 5, 6, and 7 include the wash. Lanes 8 and 9 have different elution fractions. Figure 6(B) represents the western blot analysis of the same samples. Lane 1 contains the pre-stained protein marker; Lanes 2 and 3 have the 0.5% NLS solubilised protein fraction sample; lane 4 contains flow-through; lanes 5, 6, and 7 include the wash. Lanes 8 and 9 have different elution fractions probed with anti-helicase-specific antibody in 1:3000 dilutions.

Figure 7. Effect of Substrate concentration on enzyme activity: (A) Michaelis Menten constant (K_m) was calculated from the NTPase activity of the purified HEV helicase by varying the substrate (GTP) concentrations from 0 to 2.5 mM. The graph for NTPase activity was fitted using the Mechalis-Mentenn equation, and the calculated K_m value was found to be 0.35 mM (Figure 7(A)). The unwinding activity of purified HEV helicase was also determined using double-strand RNA as a substrate. Different concentrations of dsRNA ranging from 0 to 300 nM were used to calculate the K_m value. The graph was again fitted with the Mechalis–Menten equation, and the estimated K_m value for dsRNA as a substrate was found to be ∼100 nM. All data points in the graph represent the mean or average value of the triplicate readings, and the error bars indicate the standard deviation.

Figure 8. Effect of temperature and pH on enzyme activity: The above graph represents the effect of different temperatures and pH on NTPase activity.The protein and substrate concentrations were kept constant, and the reaction was incubated for temperatures ranging from 25 to 45 °C (Figure 8(A)), Also at a constant protein and substrate concentrations and varying pH from 2 to 14 (Figure 8(B)). The enzyme activity was measured as % NTPase. All the experiments were performed in triplicates. The data points in the graph represent the mean or average values, and the error bars indicate the standard deviation.

Figure 9. Cell-free NTPase and unwinding inhibition assay and IC₅₀ determination of FDA-approved compounds. The graph represents the enzyme inhibition activity of the compounds evaluated using NTPase inhibition assay and unwinding activity inhibition assay. Figure 9(A) illustrates unwinding activity inhibition by FDA-approved compounds, and Figure 9(B) represents the inhibition profile by NTPase activity assay. The no compound control was taken as 100% HEV helicase activity, and no enzyme control was taken as 0% activity. All the experiments have been performed in triplicates. 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. All experiments were performed in triplicates, and the graph was plotted in GraphPad Prism software. Figure 9(C) represents the effect of increasing methotrexate concentration on the unwinding activity. With the increase in methotrexate concentration, the unwinding activity is decreased, as represented by the increase in intensity of the dsRNA band. The samples were run on 10% Native PAGE, and the bands were visualised on a fluorescence (FAM) based gel dock system.

Figure 10. Cell-free NTPase and IC₅₀ determination of ten novel compounds⁴¹. The graph represents the enzyme inhibition activity of the compounds evaluated using an NTPase activity inhibition assay. All the experiments have been performed in triplicates. The compounds were assessed at a concentration of 1–10 µM. The no compound control was taken as 100% HEV helicase activity, and no enzyme control was taken as 0% activity of HEV helicase. The graph’s data points represent the mean or average value of three readings, and the error bars indicate the standard deviation.

Table 3. IC50 determination from NTPase and unwinding inhibition assay.

IC50 values of different compounds using NTPase and unwinding assay
Compound name	IC50 (µM) NTPase activity	IC50 (µM) Unwinding activity
Methotrexate	1.81	9.849
Levomefolic acid	3.65	13.92
Prednisolone	4.202	25.83
Daunorubicin HCL	7.663	8.253
Disodium cromoglycate	14.84	39.76

IC50 values of different FDA-approved compounds from NTPase inhibition assay and unwinding assay.

Figure 11. Microscale thermophoresis assay. The figure represents the dose-response curve for the binding interaction between Red-Tris NTA-labelled Helicase and Methotrexate (Figure 11(A)) and compound A (Figure 11(B)). The concentration of RED-Tris-NTA-labelled helicase was kept constant, and the concentration of methotrexate and compound A varied between 500 µM and 150 nM. The Y-axis represents the ΔF_norm fluorescence, and the X-axis represents the concentration of methotrexate and compound A, respectively. Here, the values represent the mean values of three independent experiments, and the error bar indicates the standard deviation.

Figure 12. HEV Replicon Assay. Figure 12(A,B) represents the dose-dependent graphs that indicate the % inhibition of viral replication in HEV replicon-based assay in the presence of methotrexate and compound A. It was generated by quantifying luminescence, and each bar in the graph represents the mean value of triplicate, and the error bar indicates the standard deviation.

Figure 13. Effect of Methotrexate and compound A on HEV RNA Replication. The graph represents the reduction of viral RNA copy number upon varying the concentration of methotrexate (Figure 13(A)) and compound A (Figure 13(B)). Here, the values represent the mean values of three independent experiments, and the error bar indicates the standard deviation.

Figure 14. Effect of Methotrexate on HEV Replication using IFA. The cells were infected with the virus without an inhibitor as a positive control; panel (a) represents huh-7 cells stained with DAPI, (b) represents huh-7 cells stained with FITC, and (c) represents the merged image of DAPI and FITC. In another experiment, cells were similarly treated with the virus with an addition of 50 nM Mtx, where (d) represents huh-7 cells treated with DAPI, (e) represents FITC-treated cells, and (f) represents the merged image of DAPI and FITC. Further, in a similar experiment, the cells were treated with 100 nM of Mtx, and the panels (g), (h), and (i) represent DAPI, FITC, and merged images, respectively.

Figure 15. (A) Effect of Compound A on HEV Replication using IFA. The cells were infected with the virus without an inhibitor as a positive control, and panel (a) represents huh-7 cells stained with DAPI, (b) represents huh-7 cells stained with FITC, and (c) represents the merged image of DAPI and FITC. In another experiment, cells were similarly treated with the virus with 5 µM Compound A addition, where (d) represents huh-7 cells treated with DAPI, (e) represents FITC-treated cells, and (f) represents the merged image of DAPI and FITC, respectively. Further, in a similar experiment, the cells were treated with 10 µM of compound A, and the panels (g), (h), and (i) represent DAPI, FITC, and merged images, respectively. (B) Fluorescence quantification of infected and treated huh-7 cells. The graph above represents the fluorescence quantification data of the IFA experiment. The graph represents % HEV replication inhibition by the inhibitors tested. Untreated represents huh-7 cells infected with the virus without any drug treatment, while two concentrations of Mtx were tested (50 nM and 100 nM). At 50 nM Mtx concentration, ∼81% HEV replication inhibition was observed. At 100 nM Mtx concentration, ∼94% HEV replication inhibition was observed. Two concentrations of compound A, 5 µM and 10 µM were tested; at a concentration of 5 µM, compound A showed ∼14% HEV replication inhibition, while at a 10 µM concentration of compound A, ∼57% HEV replication inhibition was observed. The data points in the graph represent mean or average values from three different panels.

Lipsitch M, Krammer F, Regev-Yochay G, Lustig Y, Balicer RD. SARS-CoV-2 breakthrough infections in vaccinated individuals: measurement, causes and impact. Nat Rev Immunol. 2022;22(1):57–65.

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