Small molecule inhibitors of HCV replication from pomegranate

Abstract

Hepatitis C virus (HCV) is the causative agent of end-stage liver disease. Recent advances in the last decade in anti HCV treatment strategies have dramatically increased the viral clearance rate. However, several limitations are still associated, which warrant a great need of novel, safe and selective drugs against HCV infection. Towards this objective, we explored highly potent and selective small molecule inhibitors, the ellagitannins, from the crude extract of Pomegranate (Punica granatum) fruit peel. The pure compounds, punicalagin, punicalin, and ellagic acid isolated from the extract specifically blocked the HCV NS3/4A protease activity in vitro. Structural analysis using computational approach also showed that ligand molecules interact with the catalytic and substrate binding residues of NS3/4A protease, leading to inhibition of the enzyme activity. Further, punicalagin and punicalin significantly reduced the HCV replication in cell culture system. More importantly, these compounds are well tolerated ex vivo and'no observed adverse effect level' (NOAEL) was established upto an acute dose of 5000 mg/kg in BALB/c mice. Additionally, pharmacokinetics study showed that the compounds are bioavailable. Taken together, our study provides a proof-of-concept approach for the potential use of antiviral and non-toxic principle ellagitannins from pomegranate in prevention and control of HCV induced complications.

Figure 1. P. granatum crude extract, its different fractions and ellagitannins specifically suppress HCV NS3/4A protease activity.

(A) The purified NS3/4A protease enzyme was pre-incubated with increasing concentrations (1, 4, 6, 8 and 10 μg/mL) of P. granatum fruit peel and juice extracts followed by addition of the substrate (EGFP-NS5A/B site-CBD fusion protein). The ability of these extracts to inhibit substrate cleavage efficiency of protease was quantified by measuring fluorescence intensity. The relative enzyme activity was normalized with the DMSO vehicle control (denoted as C). (B) Experiment similar to panel ‘A' was performed with different fractions 1 to 3 and residue 3 (denoted as Fr 1–3 and Res-3) at a concentration of 10 μg/mL to identify the most active fraction. DMSO (vehicle) and crude fruit peel extract (denoted as CE) were used as mock and positive controls. (C) Experiment similar to panels ‘A' and ‘B' was performed with increasing concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 μM) of purified ellagitannins EA, PGN, PLN. Telaprevir (a known protease inhibitor) was used as positive control. ‘C' denotes DMSO vehicle control, ‘TEL' denotes telaprevir. (D) Cellular protease (trypsin) was incubated with its substrate FITC-casein in the presence of increasing concentrations (1.0, 2.5, 5.0, 10.0, 25.0 μM) of EA, PGN and PLN. Fluorescence intensity of cleaved product was quantified using fluorometer. Results shown as mean ± SD from three independent experiments and each were done in duplicates.

Figure 2. Depicting the molecular docking studies of ligand protein interactions.

(A) Left side: HCV NS3 protease complexed with punicalin: Here, punicalin (yellow) interacts with catalytic residues (green), substrate interacting residues (pink) and other residues (grey) in HCV NS3 protease. Interactions such as hydrogen bonds, hydrophobic bonds and aromatic-aromatic interactions are shown as dotted lines. All the ligand bound NS3 structures are generated using PyMOL. Right side: Zoomed in view of left side figure. (B) Left side: HCV NS3 protease complexed with punicalagin: Here, punicalagin (yellow) interacts with catalytic residues (green), substrate interacting residues (pink) and other residues (grey) in HCV NS3 protease. Interactions such as hydrogen bonds, hydrophobic bonds and aromatic-aromatic interactions are represented as dotted lines. Right side: Zoomed in view of left side figure.(C) Left side: HCV NS3 protease complexed with ellagic acid: Here, ellagic acid (yellow) interacts with catalytic residues (green), substrate interacting residues (pink) and other residues (grey) in HCV NS3 protease. Interactions such as hydrogen bonds, hydrophobic bonds, aromatic-aromatic interactions are shown as dotted lines. Right side: Zoomed in view of left side figure. The structures of protein-ligand complex figures were generated using PyMOL.

Figure 3. P. granatum principle ellagitannins inhibit HCV RNA replication.

(A) Huh7.5 cells were infected with HCV-JFH1 virus (genotype 2a) and treated with 150 μM of EA, PGN and PLN. At 48 h post treatment, HCV negative strand RNA synthesis was quantified using RT-qPCR. GAPDH (a house keeping gene) amplification was used as an endogenous control for normalization of data. Results are expressed in fold change of RNA levels. These results are representative of three independent experiments and are expressed as mean ± SD. (B) Huh 7.5 cells were transfected with HCV-H77S (genotype 1a) RNA and treated with PLN, PGN and EA (150 μM each). At 48 h post treatment, HCV RNA levels were quantified using RT-qPCR. (C) To determine the role of EA, PGN, and PLN in inhibiting HCV entry, Huh7.5 cells were infected with the HCV-JFH1 virus in absence and presence of the compounds (150 μM each). At 72 h of post treatment, total cellular RNA was extracted and subjected to RT-qPCR. ‘C' denotes control (no treatment). Data represent mean ± SD from three independent experiments.

Figure 4. Determination of cytotoxicity of P. granatum extract and ellagitannins.

(A) Huh7 cells were incubated with increasing concentrations of crude peel extract (CE) and residue-3 (0.1, 0.5, 1.0, and 2.5 mg/mL) and cytotoxicity was determined by MTT assay. Percentage cell viability was plotted considering 100% for DMSO vehicle control (denoted by C). (B) Experiment similar to panel A was performed with increasing concentrations (0.05, 0.1, 0.5, 1.0, 2.5, and 5.0 mM) of EA, PGN, and PLN. (C) JFH1 infected Huh7.5 cells were incubated with PGN, PLN and EA (150 μM) and percentage cell viability was plotted. ‘C' denotes ‘control with no infection and no treatment'. Results represent mean ± SD from three independent experiments and each was done in duplicates.

Figure 5

(A) Histopathological examination of mice liver to determine the toxicity of inhibitors. The acute toxicity effect of vehicle control (SDW, 500 μl/animal) and test samples (5000 mg/kg b.wt of CE, punicalin, punicalagin and ellagic acid) were evaluated in comparison with normal healthy mice at intra tissue level. The histological sections of healthy control mice liver retained normal tissue architecture. The liver sections of mice treated with pure compounds and CE showed near to normal arrangement of the hepatic cords, central veins and polygonal hepatocytes. Here, (a): normal control, (b) & (c): crude extract at an acute dose of 2000 and 5000 mg/kg b.wt, (d) & (e): punicalagin 2000 and 5000 mg/kg b.wt, (f) & (g): punicalin 2000 and 5000 mg/kg b.wt, (h) & (i): ellagic acid 2000 and 5000 mg/kg b.wt. Whereas, (j), (k) & (l): punicalagin, punicalin and ellagic acid at 1000 mg/kg/day for 28 days in subacute toxicity studies. An arrow indicates minimal multifocal hepatocellular necrosis observed in liver histology upon EA treatment for 28 days. (B) Systemic bioavailability of principle ellaginnins of P. granatum in BALB/c mice after oral administration. An acute dose of 1000 mg/kg b.wt of PGN, PLN and EAwas administered orally to three different groups of BALB/c mice separately. The availability of EA (an active metabolite of PGN and PLN) in plasma at different time points of post treatment (0 to 24 h) from 3 different groups was determined by HPLC analyses. Availability of these compounds in the plasma in HPLC chromatogram was identified based on the retention time of the standard reference compounds. The total amount of EA obtained in the plasma (per mL) was determined based on the standard calibration curve for EA and AUC (Area under the curve) was plotted. Pharmacokinetics parameters including maximum plasma concentration (C_max) and time (T_max) of EA was calculated. Values used to plot AUC are mean ± SD from the two biologically independent experiments.