Potential antiviral activities of chrysin against hepatitis B virus

Compound

Chrysin, a natural compound of cell culture grade was obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Dimethylsulphoxide (Sigma-Aldrich, USA, 100%) was used for dissolving chrysin and 5 mM stock concentration was prepared. Serial dilutions of drug were performed with the Dulbecco’s Modified Eagle's medium (DMEM) cell culture media to get variable concentrations used for in vitro studies. For subsequent experiments, the drug stock was stored in opaque container at 4 °C. The natural compound as well as the other reagents was of molecular biology grade.

Maintenance of cell line

Human hepatoma cell line HepG2 was obtained from National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured under standard cell culture conditions at 37 °C in a controlled humidified atmosphere with 5% CO2 supply and 100% humidity in CO2 incubator (NuAire NU-5830, USA). Culture was done in 25cm2T- flask (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle's medium (DMEM) enriched with nutrients such as glucose, sodium bicarbonate, sodium pyruvate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS), 1% (v/v) Penicillin–Streptomycin 10,000 U/mL solution (all from Invitrogen, San Diego, CA, USA). The cells were then collected from the flask using 0.25% trypsin (Gibco-BRL, Grand Island, NY, USA) and 1 mmol/L EDTA, and then revived for subsequent investigations. In the drug treatment experiments, cells with passage numbers of 2 to 15 were employed.

Cell toxicity

Prior to the study of antiviral activity against HBV, the cytotoxic effects of the experimental compound were assessed. 3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) (MTT) (Sigma, St. Louis, MO, USA) assay was employed to assess the viability and cytotoxicity, as reported earlier [32]. Briefly, 2 × 104 cells were seeded per well in 96 well plates (CoStar, Corning Inc., NY, USA) and incubated for 6–48 h. The cells were treated with variable concentrations (0, 2.5, 10 and 15 µM) of chrysin and incubated for 72 h. After treatment, MTT solution was prepared by dissolving in PBS (pH 7.4) with final concentration of 0.5 mg/mL. MTT reagent of 100 µL was added in all wells including control. Plate was again kept in culture incubator for 2 to 4 h. Dimethyl sulfoxide (100 µL) was added in each well to dissolve the formazan crystals. After 20 min of incubation, the dissolved dye was quantified spectrophotometrically by taking absorbance at OD 450 nm by an ELSIA plate reader (Bio-Rad, Hercules, CA, USA). The percent cell viability was calculated as per the formula:

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Chrysin showed its effect on cell viability. IC50 was calculated from the GraphPad Prism version 8.0 software (San Diego, CA. USA). The experiments were performed in triplicates in order to confirm reproducibility. The safer doses derived from this method were employed in subsequent studies.

Transfection with pHBV 1.3X plasmid

The pHBV 1.3X construct harbouring more than full length HBV genome was a kind gift from Dr. Joseph Kock, (Heidelberg, Germany) and Dr. Shiv Kumar Sarin (ILBS, Delhi, India). Briefly, HepG2 cells (2 × 105 cells/well) were seeded into 6-well plate and incubated for 24 h. For transfection, 1 μg of (pHBV 1.3X) wild type construct combined with 3μL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used per well (Costar, Corning Inc., NY, USA), according to the Lipofectamine 2000 manufacturer instructions.

Drug treatment to transfected HepG2 cells

The non-cytotoxic doses derived from MTT assay were used in subsequent studies. HepG2 cells were treated with safe doses (2.5, 5, 10 and 15 µM) of chrysin and incubated for 72 h. After 72 h post-transfection, culture supernatants were collected for the assessment of HBsAg, HBeAg, and extracellular HBV DNA. The cells were harvested with trypsin digestion, washed three times in phosphate buffered saline (PBS, pH 7.3). The amount of viral DNA in the cellular extract was measured.

Assessment of HBsAg and HBeAg in cell culture supernatant

To measure the HBV secretory proteins from culture supernatants of drug treated cells, the enzyme-linked immunosorbent assay (ELISA) was performed using commercially available ELISA kits-Hepalisa (J Mitra &Co, Delhi, India) and DIA. PRO, MI, (Italy) for HBsAg and HBeAg, respectively. According to the manufacturers’ instructions we performed the analysis of HBsAg and HBeAg in the culture supernatant. In order to confirm the reproducibility of independent experiment, assays were performed in triplicate. The data, presented here as % inhibition of HBsAg and HBeAg, were calculated by the following formula:

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Assessment of HBV DNA in the supernatant

After 72 h of post-treatment, we observed the effect of compound on the quantity of the extracellular HBV DNA. The culture supernatant was collected by centrifugation at 1200 rpm for 10 min at 4 °C and HBV DNA was isolated and quantified by qPCR (Roche Applied Science, Penzberg, Upper Bavaria, Germany) employing the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The oligonucleotide sequence of forward primer was 5′-CCG TCT GTG CCT TCT CAT CTG-3′, the sequence of reverse primer was 5′-AGT CCA AGA GTA CTC TTA TAG AAG ACC TT-3′, and the sequence of Taqman probe was FAM-CCG TGT GCA CTT CGC TTC ACC TCT GC. The PCR program performed included an initial denaturation at 94 °C for 2 min trailed by 40 amplification cycles with each of the two subsequent steps: 95 °C for 5 s and 60 °C for 30 s. Plasmid containing more than the full-length insert of the HBV genome was used to form a standard curve. The standard curve exhibited a satisfying linear range when around 102–107 copies of plasmid DNA were used as template. The inhibitory effects of chrysin on HBV DNA were calculated by the following formula:

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Purification and quantification of intracellular HBV cccDNA

The effect of chrysin on the level of intracellular cccDNA was observed 72 h post-treatment. For this, cccDNA was isolated from the cell pellet containing 1.0 × 106 cells using mini plasmid extraction Kit (QIAGEN Inc., Chatsworth, CA, USA) following the manufacturer’s instructions. The isolated plasmid was further treated with plasmid safe ATP-dependent DNase (PSAD, Epicentre Technologies, Madison, WI, USA), for 2 h at 37 °C, to eliminate HBV relaxed circular DNA (rcDNA), residual single-stranded viral DNA and cellular chromosomal DNA. This ATP-dependent DNase degrades linear single-stranded and double-stranded DNA, but acts moderately on closed circular double-stranded DNA. The real-time fluorescent quantitative PCR was performed with gene specific primers and Taqman TAMRA fluorescence hybridization probe to detect cccDNA. The sequence of forward primer was 5′-ACT CTT GGA CTC TCA GCA ATG-3′, sequence of reverse primer was 5′-CTT TAT AAG GGT CGA TGT CCA-3′ and sequence of Taqman probe was FAM-CTT TTT CAC CTC TGC CTA ATC ATC TCT TGT TCA- TAMRA. Because of the structural dissimilarities between cccDNA and rcDNA, only cccDNA will be amplified with the designed primers and probe set. PCR conditions were: denaturation for 2 min at 95 °C, followed by 38 cycles of denaturation at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s. The relative quantification of cccDNA was calculated using 2−∆∆Ct method as previously described [33].

Chemical structures and molecular docking studies

In the present study in silico approaches were also used  to trace the active compounds against Hepatitis B virus. It is a convenient drug screening method, where the candidate drugs can be virtually examined at low cost in a short duration. This involves computational simulation of target-ligand interaction and regulates the perfect alignment of binding of one molecule to the second molecule to generate a stable complex. This method, called docking, is used to find the activity of binding of a tiny molecule (in this case, a drug candidate- chrysin, and a positive control lamivudine) to their protein target (in this case HMGB1) by using the scoring functions.

This screening method plays a pivotal role in the functional designing of drug candidates.

Target preparation

HMGB1 (PDB ID: 1AAB) is a signalling protein present in the nucleus. Its crystal structure was downloaded from protein data bank (http://www.pdb.org/). We removed the crystallographic water molecules or ligands, in order to produce a free receptor, and to enhance the entropy of the target. The missing polar hydrogen atoms were incorporated, and the energy level of target was minimized while using Swiss_PDB viewer tool. With the help of this server, we speculated the 3D structure of HMGB1 protein. Autogrid 4 module, a bioinformatics tool used to map the protein’s 3D structure, covered all the amino acid residues of the protein. The grid three dimensions X, Y and Z were fixed to be 50, 70 and 58 Å (receptor axis coordinates), and 0.405 Å as grid space size for lamivudine-HMGB1; and 52, 83 and 58 Å (receptor axis coordinates), and 0.435Å as grid space size for chrysin–HMGB1.

Ligand preparation

The molecular structure of lamivudine (positive control) and chrysin was drawn by ChemDraw12 (PerkinElmer Informatics, Waltham, MA, USA) as shown in Fig. 1A and B, respectively. The molecular structure of compounds was converted into 3D form, and geometries were optimized in ChemBio3DUltra12 (PerkinElmer Informatics, Waltham, MA, USA). For docking studies, the tested compound chrysin and positive control lamivudine was saved in PDB format. Molecular docking was performed by using Auto dock 4.2 in order to achieve better insights into the binding mechanism of chrysin and lamivudine with HMGB1. Docking guidelines were followed in this docking simulation. To achieve molecular docking, Lamarckian Genetic Algorithm (LGA) was used to define the best potential structures of the ligands that directly interacted with the target protein. Here the ligand was allowed free to explore and interact with the protein's active site in the best possible or threshold energy configuration. Ideal docked configurations were archived and studied for further interaction between receptor-ligand, employing BOVIA Discovery Studio 4.0 to generate 2D interaction plot [34]. Docking was eventually visualized by Pymol [35]. Lamivudine, a nucleoside analogue approved by FDA for the treatment of chronic hepatitis B virus infection, was also docked with the same protein. In the molecular docking analysis, lamivudine was used as a positive control. For protein–ligand interaction, the binding constant (Kb) was calculated using equation (∆G = − RTlnKb (R = universal gas constant, 1.987 kcal/mol/; T = temperature, 298 K) [36].

Fig. 1figure 1

A and B represents optimized two-dimensional molecular geometries of anti-HBV compounds lamivudine (nucleoside analogue used as a reference drug in molecular docking analysis only) and chrysin respectively

ADMET examination

The drug-likeness analysis was performed by admetSAR and SwissADME to confirm any cytotoxicity produced by ligands in humans. Several pharmacokinetics properties such as absorption, distribution, metabolism, excretion and toxicity (ADMET) of the tested compound chrysin and the positive control lamivudine were measured with the web tools admetSAR [37] and SwissADME [38]. The physicochemical properties were also studied with the help of these servers.

Statistical analysis

All the results were presented as mean ± standard deviations (SD) of triplicate experiments standardized to control. In the present study, a standard statistical test such as one-way analysis of variance (ANOVA) was done, and data were analyzed to evaluate where precise variances were existing between the samples. Moreover, to define statistical significance Dennett’s t-test was used. GraphPad Prism software version 8.0 was used to calculate the mean and standard deviation, and draw graphs for the results of ELISA, MTT, HBsAg, HBeAg and viral DNA assays. MTT assay was expressed in terms of percentage viability, percentage cytotoxicity and IC50 value, p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

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