Summary
We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).
Abstract
Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.
Introduction
Hepatitis B virus (HBV) infection is considered a life-threatening disease worldwide. Chronic HBV infection is laden with a risk of liver cirrhosis and hepatocellular carcinoma1. Current anti-HBV treatment focuses mostly on post viral entry using nucleos(t)ide analogs (NAs) and interferon-alpha (IFN-α)2,3. The discovery of an HBV entry inhibitor, Myrcludex B, has identified a novel target for anti-HBV agents4. The combination of entry inhibitors and NAs in chronic HBV has significantly lessened the viral load compared to those targeting viral replication alone5,6. However, the classical hepatocyte model for the screening of HBV entry inhibitors is limited by low viral receptor levels (sodium taurocholate cotransporting polypeptide, NTCP). The overexpression of hNTCP in hepatoma cells (i.e., HepG2 and Huh7) improves HBV infectivity7,8. Nevertheless, these cell lines express low levels of phase I and II drug-metabolizing enzymes and exhibit genetic instability9. Hepatocyte models that can help target distinct mechanisms of candidate anti-HBV compounds such as previral entry, NTCP binding, and viral entry would expedite the identification and development of efficacious combination regimens. The study for anti-HBV activity of curcumin has elucidated the inhibition of viral entry as a new mechanism in addition to post viral entry interruption. This protocol details a host model for the screening of anti-HBV entry molecules10.
The goal of this method is to explore candidate anti-HBV compounds for viral entry inhibition, especially blocking NTCP binding and transport. As NTCP expression is a critical factor for HBV entry and infection, we optimized the hepatocyte maturation protocol to maximize NTCP levels11. In addition, this protocol can differentiate the inhibitory effect on HBV entry as inhibition of HBV attachment versus inhibition of internalization. The taurocholic acid (TCA) uptake assay was also modified using an ELISA-based method instead of a radioisotope to represent NTCP transport12,13. The receptor and ligand interaction was confirmed by their 3D structures14,15. The inhibition of NTCP function can be evaluated by measuring TCA uptake activity16. However, this technique did not provide direct evidence of NTCP binding to the candidate inhibitors. Therefore, the binding can be investigated using various techniques, such as surface plasmon resonance17, ELISA, fluorescence-based thermal shift assay (FTSA)18, FRET19, AlphaScreen, and various other methods20. Among these techniques, ITC is a goal standard in binding analysis because it can observe heat absorption or emission in almost every reaction21. The binding affinity (KD) of NTCP and candidate compounds was directly evaluated using ITC; these affinity values were more precise than those obtained using the in silico prediction model22.
This protocol covers techniques in hepatocyte maturation, HBV infection, and screening for HBV entry inhibitor. Briefly, a hepatocyte model was developed based on imHC and HepaRG cell lines. The cultured cells were differentiated into mature hepatocytes within 2 weeks. The upregulation of NTCP levels was detected using real-time PCR, western blot, and flow cytometry11. Hepatitis B virion (HBVcc) was produced and collected from HepG2.2.15. The differentiated imHC or HepaRG (d-imHC, d-HepaRG) was prophylactically treated with the anti-HBV candidates 2 h prior to the inoculation with HBV virion. The expected outcome of the experiment was the identification of the agents that decrease cellular HBV and infectivity. Anti-NTCP activity was evaluated using the TCA uptake assay. NTCP activity could be suppressed by the agents that specifically bound NTCP. The ITC technique was employed to investigate the feasibility of interactive binding that could predict inhibitors and their target proteins, determining the binding affinity (KD) of the ligand for the receptor via non-covalent interactions of the biomolecular complex23,24. For instance, KD ≥ 1 × 103 mM represents weak binding, KD ≥ 1 × 106 µM represents moderate binding, and KD ≤ 1 × 109 nM represents strong binding. The ΔG is directly correlated with binding interactions. In particular, a reaction with negative ΔG is an exergonic reaction, indicating that binding is a spontaneous process. A reaction with a negative ΔH indicates that the binding processes depend on hydrogen bonding and Van der Waals forces. Both TCA uptake and ITC data could be used to screen for anti-HBV entry agents. The outcomes of these protocols can provide a foundation for not only anti-HBV screening but also the interaction with NTCP as assessed through binding affinity and transport function. This paper describes host cell preparation and characterization, experimental design, and evaluation of the anti-HBV entry together with the NTCP binding affinity.
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Protocol
NOTE: The following procedures must be performed in a Class II biological hazard flow hood or a laminar-flow hood. The handling of HBV was ethically approved by the IRB (MURA2020/1545). See the Table of Materials for details about all solutions, reagents, equipment, and cell lines used in this protocol.
1. Preparing host cells (mature hepatocytes)
- Culture hepatocytes (3.75 × 105 cells HepaRG or imHC) and maintain in a 75 cm2 culture flask with 10 mL of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Wait for the cells to reach 80%-90% confluence (within 1 week).
- Discard the old medium from the flask and wash the cells with 4 mL of phosphate-buffered saline (PBS).
- Aspirate the PBS and add 4 mL of 0.05% trypsin-EDTA.
- Incubate the flask in the 37 °C incubator for 2-3 min, and check the adherent cells using an inverted microscope with a 10x objective lens. Ensure that the monolayer has detached from the culturing surface.
- Inhibit the trypsin by adding 4 mL of the culture medium supplemented with 10% FBS to the flask and carefully resuspend the harvested cells. Transfer the cell suspension into a 15-mL conical tube and centrifuge for 3 min at 300 × g, 25 °C.
- Aspirate the supernatant from the cell pellet and resuspend with 1-2 mL of the prewarmed culture medium supplemented with 10% FBS and antibiotics.
- Mix 10 µL each of the cell suspension and trypan blue and count the cells using a hemacytometer. Ensure that the cell viability is higher than 90% before proceeding to the next step.
- Seed 1 × 106 cells per 2 mL in each well of a 6-well plate and maintain in DME/F12 complete medium until the cells reach 100% confluence.
- For hepatic maturation, prepare hepatic maturation medium (Williams' E medium supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 50 µM hydrocortisone, 2 mM L-glutamine, and 2% DMSO).
- Replace the culture medium 2x per week.
- Maintain the hepatocytes in maturation medium for 2 weeks to obtain mature hepatocytes ready for HBV infection.
2. Quantification of cellular NTCP
- Measuring NTCP expression using real-time PCR
- Collect the pellet of mature hepatocytes through trypsinization (see Steps 1.2-1.5).
- Extract total RNA from the cell pellet using an RNA extraction kit with DNase I treatment.
- Synthesize cDNA from 200 ng of total RNA using an oligo-dT primer and reverse-transcription system following the manufacturer's instructions.
- Dilute the cDNA to 50 ng/µL and use it as a template for the PCR reaction. Mix 2 µL of the diluted cDNA with the PCR master mix reagents containing SYBR green qRT-PCR Kit solution with 5 µM NTCP-specific primers (5'-GGACATGAACCTCAGCATTGTG-3' and 5'-ATCATAGATCCCCCTGGAGTAGAT-3').
- For normalization, use GAPDH as a housekeeping gene with specific primers (5'-GAAATCCCATCACCATCTTCC-3' and 5'-AAATGAGCCCCAGCCTTCTC-3').
- Amplify the cDNA samples using a thermal cycler under the following temperature conditions: 1 cycle of 3 min at 95 °C (initial denaturation); 40 cycles of 30 s at 95 °C (denaturation), 30 s at 60 °C to 65 °C (annealing), 30 s at 72 °C (extension); 1 cycle of 5 min at 72 °C (final extension).
- Calculate the NTCP fold change in mature hepatocytes over undifferentiated cells using GAPDH as a calibrator gene based on the 2-ΔΔCT method.
- Quantifying NTCP using western blot analysis
- Harvest hepatocytes using a cell scraper and centrifuge them at 300 x g, 25 °C for 5 min to collect the cell pellet.
- Follow the RIPA buffer manufacturer's instructions to extract the cellular protein with 100 µL of RIPA buffer containing 1x protease inhibitor.
- Measure the total protein concentration using the bicinchoninic acid (BCA) method.
- Mix 12.5 µL of protein with 2x loading dye in a 1:1 ratio by volume.
- Boil the protein mixture and standard ladder at 95 °C for 5 min.
- Add 1x running buffer to the electrophoresis chamber.
- Load 5 µL of the protein standard ladder or 20 µL of the boiled protein mixture in a 10% (w/v) polyacrylamide gel.
- Perform gel electrophoresis: 50 V for 30 min followed by 90 V for 1 h at room temperature.
- Prepare a PVDF membrane by soaking it in methanol for 30 s, wash it with double-distilled water for 1-2 min, and soak it along with two pieces of filter paper in transfer buffer for 10 min or until use.
- Place the filter paper onto the anode plate of a semi-dry transfer cell; then, place the PVDF membrane, the gel, and the filter paper on top, in that order.
- Transfer the proteins from the gel to the PVDF membrane at 10 V for 25 min at room temperature.
- Block the membrane with WB blocking solution for 1 h at room temperature.
- Incubate the membrane with anti-sodium taurocholate cotransporting polypeptide (anti-NTCP) (1:100 dilution) at 4 °C overnight.
- Wash the membrane 3 x 10 min with TBST.
- Incubate the membrane with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:10,000 dilution) for 1 h at room temperature.
- Wash the membrane 3 x 10 min with TBST and then 1 x 5 min with TBS.
- Detect NTCP using an HRP substrate (see the Table of Materials).
- Add at least 0.1 mL of HRP substrate solution/cm2 of the membrane and incubate the membrane for 3-5 min in the dark.
- Visualize NTCP using a Gel Documentation System in the chemiluminescence mode, select the marker option for the membrane, adjust the exposure time with accumulative mode every 1 min, and take the photo with various exposure times for 5 min.
- Strip and probe the blot with mouse anti-GAPDH antibody (1:200,000 dilution) and HRP-conjugated goat anti-mouse secondary antibody (1:10,000 dilution).
- Measuring the NTCP level using flow cytometry
- Collect the cell pellets of mature hepatocytes by incubating the cells with 8 mM EDTA for 15 min.
NOTE: Avoid using trypsin or other proteases that can disrupt cell surface receptors. As NTCP is a cell surface receptor protein, damage due to trypsinization can give negative results. - Fix the hepatocytes with 300 µL of 3.7% paraformaldehyde in 1x PBS for 15 min. Transfer the cell suspension into a 1.5 mL microcentrifuge tube and centrifuge for 10 min at 300 × g, 25 °C.
- Wash the cells 2x with 700 µL of 1x PBS with 1% FBS (v/v), centrifuge for 10 min at 300 × g, 25 °C, add 300 µL of 0.05% Triton X-100 in PBS to the cell pellet, and incubate the cells at room temperature for 20 min to permeabilize them.
- Centrifuge for 10 min at 300 × g, 25 °C, wash the cell pellet 3 x 1 min with 700 µL of PBS containing 1% FBS (v/v). Incubate the cells with the primary antibody against NTCP (1:100 dilution) at 4 °C for 30 min. Wash the cells 3 x 1 min with Perm/Wash buffer and centrifuge for 10 min at 300 × g, 25 °C.
- Stain the cells with secondary antibody conjugated to Alexa Fluor 488 at 4 °C for 30 min. Wash the cells 3 x 1 min with Perm/Wash buffer and centrifuge for 10 min at 300 × g, 25 °C. Resuspend the cell pellet with 200 µL of PBS with 1% FBS (v/v).
- Turn on the flow cytometer, warm up the laser for 15 min, and perform routine cleaning.
- Select the measurement parameters, including forward scatter (FSC), side scatter (SSC), and fluorescein isothiocyanate (FITC) or phycoerythrin (PE), and set the auto compensation adjustment by the software. Include the compensation control samples: unstained control, FITC-stained control, and PE-stained control.
NOTE: FSC and SSC parameters are used to determine the size and granularity of individual cells to select the cell population for gating analysis. The fluorescence channel detector has FITC and PE channels. For this protocol, select the FITC channel to detect Alexa Fluor 488. - Run the unstained sample with medium fluidic flow rate (60 µL/min), adjust the threshold of FSC and SSC until they cover the cell population of interest, mark the gate region in this area, and apply to all compensation controls.
- Set the fluorescence photomultiplier tube (PMT) using the unstained control, and adjust the PMT voltage of FITC or PE in the negative quadrant. Run the positive stained sample and adjust FITC or PE in the positive quadrant scale. Run the unstained, FITC-stained, or PE-stained controls, calculate the compensation, and apply it to the sample settings. Run the hepatocyte sample to measure the NTCP level.
- Analyze the stained hepatocytes using flow cytometer software (see the Table of Materials).
- Under BD FACSuite windows, click on the control tube in the Data Sources Tab and wait for the raw data to appear.
- In the Worksheet tab on the right-hand side, click on the Ellipse Gate button, select the cell population in SSC and the FSC dot plot using the mouse cursor, and wait for the circle P1 to appear.
- Click on the Interval Gate button and mark the region in the FITC histogram, starting from the highest fluorescence intensity value to the right-hand side. Observe the line P2 that appears.
- Click on the experiment tube and observe that the data in the Worksheet tab changes. Click on the Statistics button and then on the empty space in the worksheet. Observe the statistical values of the NTCP population that appear.
- Collect the cell pellets of mature hepatocytes by incubating the cells with 8 mM EDTA for 15 min.
3. Production of the cell culture-derived HBV particles (HBVcc)
- Culture HepG2.2.15 in complete medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) in a 10 cm Petri dish for 24 h.
- Replace the complete medium supplemented with 380 µg/mL geneticin (G418) when the HepG2.2.15 reach 60%-70% confluence. Harvest the conditioned medium every 2 days; store the medium containing HBV at 4 °C.
- Remove cell debris by centrifuging the supernatant at 5,000 × g, 4 °C for 20 min. Collect the conditioned medium using a 20 mL syringe and filter it through a 0.45 µm low-protein-binding filter to remove cell debris.
- To concentrate HBV, dilute 1 volume of concentrator with 3 volumes of the filtered supernatant from Step 3.3 in a conical centrifuge tube and carefully mix the solution by tube inversion. Place the mixture at 4 °C for 1 h. Centrifuge the solution for 1 h at 1,500 × g, 4 °C and ensure that an off-white pellet is visible.
NOTE: For every 30 mL of filtered supernatant from Step 3.3, add 10 mL of the concentrator to reach 40 mL of total volume. - Evaluate the HBV titer (genome equivalent) with HBV 1.3-mer WT replicon as a standard curve ranging from 1 × 102 to 1 × 1010 using absolute real-time PCR, as described previously11.
- Gently reconstitute the pellet in FBS and store for up to 1 year at −80 °C in single-use aliquots.
4. Anti-HBV entry assay
- Maintain 100% confluent imHC or HepaRG in 6-well plates in maturation medium (2% DMSO in Williams' E medium, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 50 µM hydrocortisone, and 2 mM L-glutamine) for 2 weeks and change the medium 2x per week.
- Aspirate the medium, then add the curcumin (10-30 µM) or 4 µM cyclosporine A (CsA) diluted with William's E medium for 2 h. Aspirate the candidate HBV entry inhibitor and replace it with 1 mL of William's E medium containing 4% polyethylene glycol.
- Add HBV particles to the culture medium at a multiplicity of infection (MOI) of 100 per well and incubate at 37 °C for 18 h. Discard the supernatant, add 2 mL of cold PBS, and swirl gently to rinse off the old medium with unbound HBV.
- Add 2 mL of complete William's E medium and culture for 7 days. Aspirate the medium, wash the cells with 1x PBS, and fix the cells with 3.7% paraformaldehyde in PBS for 15 min at room temperature. Permeabilize and block the infected cells with IF blocking solution (0.2% Triton X-100 and 3% bovine serum albumin in PBS) and incubate them for 60 min at room temperature.
- Add the primary antibody (anti-HBV core antigen) at 1:200 dilution in the blocking solution and incubate overnight at 4 °C. Wash the cells 3 x 1 min with washing solution (0.5% Tween 20 in PBS).
- Add secondary antibody (1:500 dilution) to the IF blocking solution, incubate for 1 h at room temperature, and wash the cells 3 x 1 min with washing solution.
- Mount the sample with antifade mounting medium with 4',6-diamidino-2-phenylindole (DAPI), and cover with a glass coverslip. Detect the fluorescence signal using a fluorescence microscope equipped with a DAPI filter (Ex 352-402 nM and Em 417-477) and a PE filter (Ex 542-582 and Em 604-644), along with a 20x objective lens, and determine anti-HBV entry activity of the candidate compounds.
5. HBV-cell binding assay
- Maintain 100% confluent imHC or HepaRG in 6-well plates in maturation medium (2% DMSO in Williams' E medium, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 50 µM hydrocortisone, and 2 mM L-glutamine) for 2 weeks and change the medium 2x per week.
- Aspirate the medium, then add curcumin (10-30 µM) or cyclosporine A (4 µM) diluted in William's E medium for 2 h. Aspirate the HBV entry inhibitor and replace it with 1 mL of William's E medium containing 4% polyethylene glycol.
- Add HBV particles to the culture medium at an MOI of 100 per well and incubate at 4 °C for 2 h to allow HBV binding to the hepatocytes. Discard the medium containing unbound HBV, add 2 mL of cold PBS, and swirl gently to remove traces of the spent medium.
- Harvest the infected cells using a cell scraper and disrupt the cells with lysis buffer. Transfer the lysate to a collection tube and extract total DNA to evaluate the HBV DNA using real-time PCR with specific primers for HBV (forward primer: 5´- GTT GCC CGT TTG TCC TCT AATTC-3´, and reverse primer: 5´- GGA GGG ATA CAT AGA GGT TCC TTG A-3´) using PRNP (forward primer: 5´- GACCAATTTATGCCTACAGC-3´, reverse primer: 5´- TTTATGCCTACAGCCTCCTA-3´) as an internal control.
- Amplify the PCR products in a thermal cycler using the temperature conditions described in Step 2.1.
- Calculate relative HBV DNA levels/1 × 106 cells in treatment versus control using PRNP as the calibrator gene based on the 2-ΔΔCT method.
6. Taurocholic acid (TCA) uptake assay
- Maintain 100% confluent imHC and HepaRG cells in maturation medium for 14 days.
- Aspirate the medium and wash the hepatocytes with 1x PBS. Add curcumin or cyclosporine A (10-50 µM) diluted in William's E medium for 2 h. Replace the medium with the sodium taurocholic acid solution (0.5 M sodium taurocholate hydrate in 1x HEPES) and incubate for 15 min at room temperature.
- Aspirate the sodium taurocholic acid solution, and wash 3x with cold 1x HEPES. Add 300-500 µL of cold 1x HEPES and harvest the cells with cell scrapers on ice.
- Homogenize the cells by ultrasonication at a frequency of 20 kHz for 20 s and incubate on ice for 5 min. Centrifuge the lysates at 10,000 × g for 20 min to precipitate cell debris. Collect the supernatant and evaluate intracellular taurocholic acid concentration using a TCA ELISA assay kit (see the Table of Materials).
7. Determination of protein-ligand interactions using isothermal titration calorimetry
NOTE: This assay system was developed based on the MicroCal PEAQ-ITC (ITC software).
- Prepare the buffer (50 mM Tris-HCl buffer, pH 7.0).
- Prepare 15 µM of NTCP in the buffer.
- Prepare 150 µM candidate HBV entry inhibitor solutions in the buffer.
- Wash the sample cell, the reference cell, and the syringe in the ITC instrument using the buffer (Step 7.1.).
- Launch the ITC software by double-clicking on the software icon in Windows systems. Under the Run experiment highlight tab, select microCal Method for 19 Injection.itcm, and click on open. When a new window opens, click on the clean tab, and in the Cleaning Method window, select the Wash button for Cell Cleaning Method and the Rinse button for Syringe Cleaning Method. Click on Next and follow the on-screen instructions.
NOTE: The washing step can take 1.4 h to complete.
- Launch the ITC software by double-clicking on the software icon in Windows systems. Under the Run experiment highlight tab, select microCal Method for 19 Injection.itcm, and click on open. When a new window opens, click on the clean tab, and in the Cleaning Method window, select the Wash button for Cell Cleaning Method and the Rinse button for Syringe Cleaning Method. Click on Next and follow the on-screen instructions.
- Load the sample into the ITC; under the Run Experiment tab, click on the Load button, and read the on-screen instructions. Click on next and follow the video instructions until this loading step is completed.
- Fill the reference cell with solution buffer using a syringe. Fill the sample cell with 15 µM NTCP in buffer using a syringe and remove the excess NTCP solution over the sample cell. Fill the syringe with 150 µM solution of the curcumin (ligand), avoiding air bubbles in the syringe.
- Run the sample, and under the Run Experiment tab, click on the Run button. Observe the windows on the left-hand side showing experimental information and experimental setting. Enter the ligand and protein concentrations as 150 µM in [Syr], 15 µM in [Cell], and 25 °C in temperature.
- In the software, click on start to perform the injection process and wait for 1.4 h for the protein-ligand interaction to complete.
NOTE: The faded analyze button appears under the software windows. - Observe that the analyze button brightens after the injection is complete. Click on the analysis button in the control software to analyze the raw data using the analysis software. Click on overview to display the raw data and choose the fitting model as One Set of Binding Site.
- Ensure that binding status shows the experimental data (binding, no binding, control, or check data) and that the experiment values (KD, ΔG, ΔH, TΔS, and N) are presented on the software panel.
- Export the thermodynamic parameters that predict the interaction binding, including hydrogen bond, van der Waals bond, and hydrophobic interaction into tif or jpeg format.
- After the experiment is complete, wash the sample cell following Step 7.4.
8. Statistical analysis
- Perform all experiments in three independent replicates (n = 3).
- Present experimental data as means ± SD, and perform statistical analysis using the desired statistical analysis software.
- Use a two-tailed unpaired Students' t-test to compare between two mean values or apply one-way analysis of variance (ANOVA) with Dunnett for multiple comparisons between multiple values to a control value. Set the significance level at p ≤ 0.05.
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Representative Results
Hepatic maturation features were observed, including binucleated cells and polygonal-shaped morphology (Figure 1), especially in the differentiated stage of imHC (Figure 1A). A large increase in NTCP expression was measured in d-HepaRG and d-imHC at 7-fold and 40-fold, respectively (Figure 1B). The highly glycosylated form of NTCP, postulated to confer susceptibility to HBV entry, was detected more in d-imHC than in d-HepaRG (Figure 1C). The differentiated imHC contained 65.9% higher NTCP levels than did the undifferentiated cells (Figure 1D).
Figure 2A shows a summary of the prophylactic treatment. Figure 2B shows HBV immunofluorescence staining performed to evaluate the anti-HBV entry activities on day 7 post infection, while Figure 2C outlines the HBV binding assay protocol. The level of HBV binding on the cell surface receptor was evaluated by real-time PCR (Figure 2D). To determine whether NTCP was the receptor for HBV attachment, TCA uptake was determined for the candidate binding inhibitors (Figure 2E). Based on this model, the putative inhibitory activity of candidate compounds decreased HBV binding through NTCP.
The calorimetric alteration was detected with continuous injections to the sample cell (Figure 3). A standard non-linear least squares regression was plotted based on one binding site fitted well to the data. The solid line indicated the best fit to the experimental values (Figure 3A,B). Table 1 shows thermodynamic parameters correlated with the binding of NTCP with cyclosporine A (CsA) or a candidate compound.
Figure 1: Hepatic maturation of HepaRG and imHC with NTCP characterization. Both cell lines were cultured in hepatic maturation medium for 2 weeks. (A) Hepatocyte characteristics such as binucleated cells and polygonal-shaped morphology were exclusively observed in the maturation stage of imHC. Scale bars = 50 µm. (B) The expression of NTCP was upregulated in both HepaRG and imHC. NTCP normalized with GAPDH was higher in imHC than in HepaRG. (C) A highly glycosylated form of NTCP was observed after maturation. (D) Percentage of NTCP elevation in d-imHC was evaluated using flow cytometry. Data are presented as mean ± SD. *, **, and *** represent statistical difference with p < 0.05, p < 0.01, and p < 0.001, respectively. Abbreviations: NTCP = sodium taurocholate cotransporting polypeptide; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; NTCP-FITC = fluorescein isothiocyanate-labeled NTCP. Please click here to view a larger version of this figure.
Figure 2: Prophylactic CCM treatment decreased viral entry, intracellular HBV DNA, and TCA uptake activity. (A) d-imHC were pretreated with CCM for 2 h prior to the inoculation with HBV. (B) Anti-HBV entry activity was determined by immunofluorescence staining on day 7 post infection. (C) A schematic schedule of HBV binding protocol is presented. (D) The level of bound HBV DNA on hepatocytes was evaluated and compared with 4 µM CsA and the classical HBV entry inhibitor 25 units/mL heparin. (E) The effect of the 10-30 µM CCM on TCA uptake inhibition was mediated through the NTCP receptor. Abbreviations: CCM = curcumin; HBV = hepatitis B virus; TCA = taurocholic acid; d-imHC = differentiated imHC; CsA = cyclosporine A; HP = heparin. Please click here to view a larger version of this figure.
Figure 3: The affinity of NTCP to individual molecules (CsA or candidate compound) was demonstrated using ITC. The ITC profile for the combination of NTCP with either (A) CsA or (B) curcumin was generated from sequential injection of a compound (150 μM CsA or candidate) into the NTCP solution (15 µM of NTCP, pH 7.0, 25 °C). The calorimetric raw data between differential power (µcal/s) and time (min) were plotted. Data were analyzed based on a one-site binding model where the solid lines indicated the best-fit results. During the injection of ligands (CSA or curcumin) into the NTCP solution, the enthalpy (ΔH) changed after an increase in the ligand concentration in the sample cell. These data indicate that the binding reaction occurred. Abbreviations: CsA = cyclosporine A; NTCP = sodium taurocholate cotransporting polypeptide; ITC = isothermal titration calorimetry; DP = differential power. Please click here to view a larger version of this figure.
| Protein | Ligand | Binding constant (KD) | Enthalpy change (ΔH) | Entropy change (ΔTΔS) | Gibbs Free Energy change (ΔG) | Type of binding |
| (M-1) | (kcal/mol) | (kcal/mol) | (kcal/mol) | |||
| Human NTCP (SLA10A1) | CCM | 1.21 ×10-6 | -1 | 0.6 | -0.39 | Hydrogen bond |
| Human NTCP (SLA10A1) | TCA | 1 ×10-9 | 80 | -96 | -16 | Hydrophobic interaction |
Table 1: Thermodynamic parameters resulting from the interaction between NTCP and individual compounds (CCM and TCA) using ITC. Abbreviations: NTCP = sodium taurocholate cotransporting polypeptide; CCM = curcumin; TCA = taurocholic acid; ITC = isothermal titration calorimetry.
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Discussion
HBV infection is initiated via low-affinity binding to heparan sulfate proteoglycans (HSPGs) on hepatocytes25, followed by the binding to NTCP with subsequent internalization through endocytosis26. As NTCP is a crucial receptor for HBV entry, targeting HBV entry can be clinically translated to diminish de novo infection, mother-to-child transmission (MTCT), and recurrence after liver transplantation. Interrupting viral entry would be a feasible alternative cure for chronic HBV infection.
Some critical steps in the above-mentioned protocol are summarized here. While preparing host cells, ensure that hepatocytes reach 100% confluency before adding the maturation medium with 2% DMSO. Culturing subconfluent hepatocytes in 2% DMSO will lead to cell death and detachment. The maturation period can be extended up to 4 weeks to maximize NTCP expression27,28, although a 2 week maturation period is recommended.
Three techniques have been proposed for the quantification of cellular NTCP expression. It is recommended that users select the technique they are most familiar with; here, we preferred flow cytometry to western blot analysis because it is convenient, quick, and easy.
For the production of the cell culture-derived HBV particles (HBVcc), HepG2.2.15 was derived from HepG2 by transient transfection with HBV full-length genome together with the geneticin (G418) resistance gene29. The culture medium should contain 380-500 µg/mL geneticin, otherwise the cells will not produce HBVcc. The low-protein-binding, 0.45 µm filter should be used to clarify the supernatant to avoid losing HBV yield. For concentrating HBV, polyethylene glycol (PEG) was used with a standard centrifuge. HBV particles can also be concentrated using a sucrose gradient and ultracentrifugation. Multiple freeze and thaw cycles should be avoided since the infectivity will be decreased.
In the anti-HBV entry assay, the hepatocytes must reach 100% confluence before maturation induction. This protocol was developed based on prophylactic treatment. Host cells were exposed to the candidate compounds for 2 h prior to infection with HBV12. After 7 days post infection, HBV infectivity was evaluated using immunofluorescence. All components, concentrations of the blocking solution, primary and secondary antibodies, and washing steps must be optimized to achieve the best result with minimal non-specific staining. Here, we have provided optimized concentrations and techniques.
The TCA uptake assay protocol describes the gold standard for the inhibition of NTCP transport. We modified the protocol to avoid radioactive TCA. The critical steps for the TCA uptake assay are cell washing and harvesting. All these procedures must be performed on ice all the time to prevent further cellular activity-internalization of the compound. After homogenizing the cells and pelleting cell debris, the supernatant must be gently aspirated without disrupting the pellet. If the user's facility permits the use of the liquid scintillation technique, 3H-taurocholic acid is commonly used in TCA transport and uptake studies. As we validated TCA uptake using ELISA, this alternative technique can substitute the use of the radioactive compound.
ITC is a biophysical method widely used to determine the thermodynamic parameters linked to the interactions between soluble proteins and small molecular weight ligands30,31. This technique can be used to investigate the interaction of various ligands with a small molecule or protein. ITC is a direct and noninvasive method with no additional steps for signal detection, no molecular weight limitations, and no labeling, immobilization, or any other chemical modification. The limitation of ITC is the prerequisite of high concentrations of the interacting materials. Protein and ligand have to be highly purified and sufficiently soluble in water (10-100 µM) at 25 °C for 1 h with stirring. The unstable protein solution can be detected through heat signal change as a function of time.
Human enhanced-NTCP hepatocytes provide an alternative model for the in vitro study of HBV infection but are similar to HepaRG and primary human hepatocytes. These host models are hampered by their limited reliability and susceptibility8,33. The inefficient HBV propagation in HepG2-NTCP or Huh7-NTCP cells was evidenced by low HBV inoculum derived from HBVcc-producing cells. In several studies, HBV was used to infect the target cells at an MOI of 1,00033,34. The subviral particles in HBVcc contain HBV envelope protein (L/M/S) and competitively bind NTCP, resulting in decreased infectivity35. To overcome this limitation, this protocol modified HepaRG or imHC culture with a maturation protocol to maximize NTCP levels. HBVcc derived from HepG2.2.15 was concentrated before being used as inoculum. The HBV infectivity was higher than 80% in differentiated imHC based on the measurement of the HBV core antigen11.
We have described the identification of anti-HBV compounds targeting NTCP to interrupt viral entry and adopted a hepatic maturation procedure to increase NTCP levels. To identify entry inhibitors, hepatocytes were pretreated with candidate compounds for 2 h before the infection with HBV at 4 °C to allow viral binding but not entry. The decrease in HBV infectivity was evaluated on day 7 post infection. The representative data included cyclosporine A, a classical NTCP inhibitor, as a positive control to validate the model.
Since NTCP facilitates both transporter- and receptor-mediated endocytosis functions, HBV entry inhibitors could target at least one of these mechanisms. The inhibition of NTCP transport can be evaluated using a TCA uptake assay based on ELISA, eliminating the radioactive TCA from the classical protocol36. The affinity between NTCP and the entry inhibitor was measured using ITC. This technique provided essential thermodynamic parameters, including the stoichiometry (n), dissociation constant (KD), change in free energy (ΔG), enthalpy (ΔH), entropy (ΔS), and heat capacity of binding (ΔCp). Various anti-HBV agents have been identified by molecular docking, such as quercetin, rutin, hesperidin, and luperol, that could specifically bind HBV reverse transcriptase comparable to lamivudine, a nucleoside analog. The compounds were investigated using ITC and exhibited negative Gibb energy (−ΔG) ranging from −9.3 to −5.2 kcal/mol and KD of 1 × 10-6 to 1 × 10-3 M with HBV polymerase37. Taken together, the data obtained from these aforementioned assays will help screen the antiviral efficacy of candidate compounds that act as HBV entry inhibitors. The established protocol will be useful for discovering novel NTCP antagonists/inhibitors. The suppression of viral entry could be useful in prophylactic and therapeutic treatment.
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Disclosures
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
This research project is supported by Mahidol University and the Thailand Science Research and Innovation (TSRI) separately awarded to A. Wongkajornsilp and K. Sa-ngiamsuntorn. This work was financially supported by the Office of National Higher Education Science Research and Innovation Policy Council through the Program Management Unit for Competitiveness (grant number C10F630093). A. Wongkajornsilp is a recipient of a Chalermprakiat grant of the Faculty of Medicine Siriraj Hospital, Mahidol University. The authors would like to thank Miss Sawinee Seemakhan (Excellent Center for Drug Discovery, Faculty of Science, Mahidol University) for her assistance with the ITC technique.
Materials
| Name | Company | Catalog Number | Comments |
| Cell lines | |||
| HepaRG Cells, Cryopreserved | Thermo Fisher Scientific | HPRGC10 | |
| Hep-G2/2.2.15 Human Hepatoblastoma Cell Line | Merck | SCC249 | |
| Reagents | |||
| 4% Paraformadehyde Phosphate Buffer Solution | FUJIFLIM Wako chemical | 163-20145 | |
| BD Perm/Wash buffer | BD Biosciences | 554723 | Perm/Wash buffer |
| Cyclosporin A | abcam | 59865-13-3 | |
| EDTA | Invitrogen | 15575-038 | 8 mM |
| G 418 disulfate salt | Merck | 108321-42-2 | |
| Halt Protease Inhibitor Cocktail EDTA-free (100x) | Thermo Scientific | 78425 | |
| HEPES | Merck | 7365-45-9 | |
| illustraTM RNAspin Mini RNA isolation kits | GE Healthcare | 25-0500-71 | |
| illustra RNAspin Mini RNA Isolation Kit | GE Healthcare | 25-0500-71 | |
| ImProm-II Reverse Transcription System | Promega | A3800 | |
| KAPA SYBR FAST qPCR Kit | Kapa Biosystems | KK4600 | |
| Lenti-X Concentrator | Takara bio | PT4421-2 | concentrator |
| Luminata crescendo Western HRP substrate | Merck | WBLUR0100 | |
| Master Mix (2x) Universal | Kapa Biosystems | KK4600 | |
| Nucleospin DNA extraction kit | macherey-nagel | 1806/003 | |
| Phosphate buffered saline | Merck | P3813 | |
| Polyethylene glycol 8000 | Merck | 25322-68-3 | |
| ProLong Gold Antifade Mountant | Thermo scientific | P36930 | |
| Recombinant NTCP | Cloud-Clone | RPE421Hu02 | |
| RIPA Lysis Buffer (10x) | Merck | 20-188 | |
| TCA | Sigma | 345909-26-4 | |
| TCA Elisa kit | Mybiosource | MB2033685 | |
| Triton X-100 | Merck | 9036-19-5 | |
| Trypsin-EDTA | Gibco | 25200072 | Dilute to 0.125% |
| Antibodies | |||
| Anti-NTCP1 antibody | Abcam | ab131084 | 1:100 dilution |
| Anti-GAPDH antibody | Thermo Fisher Scientific | AM4300 | 1:200,000 dilution |
| HRP-conjugated goat anti-rabbit antibody | Abcam | ab205718 | 1:10,000 dilution |
| HRP goat anti-mouse secondary antibody | Abcam | ab97023 | 1:10,000 dilution |
| Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 | Invitrogen | A-11008 | 1:500 dilution |
| Reagent composition | |||
| 1° Antibody dilution buffer | |||
| 1x TBST | |||
| 3% BSA | Sigma | A7906-100G | Working concentration: 3% |
| Sodium azide | Sigma | 199931 | Working concentration: 0.05% |
| Hepatocyte Growth Medium | |||
| DME/F12 | Gibco | 12400-024 | |
| 10% FBS | Sigma Aldrich | F7524 | |
| 1% Pen/Strep | HyClon | SV30010 | |
| 1% GlutaMAX | Gibco | 35050-061 | |
| Hepatic maturation medium | |||
| Williams’ E medium | Sigma Aldrich | W4125-1L | |
| 10% FBS | Sigma Aldrich | F7524 | |
| 1% Pen/Strep | HyClon | SV30010 | |
| 1% GlutaMAX | Gibco | 35050-061 | |
| 5 µg/mL Insulin | Sigma Aldrich | 91077C-100MG | |
| 50 µM hydrocotisone | Sigma Aldrich | H0888-1g | |
| 2% DMSO | PanReac AppliChem | A3672-250ml | |
| IF Blocking solution | |||
| 1x PBS | Gibco | 21300-058 | |
| 3% BSA | Sigma | A7906-100G | Working concentration: 3% |
| 0.2% Triton X-100 | Sigma | T8787 | Working concentration: 0.2% |
| RIPA Lysis Buffer Solution | Merck | 20-188 | Final concentration: 1X |
| Protease Inhibitor Cocktail | Thermo Scientific | 78425 | Final concentration: 1X |
| Na3VO4 | Final concentration: 1 mM | ||
| PMSF | Final concentration: 1 mM | ||
| NaF | Final concentration: 10 mM | ||
| Western blot reagent | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Tween 20 | Merck | 9005-64-5 | |
| 1x TBST | 0.1% Tween 20 | ||
| 1x PBS | Gibco | 21300-058 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | A53225 | |
| Polyacrylamide gel | Bio-Rad | 161-0183 | |
| Ammonium Persulfate (APS) | Bio-Rad | 161-0700 | Final concentration: 0.05% |
| TEMED | Bio-Rad | 161-0800 | Stacker gel: 0.1%, Resolver gel: 0.05% |
| 2x Laemmli Sample Buffer | Bio-Rad | 161-0737 | Final concentration: 1X |
| Precision Plus Protein Dual Color Standards | Bio-Rad | 161-0374 | |
| WB Blocking solution/ 2° Antibody dilution buffer | |||
| 1x TBST | |||
| 5% Skim milk (nonfat dry milk) | Bio-Rad | 170-6404 | Working concentration: 5% |
| 1x Running buffer 1 L | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Glycine | Sigma | G8898 | 14.4 g |
| SDS | Merck | 7910 | Working concentration: 0.1% |
| Blot transfer buffer 500 mL | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Glycine | Sigma | G8898 | 7.2 g |
| Methanol | Merck | 106009 | 100 mL |
| Mild stripping solution 1 L | Adjust pH to 2.2 | ||
| Glycine | Sigma | G8898 | 15 g |
| SDS | Merck | 7910 | 1 g |
| Tween 20 | Merck | 9005-64-5 | 10 mL |
| Equipments | |||
| 15 mL centrifuge tube | Corning | 430052 | |
| 50 mL centrifuge tube | Corning | 430291 | |
| Airstream Class II | Esco | 2010621 | Biological safety cabinet |
| CelCulture CO2 Incubator | Esco | 2170002 | Humidified tissue culture incubator |
| CFX96 Touch Real-Time PCR Detector | Bio-Rad | 1855196 | |
| FACSVerse Flow Cytometer | BD Biosciences | 651154 | |
| Graduated pipettes (10 mL) | Jet Biofil | GSP010010 | |
| Graduated pipettes (5 mL) | Jet Biofil | GSP010005 | |
| MicroCal PEAQ-ITC | Malvern | Isothermal titration calorimeters | |
| Mini PROTEAN Tetra Cell | Bio-Rad | 1658004 | Electrophoresis chamber |
| Mini Trans-blot absorbent filter paper | Bio-Rad | 1703932 | |
| Omega Lum G Imaging System | Aplegen | 8418-10-0005 | |
| Pipette controller | Eppendorf | 4430000.018 | Easypet 3 |
| PowerPac HC | Bio-Rad | 1645052 | Power supply |
| PVDF membrane | Merck | IPVH00010 | |
| T-75 cell culture flask | Corning | 431464U | |
| Trans-Blot SD Semi-Dry Transfer Cell | Bio-Rad | 1703940 | Semi-dry transfer cell |
| Ultrasonic processor (Vibra-Cell VCX 130) | Sonics & Materials | ||
| Versati Tabletop Refrigerated Centrifuge | Esco | T1000R | Centrifuge with swinging bucket rotar |
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