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. 2018 May 4;9(34):23681-23694.
doi: 10.18632/oncotarget.25348.

Development of a cell-based assay to identify hepatitis B virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide

Affiliations

Development of a cell-based assay to identify hepatitis B virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide

Kei Miyakawa et al. Oncotarget. .

Abstract

Sodium taurocholate cotransporting polypeptide (NTCP) is a major entry receptor of hepatitis B virus (HBV) and one of the most attractive targets for anti-HBV drugs. We developed a cell-mediated drug screening method to monitor NTCP expression on the cell surface by generating a HepG2 cell line with tetracycline-inducible expression of NTCP and a monoclonal antibody that specifically detects cell-surface NTCP. Using this system, we screened a small molecule library for compounds that protected against HBV infection by targeting NTCP. We found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface NTCP with an IC50 of ~40 μM. We also found that glabridin could attenuate the inhibitory effect of taurocholate on type I interferon signaling by depleting the level of cell-surface NTCP. These results demonstrate that our screening system could be a powerful tool for discovering drugs targeting HBV entry.

Keywords: HBV-permissive cell; ISG; anti-NTCP monoclonal antibody; glabridin; innate immune signaling.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Preparation of HBV-permissive HepG2 cells with inducible NTCP expression
(A) Generation of iNTCP cells. A HepG2 Tet-On parental cell line was transduced with a retroviral vector encoding the NTCP gene fused to a tetracycline-responsive element (TRE) and then selected with puromycin. (B) iNTCP cells were treated with doxycycline (Dox) at the indicated concentrations for 24 hours. NTCP expression was then verified by western blotting (upper panels) or RT-PCR (lower panels). (C, D) NTCP expressions in indicated cells were determined by western blot (C) and qPCR (D). (E) PreS1-binding assay. iNTCP cells pretreated with 5 μg/ml of Dox for 24 hours were incubated with 400 nM FITC-conjugated PreS1 peptide for two hours before fixation and microscopy. (F, G) Induced NTCP does not affect cell proliferation. Cell cycle and cell proliferation assays of iNTCP cells. Cells were treated with Dox for 72 hours before cell cycle analysis. Nocodazole (Noc; 100 nM) was used as a control for inducing cell cycle arrest. (H) High susceptibility to HBV infection of iNTCP cells. Indicated cells were infected with HBV for 16 hours in the presence or absence of PreS1 peptide, cultured for six days, then stained with anti-HBcAg antibody (red) and DAPI (blue). iNTCP cells were treated with Dox for 24 hours before infection as well as during infection.
Figure 2
Figure 2. Development of a monoclonal antibody specifically targeting cell-surface NTCP
(A) Schematic illustration of anti-NTCP mAb generation. Using a wheat germ cell-free protein production system, large amounts of NTCP were synthesized with high solubility. Following mouse immunization, we established over 140 hybridoma clones. (B) Selection of 9A8 clones producing anti-NTCP mAb. Culture supernatants of hybridoma clones were used as primary antibodies for flow cytometry analysis of Dox-treated (red line) or -untreated iNTCP cells (blue line). (C–E) Immunofluorescence staining of cell-surface NTCP by 9A8 mAb on iNTCP cells (C), HepG2-hNTCP-C4 cells (D), and iNTCP-derived spheroid (E). Scale bars: 10 μm. (F) Epitope mapping of 9A8 mAb. Recombinant wild-type or partially truncated NTCP proteins tagged with His were generated using wheat germ extracts and subjected to western blotting using anti-His or 9A8 antibodies. The predicted epitope of 9A8 mAb is shown in pink. (G, H) 9A8 mAb fails to inhibit HBV infection. iNTCP cells (G) and primary human hepatocytes (H) were infected with HBV or its reporter virus (HBV-NL) respectively, in the presence of 9A8 mAb. Anti-HBs mAb (clone 33A4, which recognizes the PreS1 domain) was used as a control. Viral infectivity was determined by intracellular HBcAg staining (G) or NanoLuc activity (H) of infected cells.
Figure 3
Figure 3. Identification of compounds that downregulate cell-surface NTCP
(A) Schematic representation of the screening procedure. (B) Cell-surface NTCP expression (upper) and cell viability (lower) of cells treated with candidate compounds. (C) Flow cytometry analysis. Cell-surface NTCP expression of iNTCP cells treated with indicated compounds (5, 10, 25, and 50 μM) for 24 hours. **P < 0.01, ***P < 0.001, two-tailed unpaired t-test. (D) Glabridin does not affect the tetracycline-responsive promoter activity. U2OS cells stably expressing a Dox-inducible luciferase gene were treated with indicated compounds (50 μM) for 24 hours, then subjected to a luciferase assay. (E) Biomolecular binding of compounds to NTCP. Microscale thermophoresis analysis was performed to determine the interaction between glabridin and recombinant NTCP-GFP or GFP. A known NTCP-binding compound, cyclosporin A (CsA), was used as a positive control. The y-axis represents normalized fluorescence intensity.
Figure 4
Figure 4. Glabridin induces caveolar endocytosis of NTCP
(A) Flow cytometry analysis. Cell-surface NTCP expression of cells treated with glabridin (10, 25, and 50 μM) for 24 hours. *P < 0.05, **P < 0.01, two-tailed unpaired t-test. (B) Rapid down-regulation of cell-surface NTCP by glabridin. iNTCP cells were treated with glabridin (50 μM) for designated intervals and were analyzed by flow cytometer after staining with 9A8 mAb (left) or anti-transferrin receptor antibody (right). (C) Internalization of NTCP by glabridin. Subcellular localization of NTCP was determined by immunofluorescence microscopy using the 9A8 mAb. (D) Flow cytometry analysis of iNTCP cells treated concurrently with glabridin (50 μM) and genistein (a caveolar endocytosis inhibitor, 50 ng/ml) or Pitstop2 (a clathrin-mediated endocytosis inhibitor, 30 μM). (E) Glabridin leads to NTCP degradation. iNTCP cells were treated with cycloheximide (CHX) in the presence or absence of glabridin (50 μM). *P < 0.05, two-tailed unpaired t-test. (F) Glabridin does not exhibit cytotoxicity in effective concentrations. Cell viability assay of iNTCP cells treated with glabridin (25 and 50 μM) for 48 hours. Staurosporine (1 μM) was used as a control for inducing cell death.
Figure 5
Figure 5. Glabridin inhibits HBV infection in primary human hepatocytes
(A) Time-of-drug-addition experiments. Primary human hepatocytes were infected with HBV in the presence of glabridin for 3–24 hours according to the procedure schematized at the left. Culture supernatants were subjected to the HBsAg ELISA six days after infection. *P < 0.05, **P < 0.01, two-tailed unpaired t-test. (B, C) Glabridin suppresses HBV infection in iNTCP cells. iNTCP cells were infected with HBV in the presence of glabridin (25 and 50 μM). Cells were pretreated with Dox (5 μg/ml) one day before infection, and with glabridin three hours prior to infection. Cells and culture supernatants were subjected to intracellular HBcAg staining (B) and quantification of HBsAg released (C). **P < 0.01, two-tailed unpaired t-test. (D) Glabridin suppresses HBV infection in HepaRG cells. Differentiated HepaRG cells were infected with HBV-NL in the presence of glabridin (25 and 50 μM). Infectivity was determined by assaying luciferase activity six days after infection. Cell-surface NTCP expression of HepaRG cells treated with glabridin for three hours was measured by flow cytometry. **P < 0.01, two-tailed unpaired t-test.
Figure 6
Figure 6. Glabridin suppresses bile acid uptake by depleting cell-surface NTCP
(A) Glabridin inhibits taurocholate uptake. iNTCP cells were treated with glabridin (10, 25, and 50 μM) for three or 20 hours. After incubation with [3H]-taurocholate (TCA) for 15 minutes, cells were washed and intracellular radioactivity was quantified. Cyclosporin A (CsA, 10 μM) was used as a positive control in this assay. *P < 0.05, **P < 0.01, two-tailed unpaired t-test. (B) Glabridin counteracts bile acids to promote innate immune signaling. Primary human hepatocytes were sequentially treated with IFN-β (100 U/ml), TCA, and glabridin (50 μM), as shown in left panel. The expression of representative ISGs, including Mx1 and BST2, was quantified using qPCR. ns, not significant; **P < 0.01, two-tailed unpaired t-test.

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