Publications by authors named "Kento Fukano"

14 Publications

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Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Biochem Biophys Res Commun 2021 Aug 12;567:1-8. Epub 2021 Jun 12.

Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan. Electronic address:

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.
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http://dx.doi.org/10.1016/j.bbrc.2021.06.022DOI Listing
August 2021

MCPIP1 reduces HBV-RNA by targeting its epsilon structure.

Sci Rep 2020 11 27;10(1):20763. Epub 2020 Nov 27.

Department of Molecular Genetics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, 920-8640, Japan.

Hepatitis B virus (HBV) is the major causative factor of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. We previously demonstrated that a proinflammatory cytokine IL-1β reduced the level of HBV RNA. However, the mechanism underlying IL-1β-mediated viral RNA reduction remains incompletely understood. In this study, we report that immune regulator Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) can reduce HBV RNA in hepatocytes. MCPIP1 expression level was higher in the liver tissue of HBV-infected patients and mice. Overexpression of MCPIP1 decreased HBV RNA, whereas ablating MCPIP1 in vitro enhanced HBV production. The domains responsible for RNase activity or oligomerization, were required for MCPIP1-mediated viral RNA reduction. The epsilon structure of HBV RNA was important for its antiviral activity and cleaved by MCPIP1 in the cell-free system. Lastly, knocking out MCPIP1 attenuated the anti-HBV effect of IL-1β, suggesting that MCPIP1 is required for IL-1β-mediated HBV RNA reduction. Overall, these results suggest that MCPIP1 may be involved in the antiviral effect downstream of IL-1β.
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http://dx.doi.org/10.1038/s41598-020-77166-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699622PMC
November 2020

EBV-LMP1 induces APOBEC3s and mitochondrial DNA hypermutation in nasopharyngeal cancer.

Cancer Med 2020 10 20;9(20):7663-7671. Epub 2020 Aug 20.

Division of Otorhinolaryngology and Head and Neck Surgery, Kanazawa University, Kanazawa, Japan.

An Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a principal oncogene that plays a pivotal role in EBV-associated malignant tumors including nasopharyngeal cancer (NPC). Recent genomic landscape studies revealed that NPC also contained many genomic mutations, suggesting the role of LMP1 as a driver gene for the induction of these genomic mutations. Nonetheless, its exact mechanism has not been investigated. In this study, we report that LMP1 alters the expression profile of APOBEC3s(A3s), host deaminases that introduce consecutive C-to-U mutations (hypermutation). In vitro, LMP1 induces APOBEC3B (A3B) and 3F(A3F), in a nasopharyngeal cell line, AdAH. Overexpression of LMP1, A3B, or A3F induces mtDNA hypermutation, which is also detectable from NPC specimens. Expression of LMP1 and A3B in NPC was correlated with neck metastasis. These results provide evidence as to which LMP1 induces A3s and mtDNA hypermutation, and how LMP1 facilitates metastasis is also discussed.
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http://dx.doi.org/10.1002/cam4.3357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571841PMC
October 2020

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

J Gastroenterol 2020 Apr 25;55(4):441-452. Epub 2019 Nov 25.

Choju Medical Institute, Fukushimura Hospital, 19-14 Azayamanaka, Noyori-cho, Toyohashi, 441-8124, Japan.

Background: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro.

Methods: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase.

Results: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC of 14.4 ± 7.7 μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs.

Conclusions: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.
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http://dx.doi.org/10.1007/s00535-019-01643-0DOI Listing
April 2020

Concept of Viral Inhibitors via NTCP.

Semin Liver Dis 2019 Feb 17;39(1):78-85. Epub 2019 Jan 17.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an entry receptor for hepatitis B and D viruses (HBV and HDV) has not only promoted our understanding of the mechanism underlying the viral entry process, but also provided cell culture models supporting viral infection. These models have greatly facilitated cell-based chemical screening for the discovery of entry inhibitors, and mode of action studies using such inhibitors have shown the advantages of NTCP as a drug target. Furthermore, in vitro chemical screening by application of high-throughput affinity-based technologies that target NTCP has identified a variety of unique small molecules that interfere with viral entry. This review summarizes this hot topic in the development of HBV/HDV entry inhibitors, with special focus on the use of NTCP as a drug target.
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http://dx.doi.org/10.1055/s-0038-1676804DOI Listing
February 2019

Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes.

Front Microbiol 2018 8;9:3257. Epub 2019 Jan 8.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Current anti-hepatitis B virus (HBV) agents, which include nucleos(t)ide analogs and interferons, can significantly suppress HBV infection. However, there are limitations in the therapeutic efficacy of these agents, indicating the need to develop anti-HBV agents with different modes of action. In this study, through a functional cell-based chemical screening, we found that a thiazolidinedione, troglitazone, inhibits HBV infection independently of the compound's ligand activity for peroxisome proliferator-activated receptor γ (PPARγ). Analog analysis suggested chemical moiety required for the anti-HBV activity and identified ciglitazone as an analog having higher anti-HBV potency. Whereas, most of the reported HBV entry inhibitors target viral attachment to the cell surface, troglitazone blocked a process subsequent to viral attachment, i.e., internalization of HBV preS1 and its receptor, sodium taurocholate cotransporting polypeptide (NTCP). We also found that NTCP was markedly oligomerized in the presence of HBV preS1, but such NTCP oligomerization was abrogated by treatment with troglitazone, but not with pioglitazone, correlating with inhibition activity to viral internalization. Also, competitive peptides that blocked NTCP oligomerization impeded viral internalization and infection. This work represents the first report identifying small molecules and peptides that specifically inhibit the internalization of HBV. This study is also significant in proposing a possible role for NTCP oligomerization in viral entry, which will shed a light on a new aspect of the cellular mechanisms regulating HBV infection.
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http://dx.doi.org/10.3389/fmicb.2018.03257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331526PMC
January 2019

[From the Establishment of Hepatitis B Virus Cell Culture Systems to Drug Discovery].

Yakugaku Zasshi 2019 ;139(1):81-87

Department of Virology II, National Institute of Infectious Diseases.

The development of antiviral agents enables the control of chronic infectious diseases caused by infection with herpesviruses, human immunodeficiency virus, and hepatitis C virus. In contrast, antiviral treatment against hepatitis B virus (HBV) infection remains a significant area for improvement. One of the main barriers hampering the progress of HBV research has been a lack of cell culture systems efficiently reproducing the viral proliferation process. Recently, cell line-based HBV infection systems have been developed which are useful to analyze the mechanisms of HBV replication and to screen for new anti-HBV agents. In this article, we summarize the establishment of such cell models and the identification of small molecules that inhibit the HBV entry process and discuss their future potential as a novel class of anti-HBV agents.
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http://dx.doi.org/10.1248/yakushi.18-00164-4DOI Listing
April 2019

A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species Specificity.

J Virol 2019 03 19;93(5). Epub 2019 Feb 19.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan

Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and their hosts. Here, we present evidence suggesting that the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (/ ratio [ratio of nonsynonymous to synonymous evolutionary changes] of <1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7% of NTCP amino acid residues exhibit rapid evolution under positive selection (/ ratio of >1). Notably, a substitution at amino acid (aa) 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested a close association of the aa 158 positive selection with the pressure by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to the host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this NTCP residue. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing adaptive mutation in host receptor. HBV and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary arms race between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidence supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species specificity.
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http://dx.doi.org/10.1128/JVI.01432-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384088PMC
March 2019

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

Oncotarget 2018 May 4;9(34):23681-23694. Epub 2018 May 4.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan.

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 IC 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.
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http://dx.doi.org/10.18632/oncotarget.25348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955094PMC
May 2018

De Novo Macrocyclic Peptide Inhibitors of Hepatitis B Virus Cellular Entry.

Cell Chem Biol 2018 07 17;25(7):906-915.e5. Epub 2018 May 17.

Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. Electronic address:

Hepatitis B virus (HBV) constitutes a significant public health burden, and currently available treatment options are not generally curative, necessitating the development of new therapeutics. Here we have applied random non-standard peptide integrated discovery (RaPID) screening to identify small macrocyclic peptide inhibitors of HBV entry that target the cell-surface receptor for HBV, sodium taurocholate cotransporting polypeptide (NTCP). In addition to their anti-HBV activity, these molecules also inhibit cellular entry by the related hepatitis D virus (HDV), and are active against diverse strains of HBV (including clinically relevant nucleos(t)ide analog-resistant and vaccine escaping strains). Importantly, these macrocyclic peptides, in contrast to other NTCP-binding HBV entry inhibitors, exhibited no inhibition of NTCP-mediated bile acid uptake, making them appealing candidates for therapeutic development.
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http://dx.doi.org/10.1016/j.chembiol.2018.04.011DOI Listing
July 2018

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

Biochem Biophys Res Commun 2018 06 11;501(2):374-379. Epub 2018 May 11.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Department of Applied Biological Sciences, Tokyo University of Science, Noda, Japan; CREST, JST, Saitama, Japan. Electronic address:

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.
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http://dx.doi.org/10.1016/j.bbrc.2018.04.187DOI Listing
June 2018

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Sci Rep 2018 02 9;8(1):2769. Epub 2018 Feb 9.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.
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http://dx.doi.org/10.1038/s41598-018-20987-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807303PMC
February 2018

Reply to Padmanabhan and Dixit: Hepatitis C virus entry inhibitors for optimally boosting direct-acting antiviral-based treatments.

Proc Natl Acad Sci U S A 2017 06 16;114(23):E4527-E4529. Epub 2017 May 16.

Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;

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http://dx.doi.org/10.1073/pnas.1705234114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468609PMC
June 2017

Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity.

J Hepatol 2017 04 25;66(4):685-692. Epub 2016 Nov 25.

Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

Background & Aims: The sodium taurocholate co-transporting polypeptide (NTCP) is the main target of most hepatitis B virus (HBV) specific entry inhibitors. Unfortunately, these agents also block NTCP transport of bile acids into hepatocytes, and thus have the potential to cause adverse effects. We aimed to identify small molecules that inhibit HBV entry while maintaining NTCP transporter function.

Methods: We characterized a series of cyclosporine (CsA) derivatives for their anti-HBV activity and NTCP binding specificity using HepG2 cells overexpressing NTCP and primary human hepatocytes. The four most potent derivatives were tested for their capacity to prevent HBV entry, but maintain NTCP transporter function. Their antiviral activity against different HBV genotypes was analysed.

Results: We identified several CsA derivatives that inhibited HBV infection with a sub-micromolar IC. Among them, SCY446 and SCY450 showed low activity against calcineurin (CN) and cyclophilins (CyPs), two major CsA cellular targets. This suggested that instead, these compounds interacted directly with NTCP to inhibit viral attachment to host cells, and have no immunosuppressive function. Importantly, we found that SCY450 and SCY995 did not impair the NTCP-dependent uptake of bile acids, and inhibited multiple HBV genotypes including a clinically relevant nucleoside analog-resistant HBV isolate.

Conclusions: This is the first example of small molecule selective inhibition of HBV entry with no decrease in NTCP transporter activity. It suggests that the anti-HBV activity can be functionally separated from bile acid transport. These broadly active anti-HBV molecules are potential candidates for developing new drugs with fewer adverse effects.

Lay Summary: In this study, we identified new compounds that selectively inhibited hepatitis B virus (HBV) entry, and did not impair bile acid uptake. Our evidence offers a new strategy for developing anti-HBV drugs with fewer side effects.
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http://dx.doi.org/10.1016/j.jhep.2016.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7172969PMC
April 2017
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