Publications by authors named "Asao Katsume"

12 Publications

  • Page 1 of 1

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

Gastroenterology 2013 Oct 18;145(4):865-73. Epub 2013 Jun 18.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Research Division, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.

Background & Aims: Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice.

Methods: We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors.

Results: NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors.

Conclusions: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.
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http://dx.doi.org/10.1053/j.gastro.2013.06.012DOI Listing
October 2013

4E-BP1 regulates the differentiation of white adipose tissue.

Genes Cells 2013 Jul 15;18(7):602-7. Epub 2013 May 15.

Department of Experimental Phylaxiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto-City, Kumamoto 860-8556, Japan.

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1(-/-) mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1(-/-) MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues.
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http://dx.doi.org/10.1111/gtc.12059DOI Listing
July 2013

An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis.

Int J Exp Pathol 2013 Apr 11;94(2):93-103. Epub 2013 Jan 11.

Fuji-Gotemba Research Laboratories, Chugai Research Institute for Medical Science Inc, Gotemba, Shizuoka, Japan.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine-/choline-deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long-term feeding with a high-fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1-14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.
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http://dx.doi.org/10.1111/iep.12008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607137PMC
April 2013

Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent.

Bioorg Med Chem Lett 2013 Jan 30;23(1):336-9. Epub 2012 Oct 30.

Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. Electronic address:

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.
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http://dx.doi.org/10.1016/j.bmcl.2012.10.083DOI Listing
January 2013

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PLoS Pathog 2012 16;8(8):e1002860. Epub 2012 Aug 16.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.
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http://dx.doi.org/10.1371/journal.ppat.1002860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420934PMC
December 2012

An orally available, small-molecule interferon inhibits viral replication.

Sci Rep 2012 10;2:259. Epub 2012 Feb 10.

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Kanagawa, Japan.

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.
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http://dx.doi.org/10.1038/srep00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277087PMC
April 2013

Pathogenesis of hepatitis C virus infection in Tupaia belangeri.

J Virol 2010 Jan;84(1):303-11

Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-0057, Japan.

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.
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http://dx.doi.org/10.1128/JVI.01448-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798454PMC
January 2010

Host sphingolipid biosynthesis as a target for hepatitis C virus therapy.

Nat Chem Biol 2005 Nov 16;1(6):333-7. Epub 2005 Oct 16.

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan.

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.
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http://dx.doi.org/10.1038/nchembio742DOI Listing
November 2005

Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone.

Liver Int 2004 Jun;24(3):259-67

Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.

Background/aims: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or 'isolated hepatocytes' from tree shrews. In this study, we engrafted 'human liver tissue' into immunodeficient mice and investigated HCV infection using an infectious molecular clone.

Methods: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV-infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR.

Results: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection.

Conclusion: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection.
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http://dx.doi.org/10.1111/j.1478-3231.2004.0909.xDOI Listing
June 2004

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells.

J Biol Chem 2004 Apr 26;279(15):14531-41. Epub 2004 Jan 26.

Department of Microbiology and Cell Biology and Cell Physiology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.
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http://dx.doi.org/10.1074/jbc.M312822200DOI Listing
April 2004

Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice.

Cytokine 2002 Dec;20(6):304-11

Department of Medical Science I, School of Health and Sport Sciences, Osaka University, Osaka, Japan.

Transgenic mice carrying human IL-6 cDNA fused with a murine major histocompatibility class-I promoter (H-2L(d)) were serially administered with anti-interleukin-6 receptor (IL-6R) monoclonal antibody (mAb), MR16-1, from the age of 4 weeks to estimate its efficacy on a variety of disorders developed in these mice, most of which are similar to the disorders associated with Castleman's disease. In the control mice treated with isotype-matched mAb, a massive and multiple IgG1 plasmacytosis, mesangial proliferative glomerulonephritis, leukocytosis, thrombocytosis, anemia and abnormalities of blood chemical parameters have developed in accordance with the elevation of serum IL-6, and 50% of mice have died of renal failure by 18 weeks of age. In contrast, the treatment with MR16-1 prevented all these symptoms and prolonged the lifetime of the majority of the mice. Thus, the constitutive overexpression of IL-6 caused various disorders, and the treatment with anti-IL-6R mAb completely prevented from these symptoms. These results clearly confirm that IL-6 indeed plays an essential role in the pathogenesis of a variety of disorders. Furthermore, anti-IL-6R mAb could provide novel therapy for Castleman's disease and MR16-1 should be a useful tool to estimate therapeutic potential of IL-6 antagonists in a variety of murine models for human disease.
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http://dx.doi.org/10.1006/cyto.2002.2012DOI Listing
December 2002

Pax6 controls the expression of Lewis x epitope in the embryonic forebrain by regulating alpha 1,3-fucosyltransferase IX expression.

J Biol Chem 2002 Jan 23;277(3):2033-9. Epub 2001 Oct 23.

Department of Biochemical Cell Research, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

Pax6 is a transcription factor involved in brain patterning and neurogenesis. Expression of Pax6 is specifically observed in the developing cerebral cortex, where Lewis x epitope that is thought to play important roles in cell interactions is colocalized. Here we examined whether Pax6 regulates localization of Lewis x using Pax6 mutant rat embryos. The Lewis x epitope disappeared in the Pax6 mutant cortex, and activity of alpha1,3-fucosyltransferase, which catalyzed the last step of Lewis x biosynthesis, drastically decreased in the mutant cortex as compared with the wild type. Furthermore, expression of a fucosyltransferase gene, FucT-IX, specifically decreased in the mutant, while no change was seen for expression of another fucosyltransferase gene, FucT-IV. These results strongly suggest that Pax6 controls Lewis x expression in the embryonic brain by regulating FucT-IX gene expression.
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http://dx.doi.org/10.1074/jbc.M108495200DOI Listing
January 2002
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