title: Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System.
abstract: Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery.
  
  
  
  Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
authors.forename: Syo
authors.initials: S
authors.lastname: Nakajima
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Department of Applied Biological Science, Tokyo University of Science, Noda, Japan CREST, Japan Science and Technology Agency, Saitama, Japan kwatashi@nih.go.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Watashi
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ohashi
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Kamisuki
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
authors.forename: Jesus
authors.initials: J
authors.lastname: Izaguirre-Carbonell
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan.
authors.forename: Andrew Tae-Jun
authors.initials: AT
authors.lastname: Kwon
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan.
authors.forename: Harukazu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Michiyo
authors.initials: M
authors.lastname: Kataoka
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan Micro-Signaling Regulation Technology Unit, RIKEN Center for Life Science Technologies, Wako, Japan.
authors.forename: Senko
authors.initials: S
authors.lastname: Tsukuda
authors.affiliation: Department of Translational Oncology, St. Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Okada
authors.affiliation: Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Meng Ling
authors.initials: ML
authors.lastname: Moi
authors.affiliation: Department of Chemistry, University of Chicago, Chicago, Illinois, USA.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takeuchi
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Minetaro
authors.initials: M
authors.lastname: Arita
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Aizaki
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tadaki
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hasegawa
authors.affiliation: Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Takasaki
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
mesh_terms: D000470:Alkaloids / Q000302:isolation & purification / Q000378:metabolism*; D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism*; D018929:Cell Culture Techniques; D002460:Cell Line; D003716:Dengue Virus / Q000187:drug effects / Q000502:physiology; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004357:Drug Synergism; D005658:Fungi / Q000737:chemistry*; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D006801:Humans; D000071518:Liver X Receptors / Q000037:antagonists & inhibitors*; D010879:Piperazines / Q000302:isolation & purification / Q000378:metabolism*; D017955:Poliovirus / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000470:Alkaloids; D000998:Antiviral Agents; D000071518:Liver X Receptors; D010879:Piperazines; C015380:neoechinulin