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WO2007058384A1 - Procede d'inhibition de la replication du virus de l'hepatite c, inhibiteur de replication du virus et procede de selection de cet inhibiteur - Google Patents

Procede d'inhibition de la replication du virus de l'hepatite c, inhibiteur de replication du virus et procede de selection de cet inhibiteur Download PDF

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Publication number
WO2007058384A1
WO2007058384A1 PCT/JP2006/323518 JP2006323518W WO2007058384A1 WO 2007058384 A1 WO2007058384 A1 WO 2007058384A1 JP 2006323518 W JP2006323518 W JP 2006323518W WO 2007058384 A1 WO2007058384 A1 WO 2007058384A1
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fkbp8
ns5a
hsp90
hcv
replication
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PCT/JP2006/323518
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English (en)
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Yoshiharu Matsuura
Kohji Moriishi
Toru Okamoto
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Osaka University
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Publication of WO2007058384A1 publication Critical patent/WO2007058384A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae

Definitions

  • the present invention relates to a method of suppressing the replication of hepatitis C virus, a method for the prophylaxis/treatment of hepatitis C, an inhibitor of said replication of the virus and a method for screening a substance inhibiting the replication of said virus.
  • HCV Hepatitis C virus
  • Hsp90 heat shock protein 90
  • the HCV genome encodes a single polyprotein of approximately 3000 amino acids, containing the viral proteins in the order: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B .
  • the NS proteins are thought to be non-structural and are involved in the enzymatic functions of viral replication and processing the viral polyprotein.
  • the NS5A is known to show various biological activities including regulation of interferon sensitivity, transcripti ⁇ r ⁇ , ⁇ f host genes, cell, eycle control and the like (e.g., see A., and Ha-r ⁇ jfs, M. (2004). J. Gen . Virol . 85, 2485 ⁇ ;2]5fi-2 ) .
  • the present inventors have tried to identify host protein (s) interacting with NS5A using a yeast two-hybrid method, and found that FKBP8, a member of the FK506-binding protein family, specifically interacts with NS5A.
  • the present inventors have revealed that FKBP also forms a complex with Hsp90 whose inhibitors are known to suppress HCV RNA replication and processing of the HCV polyprotein (see Waxman, L. H. et al., supra). These indicate that a complex consisting of NS5A, FKBP and Hsp90 plays an important role in HCV RNA replication, and that HCV replication can be suppressed by inhibiting the formation of the complex.
  • the present inventors have conducted further investigations based on these findings, and completed the present invention.
  • the present invention provides:
  • an inhibitor of HCV replication comprising a substance inhibiting the interaction between HCV NS5A and one or more proteins of a cell infected with said virus, provided that said substance is not geldanamycin;
  • the inhibitor of HCV replication of the present invention shows a suppressive effect on HCV RNA replication by inhibiting the formation of a complex between FKBP and HCV NS5A and/or Hsp90, which results in the prophylaxis/treatment of hepatitis C.
  • FIG. 1 shows expression of FKBP8 and FKBP38 in mammalian cells.
  • A Schematic representation of FKBP8 and FKBP38. The FK506 binding domain (FBD), tetratricopeptide repeat (TPR), putative calmodulin binding motif (CaM) , and transmembrane domain (TM) are shown.
  • B N-terminally HA-tagged FKBP8 and FKBP38 were expressed in 293T cells and visualized by iininunoblotting using mouse monoclonal antibody to FKBP8 or the HA tag.
  • HA-FKBP8 was expressed together with Flag-NS5A of genotype Ib (Jl) in 293T cells and was immunoprecipitated with anti-HA antibody. Immunoprecipitated proteins were subjected to immunoblot with anti-Flag or HA antibody.
  • D Endogenous FKBP8 in HCV replicon (9-13) cells was immunoprecipitated with isotype control (lane 1) or anti-FKBP8 antibody, KDM-Il (lane 2) . Endogenous FKBP8 was co-immunoprecipitated with HCV NS5A. The data shown in each panel are representative of three independent experiments.
  • FIG. 2 shows that NS5A directly binds to FKBP8.
  • Purified Thioredoxin (Trx) , Trx-NS5A (A) and His-FKBP8 (B) in gel were stained with Coomassie brilliant blue G-250. These proteins were mixed and subjected to immunoprecipitation with anti-FKBP8 antibody. Precipitates were immunoblotted with anti-thioredoxin antibody (C) and stained with Coomassie brilliant blue G-250 (D) .
  • Figure 3 shows specific interaction between FKBP8 and NS5A.
  • HA-NS5As were obtained from several genotypes of HCV and were expressed with Flag-FKBP8 in 293T cells. Proteins immunoprecipitated with anti-HA or Flag antibody were subjected to Western blotting.
  • B Flag-NS5A was co-expressed with HA-FKBP8, -CypD, or -FKBP52 in 293T cells. Proteins immunoprecipitated with anti-HA or -Flag tag antibody were subjected to Western blotting. The data shown in each panel are representative of three independent experiments.
  • FIG. 4 shows that siRNA-mediated knockdown of FKBP8 inhibits production of infectious HCV particles.
  • Huh7.5.1 cells were transfected with siRNA for non-target control or FKBP8-Target 1 at a concentration of 80 nM, which was sufficient for knockdown of FKBP8 expression.
  • the cells were inoculated with JFHl HCVcc particles at 24 h after transfection and cells and culture supernatants were harvested everyday.
  • A Intracellular genomic RNA.
  • B HCV core protein in the supernatant .
  • Figure 5 shows determination of the NS5A-binding region in FKBP8.
  • A Schematic representation of FKBP8 and deleted mutants.
  • B Flag-NS5A was co-expressed with HA-FKBP8 and its mutants in 293T cells. Proteins immunoprecipitated with anti- HA antibody were subjected to Western blotting. The data shown in each panel are representative of three independent experiments .
  • Figure 6 shows homomultimerization of FKBP8.
  • A Flag- FKBP8 was co-expressed with HA-FKBP52, -CypD, or -FKBP8 in 293T cells, and was immunoprecipitated with anti-HA antibody. Precipitates were analyzed by Western blotting.
  • B Flag- or EE-tagged FKBP8 was co-expressed with HA-NS5A in 293T cells and was immunoprecipitated with anti-EE or Flag antibody.
  • Precipitates were analyzed by Western blotting.
  • C Flag- and EE-tagged FKBP8 were co-expressed with increasing amounts of HA-NS5A (0.1, 0.2, and 0.4 ⁇ g of expression plasmid/well) in 293T cells. Immunoprecipitates with anti-Flag antibody were analyzed by Western blotting. The data shown in each panel are representative of three independent experiments.
  • Figure 7 shows decrease in HCV RNA by FKBP8-targeted siRNA.
  • HCV replicon cells (9-13 cells) were transfected with each of three kinds of siRNA targeted to FKBP8 or non-targeted siRNA at a final concentration of 80 nM. Transfected cells were collected at 72 h post-transfection, and FKBP8 mRNA and HCV RNA levels were determined by real-time PCR after being normalized with ⁇ -actin mRNA.
  • B HCV replicon cells transfected with 80 and 160 nM of Target 1 or non-targeted siRNA were harvested at 72 h post-transfection, and the samples were analyzed by immunoblotting.
  • HCV replicon cells expressing Flag-rFKBP8 mutant (Huh7rFKBP8) or control cells (Huh7c) were transfected with Target 1 (gray bars) or non-targeted (white bars) siRNA at a concentration of 80 nM.
  • Transfected cells were harvested at 72 h post-transfection, and HCV RNA (left) and FKBP8 mRNA (right) were measured by real-time PCR and expressed as % increase after being normalized with the expression of ⁇ -actin mRNA.
  • FIG. 8 shows lack of apoptosis in FKBP8-knockdown cells.
  • Huh7 (9-13) cells were transfected with siRNA for non-target control or FKBP ⁇ -Target 1 at a concentration of 80 nM, which was sufficient for knockdown of FKBP8 expression.
  • Some cells were treated with 0.5 ⁇ g/ml staurosporin as a control for apoptosis. The cells were stained using the Vybrant apoptosis assay kit 1 (Molecular Probes, Eugene, OR) .
  • Figure 9 shows transient replication and " colony formation assays in a FKBP8-knockdown cell line.
  • A Levels of expression of FKBP8 and ⁇ -actin in Huh7N and Huh7 FKBP8KD cell lines bearing plasmids encoding shRNA for control mRNA (lane 1) and for FKBP8 mRNA (lane 2), respectively.
  • B Each cell line was transfected with in vitro-transcribed HCV replicon RNA, pFK-I 389 hRL/NS3-3'/NK5.1 (HCVRNA), or a replication- negative mutant, pFK-I 389 hRL/NS3-3'/NK5. IGND (HCV/GND) .
  • the fold increase in replication was determined by the increase in luciferase activity at 48 h compared with that observed 4 h after standardization, as based on the activity of the replication-deficient HCV/GND replicon.
  • Huh7N and Huh7 FKBP8KD cell lines were transfected with in vitro-transcribed replicon RNA (pFK-I 38 9 neo/NS3-3' /NK5.1) and the cells were incubated for 4 weeks. The remaining cells were fixed with 4% paraformaldehyde and then were stained. The data shown in each panel are representative of three independent experiments.
  • Figure 10 shows FKBP8 forms complex with NS5A and Hsp90.
  • An N-terminally myc-TEV-Flag-tagged FKBP8 was expressed in 293T cells and immunoprecipitated. The precipitated proteins were applied to SDS-PAGE and then stained with silver staining. Hsp90 and FKBP8 were identified by LC-MS/MS.
  • (B) Flag-FKBP8 was co-expressed with HA-Hsp90 in 293T cells. Hsp90-HA and Flag-FKBP8 were immunoprecipitated with antibodies to HA and Flag, respectively. Precipitates were analyzed by immunoblotting.
  • HCV replicon cells (9-13 cells) were treated with 1, 3, 10 and 30 nM of geldanamycin and after 24 h treatment, HCV RNA replication was determined by real-time PCR. Relative replication was expressed as % replication after standardized by the expression of ⁇ -actin. Cell viabilities were determined by trypan blue staining.
  • E The effect of geldanamycin on the expression of NS5A and FKBP8. The. replicon cells were examined by immunoblotting after treatment with various concentrations of geldanamycin. The data shown in each panel are representative of three independent experiments. Figure 11 shows disruption of NS5A/FKBP/Hsp90 complexes by geldanamycin.
  • Purified His-FKBP8, Hsp90 and/or Trx-NS5A were mixed with DMSO or geldanamycin (100 nM) and subjected to immunoprecipitation with anti-FKBP8 antibody. Precipitates were immunoblotted with antibody against Hsp90, thioredoxin or FKBP8.
  • the present invention provides a method of suppressing HCV replication in a host cell infected therewith, which comprises inhibiting the interaction between HCV NS5A and one or more cellular proteins.
  • the host cell infected with HCV includes a human or chimpanzee cell, preferably a human cell (e.g., hepatocyte) .
  • the host cells include cells isolated from an animal's body (cells, tissues, organs) and an animal body. In the former case, the cells can be isolated from an animal's body that is infected with HCV, or infected with HCV after the isolation.
  • the host cells infected with HCV also include a human or chimpanzee body infected therewith.
  • the "cellular protein interacting with HCV NS5A” means a protein derived from the host cell, which binds to HCV NS5A directly or indirectly via another cellular protein and facilitates HCV RNA replication.
  • the cellular proteins binding directly to NS5A include, but not limited to, FKBP8, and those binding indirectly to NS5A include, but not limited to, Hsp90.
  • Hsp90 binds to NS5A via FKBP8.
  • FKBP8 is a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 2. It may be a protein derived from any cell or tissue of a mammal (for example, human, chimpanzee, mouse, rat, rabbit, sheep, swine, bovine, horse, cat, dog, and the like, preferably human or chimpanzee) , and may also be a chemically synthesized protein or a protein synthesized using a cell-free translation system. Alternatively, this protein may be a recombinant protein produced from a transformant introduced with a polynucleotide having the nucleotide sequence that encodes the above-described amino acid sequence.
  • an amino acid sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more, to the amino acid sequence shown by SEQ ID NO: 2, and the like can be mentioned.
  • homology means the proportion (%) of the same and similar amino acid residues to all overlapping amino acid residues in the optimal alignment where two amino acid sequences are aligned using mathematic algorithm known in the relevant technical field (preferably, the algorithm is such that a gap can be introduced into one or both of the sequences for the optimal alignment) .
  • a similar amino acid means an amino acid having similar physicochemical properties; as examples, amino acids classified under the same group, such as aromatic amino acids (Phe, Trp, Tyr) , aliphatic amino acids (Ala, Leu, lie, VaI) , polar amino acids (GIn, Asn) , basic amino acids (Lys, Arg, His) , acidic amino acids (GIu, Asp) , amino acids having hydroxyl group (Ser, Thr) , and amino acids having a small side chain (GIy, Ala, Ser, Thr, Met) , can be mentioned. Substitution by such a similar amino acid is expected to produce no change in phenotype of protein (i.e., conservative amino acid substitution) . Specific examples of conservative amino acid substitution are known in the relevant technical field and described in various pieces of the literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990) ) .
  • a protein that comprises substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 2 above, and that has substantially the same quality of activity as a protein comprising the amino acid sequence shown by SEQ ID NO: 2, and the like, can be mentioned.
  • substantially the same quality of activity means that the proteins are qualitatively (e.g., physiologically or pharmacologically) equivalent to each other. Accordingly, it is preferable that the proteins be equivalent to each other in terms of transcription regulatory activity (e.g., about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times), but quantitative factors such as the extent of activity and protein molecular weight may be different.
  • transcription regulatory activity e.g., about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times
  • quantitative factors such as the extent of activity and protein molecular weight may be different.
  • FKBP8 is preferably a protein having the amino acid sequence shown by SEQ ID NO: 2, that is, the human FKBP8 protein or a homologue thereof in chimpanzee.
  • FKBP8 can be produced from a cell or tissue of the aforementioned animal by a method known per se " of protein purification. Specifically, FKBP8 can be produced by homogenizing animal tissue or cells, and separating and purifying the soluble fraction and/or nuclear fraction by a chromatography such as reversed-phase chromatography, ion exchange chromatography or affinity chromatography, and the like.
  • FKBP8 can also be produced by cultivating a transformant comprising DNA that encodes the protein, and separating and purifying the protein ' from the culture obtained.
  • genomic DNA or cDNA derived from any cell or tissue of a mammal for example, human, chimpanzee, bovine, horse, swine, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, hamster, and the like, preferably human or chimpanzee
  • the DNA can also be amplified directly by a reverse transcriptase polymerase chain reaction (hereinafter abbreviated as "RT-PCR method") using a total RNA or mRNA fraction prepared from the above-described cell or tissue.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA that encodes FKBP8 DNA comprising the nucleotide sequence shown by SEQ ID NO:1
  • DNA that comprises a nucleotide sequence hybridizing to the nucleotide sequence shown by SEQ ID N ⁇ :l under highly stringent conditions, and that encodes the aforementioned protein having substantially the same quality of activity (e.g., transcription regulatory activity and the like) as a protein comprising the amino acid sequence shown by SEQ ID NO: 2, and the like can be mentioned.
  • DNA capable of hybridizing to the nucleotide sequence shown by SEQ ID N0:l under highly stringent conditions
  • DNA that comprises a nucleotide sequence showing a homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more, to the nucleotide sequence shown by SEQ ID NO:1, and the like are used.
  • Hybridization can be conducted according to a method known per se or a method based thereon, for example, a method described in Molecular Cloning, 2nd edition (J. Sambrook et al . , Cold Spring Harbor Lab. Press, 1989) and the like. When a commercially available library is used, hybridization can be conducted according to the method described in the instruction manual attached thereto. Hybridization can preferably be conducted under highly stringent conditions.
  • High-stringent conditions refer to, for example, conditions involving a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 itiM, and a temperature of about 50 to 70°C, preferably about 60 to 65°C. -In particular, a case wherein the sodium concentration is about 19 mM and the temperature is about 65°C is preferred.
  • the DNA that encodes FKBP8 is preferably DNA comprising the nucleotide sequence shown by SEQ ID NO:1 and the like.
  • Hsp90 is a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 4. It may be a protein derived from any cell or tissue of a mammal (for example, human, chimpanzee, mouse, rat, rabbit, sheep, swine, bovine, horse, cat, dog, and the like, preferably human or chimpanzee), and may also be a chemically synthesized protein or a protein synthesized using a cell-free translation system. Alternatively, this protein may be a recombinant protein produced from a transformant introduced with a polynucleotide having the nucleotide sequence that encodes the above-described amino acid sequence.
  • amino acid sequence shown by SEQ ID NO: 4 As substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 4, an amino acid sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more, to the amino acid sequence shown by SEQ ID NO: 4, and the like can be mentioned.
  • the protein comprising substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 4
  • a protein that comprises substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO: 4 above, and that has substantially the same quality of activity as a protein comprising the amino acid sequence shown by SEQ ID NO: 4, and the like, can be mentioned.
  • Hsp90 is preferably a protein having the amino acid sequence shown by SEQ ID NO: 4, that is, the human Hsp90 protein or a homologue thereof in chimpanzee.
  • Hsp90 can be produced from a cell or tissue of the aforementioned animal by a method known per se of protein purification. Specifically, Hsp90 can be produced by homogenizing animal tissue or cells, and separating and purifying the soluble fraction and/or nuclear fraction by a chromatography such as reversed-phase chromatography, ion exchange chromatography or affinity chromatography, and the like- Furthermore, Hsp90 can also be produced by cultivating a transformant comprising DNA that encodes the protein, and separating and purifying the protein from the culture obtained.
  • genomic DNA or cDNA derived from any cell or tissue of a mammal for example, human, chimpanzee, bovine, horse, swine, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, hamster, and the like, preferably human or chimpanzee
  • synthetic DNA and the like can be mentioned.
  • the DNA can also be amplified directly by a reverse transcriptase polymerase chain reaction (hereinafter abbreviated as "RT-PCR method") using a total RNA or mRNA fraction prepared from the above-described cell or tissue.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA that encodes Hsp90 DNA comprising the nucleotide sequence shown by SEQ ID NO: 3
  • DNA that comprises a nucleotide sequence hybridizing to the nucleotide sequence shown by SEQ ID NO: 3 under highly stringent conditions and that encodes the aforementioned protein having substantially the same quality of activity (e.g., transcription regulatory activity and the like) as a protein comprising the amino acid sequence shown by SEQ ID NO: 4, and the like can be mentioned.
  • DNA capable of hybridizing to the nucleotide sequence shown by SEQ ID NO: 3 under highly stringent conditions
  • DNA that comprises a nucleotide sequence showing a homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more, to the nucleotide sequence shown by SEQ ID NO: 3, and the like are used.
  • the DNA that encodes Hsp90 is preferably DNA comprising the nucleotide sequence shown by SEQ ID NO: 3 and the like.
  • Inhibition of the interaction between HCV NS5A and a cellular protein can be achieved, for , example, by adding a substance that suppresses the expression of the cellular protein.
  • substances that suppress the expression of the cellular protein include transcription suppression factors, RNA polymerase inhibitors, RNases, protein synthesis inhibitors, proteases, and protein denaturants; to minimize the adverse effects on other genes and proteins that are expressed in host cells, it is important that the substance should be capable of specifically acting on the target molecule. Accordingly, a preferred embodiment of a substance that suppresses the expression of the cellular protein
  • an antisense nucleic acid (preferably, FKBP or Hsp90) is an antisense nucleic acid of the mRNA encoding the protein or its primary transcript.
  • An antisense nucleic acid refers to a nucleic acid that consists of a nucleotide sequence capable of hybridizing to target mRNA (primary transcript) under the physiological conditions of cells that express the target mRNA (primary transcript) , and that is capable of inhibiting the translation of the polypeptide encoded by the target mRNA (primary transcript) while in the hybridized state.
  • the antisense nucleic acid may be DNA or RNA and may be a DNA/RNA chimera.
  • Other important requirements for designing an antisense nucleic acid include increasing the water solubility and cell membrane permeability; these goals can also be achieved by improving the dosage form such as through the use of liposome or microspheres.
  • the length of the antisense nucleic acid of the present invention is not subject to limitation, as long as the antisense nucleic acid is capable of specifically hybridizing to the mRNA encoding the cellular protein or its primary transcript, and the antisense nucleic acid may be a sequence comprising a sequence of about 15 bases at the shortest in length or complementary to the entire sequence of the mRNA (primary transcript) at the longest. From the viewpoint of ease of synthesis, antigenicity concern, and other aspects, there may be mentioned, for example, oligonucleotides consisting of preferably about 15 to about 30 bases.
  • the nucleotide sequence capable of hybridizing to the mRNA encoding the cellular protein under physiological conditions may be any one, as long as it possesses about 80% homology or more, depending on the base composition of the target sequence.
  • the target sequence for the antisense nucleic acid of the present invention is not subject to limitation, as long as it is a sequence such that the translation of the cellular protein is inhibited as a result of hybridization of the antisense nucleic acid, and may be the entire sequence or a partial sequence of the mRNA or may be the intron portion of the primary transcript.
  • the target sequence when using an oligonucleotide as the antisense nucleic acid, it is desirable that the target sequence .should be located between the 5' -terminus of the mRNA and the C-terminus of the coding region.
  • the target sequence is a region on the N-terminus side of the coding region from the 5' -terminus, with greatest preference given to a nucleotide sequence in the vicinity of the initiation codon.
  • the target sequence should be selected such that an antisense nucleic acid complementary thereto does not form a secondary structure such as a hairpin structure.
  • the antisense nucleic acid of the present invention may be capable of not only hybridizing to the mRNA encoding the cellular protein or its primary transcript to inhibit the translation, but also binding to the gene encoding the cellular protein, a double-stranded DNA, to form a triple- strand (triplex) to inhibit the transcription into mRNA.
  • a substance that suppresses the cellular protein expression is a ribozyme capable of specifically cleaving the mRNA encoding the cellular protein or its primary transcript in the coding region (including the intron portion in case of the primary transcript) .
  • the term ribozyme refers to an RNA possessing an enzyme activity to cleave nucleic acid. Since it has recently been shown that oligo DNA having the nucleotide sequence at the enzyme activity site also possesses nucleic acid cleavage activity, the term ribozyme is used herein to include DNA, as long as it possesses sequence-specific nucleic acid cleavage activity.
  • the most widely applicable ribozyme is self-splicing RNA found in infectious RNA of viroids, virusoids, etc.; such ribozymes include the hammerhead type and the hairpin type.
  • the hammerhead type is capable of specifically cleaving the target mRNA alone by exhibiting enzyme activity with about 40 bases, and rendering several bases at each end adjoining to the hammerhead structure (about 10 bases in total) complementary to the desired cleavage site of the mRNA.
  • This type of ribozymes is also advantageous in that they do not attack genomic DNA because their substrate is RNA alone.
  • the ribozyme when using the ribozyme in the form of an expression vector containing DNA that encodes the ribozyme, the ribozyme may be a hybrid ribozyme resulting from the further joining of a sequence with tRNA modified to promote the transfer to cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].
  • a substance that suppresses the expression of the cellular protein is a double-stranded oligo RNA (siRNA) that is complementary to a partial sequence in the coding region of the mRNA or its primary transcript (including the intron portion in the case of the primary transcript) .
  • RNA interference a phenomenon wherein upon intracellular introduction of a short double-stranded RNA, an mRNA complementary to that RNA is .degraded, has long been known to occur in nematodes, insects, plants, and other organisms. Since this phenomena has recently been found to occur in animal cells as well [Nature, 411 (6836) : 494-498 (2001)], RNAi has been widely applied as an alternative to ribozyme.
  • an siRNA against the mRNA encoding the cellular protein is used as an inhibitor of the interaction between HCV NS5A and the cellular protein.
  • the antisense oligonucleotide and ribozyme of the present invention can be prepared by determining the target sequence for the mRNA or its primary transcript on the basis of the cDNA sequence (e.g., SEQ ID N0:l in the case of hFKBP ⁇ and SEQ ID NO: 3 in the case of hHsp90) or genomic DNA sequence of the cellular protein, and synthesizing a complementary sequence using a commercially available DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.) .
  • the target sequence for the mRNA or its primary transcript on the basis of the cDNA sequence (e.g., SEQ ID N0:l in the case of hFKBP ⁇ and SEQ ID NO: 3 in the case of hHsp90) or genomic DNA sequence of the cellular protein, and synthesizing a complementary sequence using a commercially available DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.) .
  • An siRNA can be prepared by synthesizing a sense strand and an antisense strand using a DNA/RNA synthesizer, denaturing each strand in the appropriate annealing buffer solution at about 90°C to about 95°C for about 1 minute, and subsequently annealing them at about 30°C to about 70 0 C for about 1 to 8 hours. Additionally, a longer double-stranded polynucleotide can be prepared by synthesizing complementary oligonucleotide strands in alternative overlaps, annealing them, and subsequently subjecting them to ligation with ligase.
  • a preferred embodiment of a substance that inhibits the binding activity of the cellular protein to HCV NS5A is a neutral antibody against the protein. When the cellular protein is FKBP8, since TPR domain is responsible for the binding to HCV NS5A, a neutral anti-FKBP antibody of the present invention preferably recognizes said domain as an epitope.
  • This antibody may be a polyclonal antibody or monoclonal antibody, and can be prepared by a well-known immunological technique. Any fragment of the antibody serves for the purpose, as long as it has an antigen-binding site (CDR) for the cellular protein, and is exemplified by Fab, F(ab') 2/ ScFv, minibody, etc.
  • CDR antigen-binding site
  • a polyclonal antibody can be obtained by immunizing the cellular protein or a fragment thereof [may be prepared as a complex cross-linked with a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) , if necessary] as the immunogen, along with a commercially available adjuvant (e.g., complete or incomplete Freund' s adjuvant) , to an animal by subcutaneous or intraperitoneal administration about 2 to 4 times at intervals of 2 to 3 weeks (the antibody titer of serum separated from drawn blood determined by a commonly known antigen-antibody reaction, and its elevation confirmed in advance) , collecting whole blood about 3 to about 10 days after final immunization, and purifying the antiserum.
  • Animals to be administered with the antigen include mammals such as rats, mice, rabbits, goat, guinea pigs and hamsters.
  • a monoclonal antibody can also be prepared by a cell fusion method (e.g., Takeshi Watanabe, saibouyugouhou no genri to monok ⁇ ronaru kotai no sakusei, Akira Taniuchi and Toshitada Takahashi, eds . , "monokuronaru kotai to gan - kiso to rinsho-", pp. 2-14, Science Forum Publishing, 1985) .
  • a cell fusion method e.g., Takeshi Watanabe, saibouyugouhou no genri to monok ⁇ ronaru kotai no sakusei, Akira Taniuchi and Toshitada Takahashi, eds . , "monokuronaru kotai to gan - kiso to rinsho-", pp. 2-14, Science Forum Publishing, 1985
  • a mouse is given this factor, along with a commercially available adjuvant, 2 to ' 4 " times by subcutaneous or intraperitoneal administration/ its spleen or lymph node is collected about 3 days a'fteh ⁇ final administration, and leukocytes are separated.
  • leukocytes are 'fused with myeloma cells (e.g., NS-I, , ,P3X63Ag8, etc.) to yield a hybridoma that produce's 'a! monoclonal antibody against this factor.
  • the cell fusion may be achieved by the PEG method [J. Immunol.
  • a hybridoma that produces the desired monoclonal antibody can be selected by detecting in the culture supernatant an antibody that specifically binds to an antigen using well-known EIA, RIA, or the like. Cultivation of a hybridoma that produces a monoclonal antibody can be conducted in vitro, or in vivo in mice or rats, preferably in mouse ascites fluid, and the resulting antibody can be obtained from a hybridoma culture supernatant or animal ascites fluid, respectively.
  • the antibody of the present invention is preferably a chimeric antibody between a human and another animal (e.g., mice etc.), more preferably a humanized antibody, most preferably a human antibody.
  • chimeric antibody refers to an antibody having a variable region (V region) from an immunized animal and a constant region (C region) from a human; "humanized antibody” refers to an antibody wherein all regions except CDR have been replaced with a human antibody.
  • a chimeric antibody or a humanized antibody can, for example, be obtained by cutting out a sequence that encodes a V region or CDR from the gene for a mouse monoclonal antibody prepared in the same manner as above, cloning a chimeric gene resulting from fusion with DNA that encodes a C region of an antibody from human myeloma into an appropriate expression vector, and introducing the vector to an appropriate host cell to express the chimeric gene.
  • a human antibody can, for example, be obtained using a human antibody- producing mouse such as KM mouseTM.
  • Another preferred embodiment of a substance that inhibits the binding activity of the cellular protein to HCV NS5A is a compound showing an antagonistic action on the binding of cellular protein to NS5A (the other protein) .
  • Such a compound can be obtained using any of the below-mentioned screening methods.
  • a substance showing an antagonistic action on the binding of the cellular protein to NS5A can be a dominant negative mutant of the cellular protein, which can bind to NS5A but cannot facilitate HCV RNA replication.
  • the cellular protein is FKBP8
  • a dominant negative mutant which cannot bind to NS5A but can bind to Hsp90 is also useful, because the mutant inhibits the formation of NS5A-FKBP8-Hsp90 complex by binding competitively to Hsp90.
  • Such a mutant for example, can be prepared by introducing mutation into TPR domain of FKBP8 using a site- directed mutagenesis.
  • the method of suppressing HCV replication comprises applying, to host cells infected with HCV, an effective amount of any of the above-mentioned ' substances inhibiting the expression of the cellular protein or the binding activity of the cellular protein to HCV NS5A (including indirect binding) .
  • an effective amount of any of the above-mentioned ' substances inhibiting the expression of the cellular protein or the binding activity of the cellular protein to HCV NS5A (including indirect binding) When the host cells are isolated and cultured in a medium, such a substance may be added to the medium.
  • the host cells are a human or chimpanzee (preferably human) individual infected with HCV.
  • these substances are mixed with a pharmacologically acceptable carrier required to yield a pharmaceutical composition, and then administered to the animal.
  • the present invention also provides a method for the prophylaxis/treatment of hepatitis C in a patient infected with HCV, which comprises administering to said patient an effective amount of substance (s) inhibiting the expression of the cellular protein (s) (preferably, FKBP8 and Hsp90) or these binding activity to NS5A or another cellular protein mediating the formation of a complex with NS5A (FKBP8, in the case of Hsp90) .
  • s cellular protein
  • FKBP8 and Hsp90 another cellular protein mediating the formation of a complex with NS5A
  • various organic or inorganic carrier substances conventionally used as pharmaceutical preparation materials can be mentioned, and these are formulated as excipients, lubricants, binders and disintegrants, in solid preparations; as solvents, solubilizing agents, suspending agents, isotonizing agents, buffering agents and soothing agents, in liquid preparations, and the like.
  • pharmaceutical preparation additives such as antiseptics, antioxidants, coloring agents, sweetener ' s and the like can be used.
  • lactose lactose, saccharose, D-mannitol, D-sorbitol, starch, gelatinized starch, dextrin, crystalline cellulose, low substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, light silicic anhydride, synthetic aluminum silicate, magnesium metasilicate aluminate and the like can be mentioned.
  • suitable lubricants magnesium stearate, calcium stearate, talc, colloidal silica and the like can be mentioned.
  • suitable binders gelatinized starch, sucrose, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, saccharose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone and the like can be mentioned.
  • suitable disintegrants lactose, saccharose, starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium crosscarmellose, sodium carboxymethyl starch, light silicic anhydride, low substituted hydroxypropyl cellulose and the like can be mentioned.
  • suitable solvents water for injection, physiological saline, Ringer's solutions, alcohols, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like can be mentioned.
  • solubilizing agents polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like can be mentioned.
  • surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose; polysorbates, polyoxyethylene hardened castor oil and the like can be mentioned.
  • surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glyceryl monostearate
  • hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxye
  • sodium chloride sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like can be mentioned.
  • buffer solutions of a phosphate, an acetate, a carbonate, a citrate and the like, and the like can be mentioned.
  • benzyl alcohol and the like can be mentioned.
  • Suitable antiseptics paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like can be mentioned.
  • antioxidants examples include sulfides, ascorbates and the like.
  • water-soluble food tar colors e.g., food colors such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2
  • water-insoluble lake pigments e.g., aluminum salts of the aforementioned water-soluble food tar colors and the like
  • natural pigments e.g., ⁇ -carotene, chlorophyll, red iron oxide and the like
  • sweeteners sodium saccharide, dipotassium glycyrrhizinate, aspartame, stevia and the like can be mentioned.
  • oral formulations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions and suspensions; non-oral formulations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections and the like) , external formulations (e.g., nasal preparations, transdermal preparations, ointments and the like), suppositories (e.g., rectal suppositories, vaginal suppositories and the like) , pellets, drops, sustained-release preparations (e.g., sustained-release microcapsules and the like) and the like can be mentioned; these can be safely administered orally or non- orally.
  • injections e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections and the like
  • external formulations e.g., nasal preparations, transdermal preparations,
  • the pharmaceutical composition can be produced by a method conventionally used in the field of pharmaceutical preparation making, for example, a method described in the Japanese Pharmacopoeia and the like. A specific method of producing a preparation is hereinafter described in detail.
  • the content of the substance inhibiting the interaction between NS5A and cellular protein in the pharmaceutical composition varies depending on the dosage form, the dose of the compound and the like; and is, for example, from about 0.1 to 100% by weight.
  • an oral formulation is produced by adding to an active ingredient an excipient (e.g., lactose, saccharose, starch, D-mannitol and the like), a disintegrant (e.g., calcium carboxymethyl cellulose and the like), a binder (e.g., gelatinized starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like) , a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000 and the like) and the like, compression-molding the resultant mixture, and subsequently, as required, coating the resulting material with a coating base by a method known per se for the purpose of taste masking, enteric solubility or sustained release.
  • an excipient e.g., lactose, saccharose, starch, D-mannitol and the like
  • a disintegrant e.g., calcium carboxymethyl cellulose and
  • the coating base a sugar-coating base, a water-soluble film coating base, an enteric film coating base, a sustained-release film coating base and the like can be mentioned.
  • saccharose is used, which may be used in combination with one species or two or more species selected from among talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like.
  • cellulose polymers such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose and methylhydroxyethyl cellulose; synthetic polymers such as polyvinylacetal diethyianimoacetate, aminoalkylmethacrylate copolymer E [Eudragit-E (trade name), Rohm Pharma Corp.] and polyvinyl pyrrolidone; polysaccharides such as pullulan; and the like can be mentioned.
  • cellulose polymers such as hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, carboxymethylethyl cellulose, and cellulose acetate phthalate; acrylic polymers such as Methacrylic Acid Copolymer L [Eudragit-L (trade name), Rohm Pharma Corp.], Methacrylic Acid Copolymer LD [Eudragit-L-30D55 (trade name) , Rohm Pharma Corp.], and Methacrylic Acid Copolymer S [Eudragit-S (trade name), Rohm Pharma Corp.]; natural substances such as shellac, and the like can be mentioned.
  • Methacrylic Acid Copolymer L Eudragit-L (trade name), Rohm Pharma Corp.]
  • Methacrylic Acid Copolymer LD Methacrylic Acid Copolymer LD [Eudragit-L-30D55 (trade name) , Rohm Pharma Corp.]
  • Methacrylic Acid Copolymer S Eudragit-S (trade
  • cellulose polymers such as ethyl cellulose; acrylic polymers such as aminoalkyl methacrylate copolymer RS [Eudragit-RS (trade name), Rohm Pharma Corp.], and an ethyl acrylate- methylmethacrylate copolymer suspension [Eudragit-NE (trade name), Rohm Pharma Corp.]; and the like can be mentioned.
  • the above-mentioned coating bases may also be used in a mixture of two or more kinds thereof in a suitable ratio.
  • a shading agent for example, titanium oxide or iron sesquioxide
  • An injection is produced by dissolving, suspending or emulsifying an active ingredient in an aqueous solvent (e.g., distilled water, physiological saline, Ringer's solution and the like), an oily solvent (e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil and corn oil, propylene glycol, and the like) , or the like, along with a dispersing agent
  • an aqueous solvent e.g., distilled water, physiological saline, Ringer's solution and the like
  • an oily solvent e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil and corn oil, propylene glycol, and the like
  • polysorbate 80 polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate and the like
  • a preservative e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol and the like
  • an isotonizing agent e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like
  • additives such as a solubilizing agent (e.g., sodium salicylate, sodium acetate and the like), a stabilizer (e.g., human serum albumin and the like), a soothing agent (e.g., benzyl alcohol and the like) and the like may also be used.
  • a solubilizing agent e.g., sodium salicylate, sodium acetate and the like
  • a stabilizer e.g., human serum albumin and the like
  • a soothing agent e.g., benzyl alcohol and the like
  • the preparation thus obtained is safe and of low toxicity, it can be administered orally or non-orally to a mammal (i.e., human or chimpanzee).
  • the antisense nucleic acid, ribozyme or siRNA can also be administered after insertion to an appropriate vector, for example, retrovirus vector, adenovirus vector, adenovirus- associated virus vector and the like.
  • nucleic acids can be administered using a gene gun or a catheter like a hydrogel catheter, and can also be administered locally into the trachea as an inhalant after conversion to an aerosol.
  • the dosage of the inhibitor of the interaction between HCV NS5A and one or more cellular proteins varies depending on the subject of administration, route of administration and the like; in an adult human patient infected with HCV (body weight 60 kg) , for example, the dosage is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg, per day, based on the active ingredient compound ⁇ obtained by the screening method of the present invention.
  • the present invention provides a method for screening for a substance inhibiting HCV replication.
  • the method comprises bringing FKBP8 into, contact with the HCV NS5A and/or Hsp90 in the presence and absence of a test substance, and comparing the degrees of the binding of FKBP8 to NS5A and/or Hsp90 under the both conditions.
  • FKBP8 and Hsp90 used in the method can be obtained from cells producing same in such a manner as described in the above (1) .
  • these proteins can be obtained by transforming a suitable host with expression vectors harboring the nucleic acids encoding the proteins described in the above (1), culturing the transformant in a medium and recovering the proteins from the culture.
  • FKBP8 (or Hsp90) is provided, for example, in a form of cell extract from cells containing a gene encoding FKBP8 (or Hsp90) .
  • the gene may be endogenous, or foreign DNA introduced into a given host cell using a known method.
  • the host cell can further express Hsp90 (or FKBP8) and/or HCV NS5A.
  • NS5A may be expressed by infecting the cell with HCV, or transfecting the cell with a nucleic acid encoding NS5A.
  • the nucleic acid encoding NS5A can be obtained by a known method, such as RT-PCR, using HCV genome as a % template.
  • FKBP8 and NS5A and/or Hsp90 may be provided as a cell genetically engineered so as to express them.
  • the cell itself constitutes a reaction system.
  • the screening method of the present invention can be performed in various embodiments by changing the means for providing FKBP8 and NS5A and/or Hsp90 into the reaction system and/or the means for determining the degree of the binding of FKBP8 to NS5A and/or Hsp90.
  • the binding of FKBP8 to NS5A and/or Hsp90 is assayed by directly detecting the complex using labeled FKBP8, NS5A or Hsp90 and at least one of anti- FKBP antibody, anti-NS5A antibody and anti-Hsp90 antibody.
  • This is an assay system comprising a B/F separation step.
  • FKBP8, NS5A or Hsp90 is provided as a fused protein having a tag (e.g., histidine-tag, GST-tag, etc), a carrier having an affinity for the tag (e.g., magnetic nickel beads, glutathione-immobilized resin, etc) can be used in the place of a solid-phased antibody.
  • a tag e.g., histidine-tag, GST-tag, etc
  • a carrier having an affinity for the tag e.g., magnetic nickel beads, glutathione-immobilized resin, etc
  • the following methods are exemplified.
  • FKBP8 is contacted with NS5A and/or Hsp90.
  • the reaction mixture is incubated and then subjected to a native gel electrophoresis to separate the FKBP8-NS5A, FKBP8-Hsp90 or NS5A-FKBP8-Hsp90 complex from free FKBP8 and free NS5A and/or Hsp90.
  • Immunoblotting using a labeled antibody is carried out, the intensities of the band corresponding to the complex are compared under the both conditions.
  • an anti-FKBP antibody, anti-NS5A antibody or anti-Hsp90 antibody is added to the reaction mixture to immunoprecipitate the complex, the precipitate is electrophoresed, which is followed by western blotting using an antibody labeled with RI or non-RI (enzyme, fluorescent, etc) .
  • the determination of the complex can be performed using a conventional immunoassay system.
  • an anti- FKBP antibody, anti-NS5A antibody or anti-Hsp90 antibody is added to the reaction mixture to immunoprecipitate the complex.
  • RIA, EIA or FIA using a labeled antibody is carried out to detect the complex immobilized on the solid phase.
  • the complex in the reaction mixture can be directly determined using a homogenous immunoassay which does not require B/F separation, such as surface plasmon resonance, fluorescence polarization immunoassay, fluorescence quenching immunoassay, fluorescence resonance energy transfer immunoassay, and the like.
  • a homogenous immunoassay which does not require B/F separation, such as surface plasmon resonance, fluorescence polarization immunoassay, fluorescence quenching immunoassay, fluorescence resonance energy transfer immunoassay, and the like.
  • the binding of FKBP8 to NS5A and/or Hsp90 is assayed by so-called two-hybrid method, using the expression of a reporter gene or the like as an index.
  • either a DNA encoding FKBP or a DNA encoding NS5A (or Hsp90) is fused with a DNA encoding DNA binding domain (BD) of a transcription factor such as GAL4, and the other DNA is fused with a DNA encoding transcription activating domain (AD) of a transcription factor such as V16.
  • BD DNA binding domain
  • AD DNA encoding transcription activating domain
  • chimeric DNAs are respectively inserted into suitable expression vectors, and co-transfected into a host cell, wherein said cell contains a reporter gene under the control of a promoter containing a cis-element capable of binding to the BD.
  • the host cell may include, but not limited to, a cell line derived from human (e.g., HeLa, HEK293, HepG2, etc), monkey (e.g., COS-I, COS-7, Vero, etc), mouse (COP, L, C127, Sp2/0, NS-I, NIH3T3, ST2, etc), rat, hamster (CHO, BHK) and the like.
  • a yeast two-hybrid system is also available.
  • the reporter gene may include, but not limited to, luciferase gene, green fluorescent protein gene, peroxidase gene, and the like.
  • the resulting transformant is cultured in the presence and absence of a test substance, and the expression amounts of the reporter gene are determined and compared under the both conditions. When the expression of the reporter gene is reduced in the presence of a test substance, the test substance is a possible candidate of inhibitor of HCV replication.
  • an amount of HCV RNA in an HCV replicon cell is also used as an index for the validation of a test substance, as well as the degree of the binding of FKBP to NS5A and/or Hsp90. According to this method, a substance inhibiting HCV replication can be selected more definitely.
  • HCV RNA amount can be determined by a method known per se. For example, HCV RNA amount can be determined by extracting total RNA from HCV replicon cells and carrying out RT-PCR or northern blot analysis.
  • the substance obtained by the screening method of the present invention mentioned above inhibits HCV RNA replication, the substance is useful for the prophylaxis and/or treatment of hepatitis C.
  • the substance can be formulated into a pharmaceutical composition and administered to a mammal such as human and chimpanzee, in the same manner as described above.
  • the present invention is hereinafter described in more detail by means of the following examples, which, however, are not to be construed as limiting the present invention.
  • HCV NS5A Screening for the gene-encoding host protein that interacts with HCV NS5A was performed with a yeast two-hybrid system, Matchmaker two-hybrid system 3 (Clontech, Palo Alto, CA), according to the manufacturer's protocol. Human fetal brain and liver libraries were purchased from Clontech.
  • the cDNA of NS5A-encoding amino acids 1973 to 2419 of an HCV polyprotein of the Jl strain (genotype Ib) .(Aizaki et al., 1998, Hepatology 27, 621-627) was amplified by polymerase chain reaction (PCR) and was cloned into the pGBKT7 vector (Clontech) (Hamamoto et al . , 2005, J.
  • Plasmids DNA fragments encoding NS5A were amplified from HCV genotype Ib strains Jl and Conl (provided by Dr. Bartenschlager) , genotype Ia strain H77C (provided by Dr. Bukh) , and genotype 2a strain JFH-I ,(provided by Dr. Wakita) by PCR using Pfu turbo DNA polymerase (Stratagene, La Jolla, CA) .
  • the fragments were cloned into pCAGGs-PUR/N-HA, in which the sequence encoding an HA tag is inserted at the 5' -terminus of the cloning site of pCAGGs-PUR (Niwa et al., 1991, Gene 108, 193-199) .
  • the DNA fragment encoding human FKBP8 was amplified from the total cDNA of Huh7 cells by PCR, and this fragment was introduced into pEF-FLAG pGBK puro (Huang et al . , 1997,
  • pCAGGs-PUR/NHA pcDNA3.1-N-HA
  • pcDNA3.1-N-HA Hamamoto et al., 2005, J. Virol, in press.
  • pcDNA3.1-N-EE in which an Glu-Glu (EE) tag is inserted in the 5' -terminus of the cloning site of pcDNA3.1 (+) (Invitrogen, Carlsbad, CA).
  • the DNA fragments encoding human FKBP52 and CypD were amplified from a human fetal brain library (Clontech) by PCR, and were introduced into pcDNA3.1-N-HA.
  • DMEM Dulbecco' s modified Eagle's medium
  • FCS fetal calf serum
  • Huh 9-13 cell line which possesses an HCV subgenomic replicon (Lohmann et al., 1999, Science 285, 110-113)
  • FCS fetal calf serum
  • Glutathione-S-transferase-fused human FKBP8 (GST-FKBP8) was expressed in E. coli strain JM109 transformed with pGEX- 4T3 containing FKBP8 gene. GST-FKBP8 was purified with
  • Glutathione-conjugated Sepharose Affinity Matrix (Amersham Pharmacia Biotech, Franklin Lakes, NJ) .
  • Purified GST-FKBP8 was immunized to BaIb/c mice. Lymphonodus cells were obtained after 5 boost immunizations and were fused to mouse myeloma PAI cells. The resulting hybridomas were screened by ELISA using GST and GST-FKBP8. The selected clones were further screened by flow cytometry using 293T cells expressing HA- FKBP8 (O'Reilly et al., 1998, Biotechniques 25, 824-830). Among several positive clones, two clones strongly reactive to human FKBP8 were designated as KDM-Il and 19 (IgG2b) .
  • Antibodies were purified from supernatants of cell culture by Protein G Sepharose 4B beads (Amersham) .
  • Mouse monoclonal antibodies to the HA and EE tags were purchased from Covance (Richmond, CA) .
  • Anti-Flag mouse antibody M2, horseradish peroxidase-conjugated M2 antibody, and anti- ⁇ - actin mouse monoclonal antibody were purchased from Sigma.
  • Mouse monoclonal antibody to NS5A was from Austral Biologicals (San Ramon, CA) .
  • Mouse monoclonal antibodies to NS4B and NS5B have been described previously (Kashiwagi et al., 2002, J. Biol.Chem. 277, 28700-28705).
  • Rabbit polyclonal antibody to NS5A was prepared as described previously (Hamamoto et al., 2005, supra) .
  • Rabbit polyclonal antibody to thioredoxin was described previously (Moriishi et al . , 1999, Biol. Pharm. Bull. , 22, 1167-1172) .
  • His 6 -tagged FKBP8 His-FKBP8
  • thioredoxin-fused NS5A aa 25-213, domain I
  • Trx-NS5A thioredoxin-fused NS5A
  • Ten milliliter of overnight culture was added into 1 L of 2 x YT medium and was incubated at 37 0 C. When the absorbance of culture supernatant indicated 0.4 OD 6 oo?
  • IPTG isopropyl beta-thiogalactoside
  • PBS phosphate buffered saline
  • the washed cell pellet was suspended in 40 ml lysis buffer (5OmM phosphate buffer [pH 8.0] containing 150 mM NaCl, 1% Triton X- 100 and 0.2 ⁇ g/ml lysozyme) and was incubated at 4°C for 2h.
  • Cells were seeded onto 6-well tissue culture plates at 24 h before transfection.
  • the plasmids were transfected into the cells by liposome-mediated transfection using LipfectAMINE 2000 (Invitrogen) .
  • the cells were harvested at 48 h post- transfection, washed with 1 ml of ice-cold phosphate-buffered saline (PBS), suspended in 0.5 ml of lysis buffer (20 mM Tris- HCl pH 7.4 containing 135 mM NaCl, 10% Glycerol and 1% TritonX-100) supplemented with 1 mg/ml leupeptin, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 5 mM NaVO 4 , and then the cells were incubated for 20 min at 4°C.
  • the cell lysates were sonicated for 5 min and centrifuged at 14,000 x g for 5 min at
  • the immunoprecipitation test was carried out by a previously described method (Moriishi et al . , 2003, Antivir. Chem. Chemother. , 14, 285-297) .
  • the immuno-precipitates boiled in the loading buffer were subjected to '12.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE) .
  • the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and were reacted with the appropriate antibodies.
  • the immune complexes were visualized with Super Signal West Femto substrate (Pierce, Rockford, IL) and they were detected by an LAS-3000 image analyzer system (Fujifilm, Tokyo, Japan) .
  • the density of protein band was determined by using IMAGE-PRO PLUS 5.1 software (Media Cybernetics, Silver Springs, MD) . (7) Gene silencing by siRNA
  • Negative control siRNA i.e., siCONTROL Non-Targeting siRNA-2, was purchased from Dharmacon.
  • the Huh7 cells harboring a subgenoitiic HCV replicon grown on 6-well plates were transfected with 80 nM or 160 nM of siRNA with siFECTOR (B- Bridge International, Sunnyvale, CA) .
  • the cells were grown in DMEM containing 10% FCS and were then harvested at 48 h or 72 h post-transfection.
  • First-strand cDNA was synthesized by using a first-strand cDNA synthesis kit (Amersham) and random primers. Each cDNA was estimated by Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) according to the manufacturer's protocol. Fluorescent signals were analyzed by an ABI PRISM 7000 (Applied Biosystems) .
  • the HCV NS5A, human ⁇ -actin, and human FKBP8 genes . were amplified using the primer pairs of 5' -AGTCAGTTGTCTGCGCTTTC-3 ' (SEQ ID NO : .8 ) and
  • the FKBP8 primers are located at different exons in order to prevent the false-positive amplification of contaminated genomic DNA.
  • the values of the HCV genome and FKBP8 mRNA were normalized with those of ⁇ -actin mRNA. Each PCR product was detected as a single band of the correct size upon agarose gel electrophoresis (data not shown) . (9) Generation of infectious HCV particles
  • the viral RNA of JFHl was introduced into Huh7.5.1 according to the method of Wakita et al . (Wakita et al., 2005, Nat. Med., 11, 791-796). The supernatant was collected at 7 days post-transfection and used as HCVcc particles.
  • the naive Huh7.5.1 cells were transfected with siRNA of non-target control or FKBP8-Target 1 at a concentration of 80 nM.
  • the siRNA-treated Huh7.5.1 cells were inoculated with HCVcc particles at 24 h post-transfection. Infected cells and culture supernatants were harvested every day until 5 days post-infection. (10) Establishment of cell lines expressing an siRNA-resistant FKBP8 mutant and knockdown FKBP8 expression
  • A, G, and T at nucleotides 273, 276, and 288 from the 5' end of the open-reading frame of human FKBP8 were replaced with G, A, and C, respectively, according to a splicing method achieved by overlap extension ; these silent mutations were then cloned into pEF-Flag pGBKpuro.
  • the resulting plasmid encoding a mutant FKBP8 resistant to knockdown- by siRNA was transfected into Huh7 cells harbouring the HCV RNA replicon.
  • the culture medium was replaced with DMEM supplemented with 10% FCS and 2 ⁇ g/ml of puromycin (Nakarai Tesque, Tokyo, Japan) at 24 h post-transfection, and the cells were cultured for 7 days.
  • the surviving cells were used for the FKBP8-knockdown experiments.
  • the plasmid pFK-I 38 9 neo/NS3-3' /NK5.1 (Pietschmann et al . , 2002, J. Virol., 75, 1252-1264) was obtained from R. Bartenschlager .
  • the plasmid cleaved at the Seal site was transcribed in vitro using the MEGAscript T7 kit (Ambion) according to the manufacturer's protocol.
  • the linearized plasmid (10 ⁇ g) was introduced into Huh7 cells at 4 million cells per 0.4 ml by electroporation at 270 V and 960 ⁇ F using a Gene PulserTM (Bio-Rad, Hercules, CA) .
  • Electroporated cells were suspended at a final volume of 10 ml of culture medium. Three-ml aliquots of cell suspension were mixed with 7 ml of culture medium and then the cells were seeded on culture dishes (diameter: 10 cm) . The culture medium was replaced with DMEM containing 10% FCS and 1 mg/ml of G418 (Nakarai Tesque) at 24 h post-transfection. The medium was exchanged weekly for fresh DMEM containing 10% FCS and 1 mg/ml G418. The remaining colonies were fixed with 4% paraformaldehyde at four weeks after electroporation, and the cells were stained with crystal violet. (12) Transient replication assay
  • the cDNA encoding Renilla luciferase was introduced between the Ascl and Pmel sites of the plasmid pFK-I 3 89 neo/NS3- 3'/NK5.1, in place of the neo gene.
  • the resulting plasmid, pFK-I 38 9 hRL/NS3-3' /NK5.1, was cleaved with Seal and was transcribed in vitro using a MEGAscript T7 kit (Ambion) .
  • Huh7 cells were suspended at 10 million cells per ml and the suspensions were mixed with 10 ⁇ g of in vitro-transcribed RNA at a 400- ⁇ l volume; the cells were then electroporated at 270 V and 960 ⁇ F by a Gene PulserTM (Bio-Rad) .
  • the electroporated cells were suspended in 25 ml of culture medium and then were seeded at 1 ml per well on 12-well culture plates. Luciferase activity was measured at 4 and 48 h post-transfection using a Renilla Luciferase assay system (Promega) according to the manufacturer's protocol. Luciferase activity at 4 h after electroporation was used to determine the transfection efficiency. (13) Determination of FKBP ⁇ -binding proteins
  • MEF purification was carried out by a previously described method (Ichimura et al . , 2005, J. Biol. Chem. 280, 13187- 13194) .
  • the FKBP8 gene was amplified by PCR and introduced into pcDNA3.1 encoding the myc-TEV-Flag epitope tag (Ichimura et al., 2005, J. Biol. Chem. 280, 13187-13194).
  • the resulting plasmid was transfected into 293T cells, which were then subjected to MEF purification.
  • FKBP ⁇ -binding proteins were separated by SDS-PAGE and visualized by silver staining. The stained bands were excised, digested in gels with Lys-C, and analyzed by the direct nanoflow LC-MS/MS system (Ichimura et al., 2005, J. Biol. Chem. 280, 13187-13194).
  • FK506-binding protein 38kDa Although FKBP38 has been isolated from human and mouse mRNA (Lam et al., 1995, Gene 160, 297- 302), Nielsen and colleagues revealed an additional sequence at the N-terminus of FKBP38, based on an analysis of the transcriptional start site in the genomic sequences of FKBP38 (Nielsen et al . , 2004, Genomics 83, 181-192). The isoforms of FKBP38 were designated as FKBP8, which includes splicing variants of 44 and 46 kDa in mice, and 45 kDa in humans corresponds to the 44 kDa of the mouse FKBP8 (Nielsen et al .
  • Human FKBP8 is identical to FKBP38 except for the extra 58 amino acid residues at the N- terminal, and the FK506-binding domain in the N-terminal half, followed by three sets of tetratricopeptide repeats (TPRs) , a calmodulin binding site, and a transmembrane domain (Fig. IA) . Because the levels of expression of FKBP8 and FKBP38 have not been well characterized in human cell lines, we generated a mouse monoclonal antibody against human FKBP8, and we designated it as clone KDM19.
  • This antibody recognizes a 50- kDa of endogenous FKBP8 in 293T cells, as well as exogenous HA-tagged FKBP8 (HA-FKBP8), which has slightly greater molecular weight (Fig. IB) .
  • the KDM19 antibody detected an exogenous HA-tagged FKBP38 (HA-FKBP38) in 293T cells, no protein band corresponding to endogenous FKBP38 was detected.
  • Similar results were obtained in human liver tissue and in the hepatoma cell lines Huh7, HepG2, and FLC-4 (data not shown) .
  • Flag- tagged NS5A (Flag-NS5A) was expressed together with HA-FKBP8 in 293T cells. Cells transfected with the expression plasmids were harvested at 48-h posttransfection, lysed, and subjected to immunoprecipitation. Flag-NS5A was co-precipitated with HA- FKBP8 by anti-HA antibody (Fig. 1C) . Flag-NS5A was also immunoprecipitated' together with HA-FKBP38, suggesting that the extra N-terminal sequence of FKBP8 is not critical for NS5A binding (data not shown) .
  • HCV NS5A Huh7(9-13) cells harboring subgenomic HCV RNA replicon.
  • Endogenous FKBP8 was co- precipitated with HCV NS5A by anti-FKBP8 antibody (Fig. ID) .
  • His-FKBP8 His s -tagged FKBP8
  • Trx-NS5A thioredoxin-fused domain I of NS5A
  • HA-tagged NS5A (HA-NS5A) proteins of genotype Ia (H77C) , Ib (Conl and Jl), or 2a (JFHl) were expressed together with Flag-tagged FKBP8 (Flag-FKBP8) in 293T cells (Fig. 3A) .
  • Flag-FKBP8 was co-immunoprecipitated with the HA-NS5AS of all of the genotypes examined here by anti-HA antibody, although the interaction between Flag-FKBP8 and the HA-NS5A of genotype 2a was weaker than that of the other genotypes tested [Since siRNA-mediated knockdown of FKBP8 impaired production of infectious HCV particles in JFHl cell culture system (Fig. 4), a high affinity of interaction between FKBP8 and NS5A may not be necessary for JFHl replication] . Furthermore, the HA-NS5As were co-precipitated with Flag-FKBP8 by anti-Flag antibody (Fig. 3A, bottom panel) .
  • the TPR domain of FKBP8 is known to be responsible for protein-protein interactions.
  • FKBP8 shares high homology with CypD and FKBP52, both of which ⁇ contain three tandem repeats of TPR, ' as does FKBP8 (Boguski et al., 1990, Nature, 346, 114; Hirano et al., 1990, Cell, 60, 319-328) .
  • FKBP8 co-immunoprecipitation of Flag-NS5A with HA-FKBP52 and HA-CypD by anti-Flag or anti-HA antibody was not successful (Fig. 3B) .
  • TPR domain is required for the interaction between NS5A and FKBP8
  • FKBP8, CypD, and FKBP52 have high similarity and identity to each other within the TPR domain (Lam et al., 1995, Gene, 160, 297-302) .
  • FKBP8 mutants lacking the transmembrane region, the calmodulin-binding region, the TPR domains, and/or the FK506-binding domain were generated in order to identify the region responsible for the interaction with NS5A (Fig. 5A) .
  • HA-tagged FKBP8 mutants were co-expressed with Flag-NS5A in 293T cells and were imitiunoprecipitated with anti-HA antibody.
  • Flag-NS5A was co-immunoprecipitated with the FKBP8 mutants, except in the case of a dTPR mutant lacking the transmembrane, calmodulin binding, and TPR domains (Fig. 5B) .
  • a dTPR mutant lacking the transmembrane, calmodulin binding, and TPR domains
  • Fig. 5B TPR domains
  • FKBP8 forms a homomultimer and a heteromultimer with NS5A
  • FKBP8 is similar to FKBP52 and CypD with respect to their amino acid sequences and functional domains.
  • Flag- FKBP8 was co-expressed with HA-FKBP52, HA-CypD, or HA-FKBP8 in 293T cells and it was immunoprecipitated with anti-Flag or anti-HA antibody.
  • Flag-FKBP8 and HA-FKBP8 were co- immunoprecipitated with each antibody, but not with HA-FKBP52 or HA-CypD. It is known that Hsp90 forms a homodimer and
  • FKBP52 also interacts with Hsp90 through TPR domain as FKBP8 (Chadli et al., 2000, Proc Natl Acad Sci U S A, 91, 12524- 12529) . If homodimer of FKBP8 may reflect the interaction of FKBP8-Hsp90-Hsp90-FKBP8 complex, FKBP52 would be co- precipitated with FKBP8. However, we could not detect any association of FKBP8 and FKBP52 in the iiranunoprecipitation analysis (Fig. 6A) . These data suggest that FKBP8 can form a homomultimer without Hsp90 and associate with neither FKBP52 nor CypD.
  • HA-NS5A was co-expressed with Flag-FKBP8 and Glu-Glu-tagged FKBP8 (EE-FKBP8) in 293T cells, and was then immunoprecipitated with anti-Flag or anti- EE antibody.
  • HA-NS5A was co-immunoprecipitated with Flag-FKBP8 and EE-FKBP8 by anti-Flag or anti-EE antibody (Fig. 6B) .
  • siRNA targeted to FKBP8 or control siRNA was transfected into Huh7 (9-13) cells harboring subgenomic HCV replicon RNA.
  • Huh7 9-13 cells harboring subgenomic HCV replicon RNA.
  • siRNAs targeted to different regions of FKBP8 Targets 1 to 3 .
  • the total RNA was extracted from the transfected cells, and HCV RNA and FKBP8 mRNA levels were determined by real-time PCR.
  • HCV subgenomic RNA and FKBP8 mRNA levels in the cells transfected with each of the FKBP8 siRNAs were reduced by more than 60%, as compared to the levels in cells treated with the control siRNA at 72 h post-transfection (Fig. 7A) .
  • the levels of expression of FKBP8 and the HCV proteins i.e., NS4B, NS5A, and NS5B
  • the levels of expression of FKBP8 and the HCV proteins decreased in HCV replicon cells transfected with 80 or 160 nM of the FKBP8 siRNA (Target 1), but this was not observed in the cells with the control siRNA (Fig. 7B) .
  • a plasmid encoding Flag-FKBP8 containing either a silent mutation within the siRNA target sequence (Flag-rFKBP8) or empty plasmid was transfected into the HCV replicon cells and then selection was carried out with the appropriate antibiotics.
  • Huh7 was transfected with pSilencer 2.1 U6 hygro containing the cDNA of shRNA to FKBP8, and then selection was carried out with hygromycin.
  • FKBP8 was detected in Huh7 cells harboring a control plasmid (Huh7N) , whereas decreased expression of FKBP8 was clearly observed in cells expressing the shRNA to FKBP8 (Huh7FKBP8KD) (Fig. 8A) .
  • a chimeric HCV RNA containing the Renilla luciferase gene was transfected into these cell lines.
  • HCV RNA containing a neomycin-resistant gene was transfected into the cell lines in order to examine the role played by FKBP8 in HCV RNA replication.
  • the efficiency of colony formation in Huh7N and Huh7FKBP8KD cells with the HCV RNA were 1700 and 23 colonies per ⁇ g RNA, respectively (Fig. 9C) .
  • the visible protein bands were excised and determined by a nanoflow LC-MS/MS system.
  • Major protein bands with a molecular size of 94 kDa and 53kDa were identified as Hsp90 and FKBP8, respectively, although it should be noted that the remaining bands detected in the samples could not be reliably identified (Fig. 10A) .
  • Flag-FKBP8 was co-expressed with Hsp90-HA and immunoprecipitated by anti-Flag or anti-HA antibody.
  • Hsp90-HA and Flag-FKBP8 were co-precipitated with each other by either of the antibodies (Fig. 10B) .
  • EE-FKBP8 was co-expressed with Hsp90- HA and Flag-NS5A.
  • Hsp90-HA and Flag-NS5A were co- immunoprecipitated with EE-FKBP8 by anti-EE antibody.
  • the increase in Flag-NS5A expression had no effect on the interaction between EE-FKBP8 and Hsp90-HA, thus suggesting that the binding site of FKBP8 to NS5A differs from that of FKBP8 to Hsp90.
  • Geldanamycin is known to bind to the ATP/ADP binding site of Hsp90 and specifically inhibits the enzymatic activity of Hsp90, resulting in the promotion of the degradation of client proteins for Hsp90 (Neckers, 2002, Trends MoI. Med. 8, S55-61).
  • HCV replicon cells were treated with various concentrations of geldanamycin.
  • Treatment with geldanamycin clearly reduced the levels of HCV RNA replication (Fig. 10D) ; moreover, this treatment led to the slight suppression of NS5A without reducing the levels of FKBP8 expressed in the HCV replicon cells (Fig. 10E) .
  • RNA replication in the HCV replicon cells by treatment with geldanamycin was not due to an inhibition of HCV polyprotein processing.
  • In vitro pull down assays revealed that geldanamycin inhibited the binding of FKBP8 to Hsp90 and/or NS5A domain I (Fig. 11) .
  • geldanamycin may inhibit HCV replication by disruption of NS5A/FKBP8/Hsp90 complex.
  • Hsp90 was found to be able to bind to FKBP8 and form a complex with HCV NS5A.
  • several host proteins such as VAPs and FBL2 interact with the HCV replication complex and regulate HCV RNA replication (Evans et al., 2004, Proc. Natl. Acad. Sci. USA 101, 13038-13043; Gao et al., 2004, J. Virol. 78, 3480-3488; Hamamoto et al . , 2005, J.
  • FKBP52 possesses PPIase activity and chaperone activity in domain I (amino acids 1-148) and domain 3 (TPR domain, amino acids 264-400) ,' respectively (Pirkl et al., 2001, J. Biol. Chem. 276, 37034-37041) ., Therefore, it is reasonable to speculate that the TPR, domain is responsible for the chaperone activity of FKBP8, and that the FKBP8 and NS5A complex transports Hsp90 to the appropriate clients, including viral and host proteins, which in turn leads to the stabilization of the replication complex and the enhancement of HCV RNA replication.

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Abstract

La présente invention concerne un procédé d'inhibition de la réplication d'un virus de l'hépatite C (HCV) dans une cellule hôte qu'il infecte, lequel procédé comprend l'inhibition des interactions entre le NS5A viral et une ou plusieurs protéines cellulaires, en particulier FKBP8 et Hsp90. L'invention concerne également un procédé de prophylaxie et/ou de traitement de l'hépatite C chez un patient infecté par le HCV, lequel procédé comprend l'administration au patient d'une quantité efficace d'une substance qui inhibe l'expression de FKBP8 ou de son activité de liaison vis-à-vis de NS5A et/ou d'une quantité efficace d'une substance qui inhibe l'expression de Hsp90 ou de son activité de liaison vis-à-vis de FKBP8. La présente invention concerne en outre un procédé de sélection d'une substance qui inhibe la réplication de HCV et qui comprend les étapes qui consistent à mettre FKBP8 en contact avec le NS5A viral et/ou Hsp90 en présence et en l'absence d'une substance de test et à comparer les degrés de liaison de FKBP8 à NS5A et/ou Hsp90 dans les deux cas.
PCT/JP2006/323518 2005-11-17 2006-11-17 Procede d'inhibition de la replication du virus de l'hepatite c, inhibiteur de replication du virus et procede de selection de cet inhibiteur WO2007058384A1 (fr)

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