WO2011083637A1 - Therapeutic agent for hepatic disorder - Google Patents

Therapeutic agent for hepatic disorder Download PDF

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WO2011083637A1
WO2011083637A1 PCT/JP2010/071186 JP2010071186W WO2011083637A1 WO 2011083637 A1 WO2011083637 A1 WO 2011083637A1 JP 2010071186 W JP2010071186 W JP 2010071186W WO 2011083637 A1 WO2011083637 A1 WO 2011083637A1
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keap1
nrf2
liver
binding
agent
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PCT/JP2010/071186
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French (fr)
Japanese (ja)
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小松雅明
田中啓二
丹羽眞一郎
牧野泰孝
笠間隆志
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財団法人東京都医学総合研究所
リンク・ジェノミクス株式会社
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Priority to JP2011548929A priority Critical patent/JP5814128B2/en
Publication of WO2011083637A1 publication Critical patent/WO2011083637A1/en

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    • 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
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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.

Definitions

  • the present invention relates to a protein interaction between p62 and Keap1 that controls the formation of ubiquitin-positive inclusion bodies in cells, a therapeutic agent for liver disease and an anticancer agent having a substance that inhibits or suppresses the interaction, and uses the interaction Related to screening methods.
  • the liver is the largest metabolic organ in humans and an essential organ for life support. This organ causes liver enlargement, fatty liver, etc. due to high calorie / high fat eating and drinking, stress, etc., leading to functional deterioration. Moreover, viral hepatitis and alcoholic hepatitis are caused by viral infection and alcohol consumption, etc., and these disorders lead to cirrhosis and liver cancer over a long period of time. However, the present condition is that the effective drug with respect to these liver diseases is not developed. Therefore, development of an effective drug for various liver diseases is desired. Among these, promising therapeutic agents for liver diseases and anticancer agents are provided from our autophagy research.
  • Autophagy is a large-scale intracellular degradation pathway involved in the turnover of cellular components, removal of unwanted organelles, removal of aggregation-prone mutant proteins, and the like.
  • the isolation membrane extends to form a lipid bilayer structure (autophagosome) surrounding the cytoplasmic components.
  • This autophagosome fuses with the lysosome and degrades the cytoplasmic component enclosed by the lysosomal hydrolase.
  • Yeast genetics studies have revealed that many ATG genes play an essential role in autophagy.
  • LC3 localized in the autophagosome has been identified as a human homologue of yeast Atg8 and plays an essential role in the elongation of the isolation membrane.
  • Non-patent Document 1 Komatsu et al. 2007 Cell
  • p62 interacts with a diverse group of molecules responsible for signal transduction. Further, it has a ubiquitin-associated domain at the C-terminus, and is known as a shuttle molecule for ubiquitinated proteins to the proteasome (Non-Patent Document 2: Seibenhener et al. 2004 MCB).
  • Non-Patent Document 2 Seibenhener et al. 2004 MCB.
  • p62 is decomposed
  • Non-Patent Document 5 Ichimura et al. 2008 JBC).
  • p62 knockout mice develop type 2 diabetes, but hepatic damage and neurodegeneration observed in autophagy-deficient mice have not been confirmed (Non-patent Document 6: Rodriguez A 2006 Cell Metab).
  • Non-patent Document 6 Rodriguez A 2006 Cell Metab.
  • p62 accumulates and becomes insoluble, and finally p62-positive inclusion bodies are confirmed.
  • Non-patent Document 7 Komatsu et al. 2005 JCB, Non-patent Document 1). : Komatsu et al. 2007 Cell).
  • the formation process of the inclusion body is composed of three stages: abnormal folding of the mutant protein, formation of soluble oligomer, and insolubilization / accumulation of soluble oligomer. Formation of this inclusion body has been observed in various pathological conditions (neurodegenerative diseases such as alcoholic hepatitis, Parkinson's disease, amyotrophic lateral sclerosis, and ALS).
  • Non-patent Document 1 Komatsu et al. 2007 Cell.
  • the accumulation of ubiquitin / p62-positive inclusions is associated with neurological diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, astrocytoma, alcoholic hepatitis, fatty liver, liver of liver cancer patients.
  • Non-patent document 8 Stumptner et al., 2007 Hpatology
  • Non-patent document 9 Zatloukal K et al. AM J Pathol 2002.
  • GST glutathione S-transferase
  • P NAD
  • NQO1 quinine 1
  • Nrf2 is captured by the ubiquitin ligase Keap1 (Kelch-like ECH-associated protein 1) and degraded by the proteasome by undergoing ubiquitination.
  • ubiquitin ligase Keap1 Kerch-like ECH-associated protein 1
  • a specific cysteine residue of Keap1 is oxidized and loses binding to Nrf2.
  • Nrf2 that has escaped the degradation moves into the nucleus and activates transcription of detoxifying enzymes such as GST and NQO1 (Non-patent Document 10: Ton KI et al. Mol Cell Biol. 2007).
  • Nrf2 In the autophagy-deficient liver, Nrf2 accumulates in the nucleus, and the accumulation is suppressed by the simultaneous deficiency of p62.
  • the main object of the present invention is to identify a control mechanism of p62 inclusion body formation and to provide a novel therapeutic agent for liver disease and anticancer agent or diagnostic agent based on the mechanism.
  • the present inventors have found for the first time that p62 binds to Keap1. It was also clarified that the binding site of p62 in Keap1 uses the same pocket as that of Nrf2. When p62 accumulates due to autophagy deficiency, the binding between Keap1 and p62 increases, and free Nrf2 increases accordingly. The accumulated Nrf2 moves into the nucleus and increases the transcription of the detoxification enzyme group. Further, according to the experiments by the present inventors, when Nrf2 was also simultaneously deficient in the autophagy-deficient liver, formation of intracellular inclusion bodies, accompanying hepatic hypertrophy, and liver damage could be suppressed.
  • the present invention provides the following agents, diagnostic methods, diagnostic kits, diagnostic agents, and screening methods. (1) An inhibitor or inhibitor of binding between Keap1 and p62.
  • a molecule that specifically binds to p62 is i) a substance that specifically binds to the Keap1 interaction region of p62; ii) a substance that specifically binds to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3); or iii ) An antisense nucleic acid or siRNA against the p62-encoding nucleotide sequence, wherein the p62 expression inhibitor is The agent according to (2) or (3) above.
  • the agent according to (2) above, wherein the molecule that specifically binds to p62 is LC3 or an anti-p62 antibody.
  • (6) A method for determining an application patient of the therapeutic agent for liver disease described in (3) above, i) measuring the expression level of p62 or Nrf2 by liver biopsy of the subject, or ii) measuring the activity of Nrf2 by liver biopsy of the subject, Including a method.
  • a screening method for an agent that inhibits or suppresses the interaction between Keap1 and p62 i) measuring the amount of Keap1 binding to p62 in the presence and absence of the test substance, and ii) determining the amount of Keap1 binding to p62 between the presence and absence of the test substance.
  • a method comprising the step of selecting as (10) In the step (9), in the step of measuring the binding amount, any one of a pull-down method, a mass spectrometry method, a method of detecting protein-protein interaction as a fluorescence signal, or imaging using a fluorescent label is used. The method described. (11) The method according to (9) or (10) above, which is used for screening for a substance that inhibits or suppresses ubiquitin-positive inclusion body formation.
  • the step of measuring the amount of binding comprises i) Keap1 interaction region of p62, or ii) amino acid sequence from position 345 to position 359 in the amino acid sequence of mouse p62 (SEQ ID NO: 3): SKEVDPSTGELQSLQ (SEQ ID NO: 5)
  • the above substance is specific to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3).
  • the method according to any one of (9) to (13), wherein the binding is performed.
  • the present invention provides a method for controlling the interaction between p62 and Keap1 that controls the formation of ubiquitin positive inclusion bodies. It is known that deletion of autophagy causes accumulation and insolubilization of intracellular p62 protein, accumulation of p62-ubiquitin positive inclusion bodies, and accompanying liver damage. It is also known that this inclusion body accumulation is eliminated by deletion of p62.
  • the binding site is a region consisting of amino acid residues 345 to 359 in the KIR (Keap1 Interacting Region) sequence (mouse p62 amino acid sequence (SEQ ID NO: 3)).
  • KIR Keap1 Interacting Region
  • Nrf2 a transcription factor of detoxification enzymes in hepatocytes, expression of detoxification enzymes (antioxidant proteins, etc.), intracellular inclusions was found to induce the accumulation of liver and decrease in liver function. Therefore, by controlling (suppressing or promoting) the interaction (or binding) between p62 and Keap1, insolubilization of Keap1, stabilization and activation of Nrf2 which is a transcription factor of detoxification enzymes in hepatocytes, detoxification enzymes It is possible to control the expression of (antioxidant proteins, etc.), accumulation of intracellular inclusions, decrease in liver function, and the like.
  • the present invention has an important significance in that a new role of selective autophagy has been found in a mechanism for controlling transcription of a cytoprotective enzyme gene. Subsequently, the present invention provides a therapeutic agent for liver disease that inhibits or suppresses the interaction between p62 and Keap1. As described above, attenuation of autophagy causes hepatocellular carcinoma and liver damage accompanied by ubiquitin / p62-positive inclusions confirmed in various diseases. Since this is eliminated by suppressing the interaction between p62 and Keap1, a substance that inhibits or suppresses the interaction between p62 and Keap1 is likely to be applicable to a therapeutic agent for liver disease and an anticancer agent.
  • this therapeutic agent and anticancer agent are unlikely to cause autophagy abnormalities.
  • the interaction between Keap1 and p62 leads to stabilization and activation of Nrf2.
  • Nrf2 activation system associated with the binding of p62-Keap1 is considered to hardly function outside the liver, the interaction between p62 and Keap1 Anticancer agents that inhibit or suppress the action are considered to have few side effects.
  • this invention provides the therapeutic agent and anticancer agent of a liver disease which inhibit or suppress the interaction of Keap1 and p62 by suppressing the quantity of p62 in a cell.
  • the present invention can be applied to liver disease therapeutic agents and anticancer agents, diagnostic agents or screening methods thereof based on a novel mechanism of action.
  • the therapeutic agent for liver disease of the present invention has an advantage that there are few side effects.
  • FIG. 6 shows a list of p62 point mutation constructs and the corresponding pull-down assay.
  • a mutant (p62 T352A) that cannot interact with FLAG-tagged p62 and Keap1 was overexpressed in wild-type primary mouse hepatocytes using the adenovirus system. 48 hours after infection, cells were cultured for 4 hours in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 10 ⁇ M lactacystin. Cell lysates were analyzed by immunoprecipitation with anti-Nrf2 antibody, followed by SDS-PAGE and immunoprecipitation with anti-Nrf2 antibody and anti-ubiquitin antibody. Bands corresponding to Nrf2, ubiquitinated Nrf2, FLAG-p62, endogenous p62, and actin are shown. Results from three independent experiments are shown.
  • FIG. B It is a figure which shows the result of the immunoblot analysis of an Atg7 defect
  • the lower panel is an overlay of Keap1 (green) and p62 (red) images.
  • Each inset in the panel of Atg7 deficient liver is an enlarged view of the area enclosed by a square.
  • the bar is 20 ⁇ m.
  • F / F: Mx1 Atg7 / Nrf2 deficiency
  • RNA was prepared from the liver of the genotype shown in the figure on day 28 after pIpC injection. Values are normalized to the amount of mRNA in Atg7 F / F liver. The experiment was performed three times.
  • Panel (d)-(h): Atg7 F / F (control) (panel (d)) and Atg7 / Nrf2-deficient (Atg7- / Nrf2-) (panel (e)-(h)) is there.
  • the squared regions in panels (e) and (g) are enlarged and shown as panels (f) and (h), respectively.
  • the glycogen region (asterisk) that is easily detected in the control liver is occupied by a large amount of smooth ER and a large number of peroxisomes, some of which are indicated by arrows in panels (f) and (g) Note that is accumulated in the surrounding area.
  • Panels (g) and (h) show typical inclusion bodies observed in Atg7 / Nrf2-deficient liver. Bars: (d) and (e), 5 ⁇ m; (f) and (g), 1 ⁇ m; (h), 0.2 ⁇ m.
  • (A) It is a figure showing the measurement result of the weight of the mouse liver of a different genotype in the 28th day after pIpC injection
  • FIG. 1 shows typical liver histology of mice of the genotype shown in the figure.
  • the liver on day 28 after pIpC injection was treated with H & E staining. Higher-order magnified pictures are shown in the lower panel.
  • CV is the central vein and P is the portal vein. Bar: 100 ⁇ m.
  • Keap1 green
  • p62 red
  • the bar is 10 ⁇ m.
  • (A) shows photographs of livers at each age of Apg7 f / f mice (control) and Apg7 f / f; Alb-Cre mice (liver-specific autophagy-deficient mice).
  • (B) is a figure which shows the result of the immunoblotting of the extract adjusted from the liver of each 12-month-old mouse
  • (C) is a figure which shows the result of quantitative PCR using RNA adjusted from the liver of each 12-month-old mouse
  • the present invention provides an agent that inhibits or suppresses the interaction between p62 and Keap1.
  • a substance that inhibits or suppresses the interaction between p62 and Keap1 may be applicable as a therapeutic agent for liver disease and an anticancer agent.
  • a substance that inhibit or suppress the interaction between p62 and Keap1 For example (1) A substance that specifically binds to a binding site with Keap1 in the three-dimensional structure of p62, (2) a substance that inhibits or suppresses the interaction by binding in the vicinity of the binding site with Keap1 in the three-dimensional structure of p62; (3) a substance that binds to p62 and changes the three-dimensional structure of p62, (4) a substance that inhibits or suppresses interaction by inhibiting the expression of p62, (5) Substances that inhibit or suppress the interaction by decomposing p62 by autophagy are included.
  • a substance that inhibits or suppresses the interaction between p62 and Keap1 I) a molecule that specifically binds to p62, or Ii) p62 expression inhibitor Is included.
  • P62 is known as a ubiquitin-binding protein. Information on the amino acid sequence of p62 or the nucleotide sequence encoding it can be obtained from publicly available sequence databases. Some of these database accession numbers are listed here.
  • NCBI Accession number (species name)]: NCBI: NP_003891.1 (human), NCBI: NM_003900.4 (human), NCBI: NP_035148.1 (mouse), NCBI: NM_011018.2 (mouse) Swiss- Prot: 008623.1 (rat), NCBI: NP_78737.2 (rat), NCBI: NM_175843.3 (rat), Swiss-Prot: Q5RBA5.1 (orangutan).
  • p62 is not limited to human p62, but other species of p62 (eg, other mammalian species (eg, monkeys, cows, pigs, mice, rats, etc.), reptiles, amphibians, Homologs of fish, etc.).
  • other mammalian species eg, monkeys, cows, pigs, mice, rats, etc.
  • reptiles eg., amphibians, Homologs of fish, etc.
  • the amino acid sequence of such a homologue is, for example, 80% or more, 85% or more, 90% or more with respect to the amino acid sequence of human p62 (SEQ ID NO: 1) or the amino acid sequence of mouse p62 (SEQ ID NO: 3), Alternatively, one to several amino acids (for example: 2, 3, 4, 5) in the amino acid sequence having a sequence identity of 95% or more, the amino acid sequence of human p62 (SEQ ID NO: 1) or the amino acid sequence of mouse p62 (SEQ ID NO: 3) , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) deletion, substitution, insertion, or addition of amino acid residues It consists of an amino acid sequence having “Keap1 (Kelch-like ECH-associated protein 1)” interacts with NF-E2-related factor 2 (NF-E2-related factor 2 (Nrf2)) in a redox-sensitive manner, and when this protein diverges in the cytoplasm.
  • Keap1 Kerch-like E
  • Nrf2 is known to migrate to the nucleus. This interaction is known to control the expression of the catalytic subunit of ⁇ -glutamylcysteine synthetase.
  • Information on the amino acid sequence of Keap1 or the nucleotide sequence encoding it can be obtained from publicly available sequence databases. Some of the accession numbers in those databases are listed here. [Database name: Accession number (species name)]: Swiss-Prot: Q14145.2 (human), Swiss-Prot: Q9Z2X8.1 (mouse), GenBank: BAA46339.1 (mouse), GenBank: AB020063.1 ( Mouse), Swiss-Prot: Q684M4.1 (pig).
  • Keap1 is not limited to human Keap1, but other species of keap1 (eg, other mammalian species (eg, monkeys, cows, pigs, mice, rats, etc.), reptiles, amphibians, Homologs of fish, etc.).
  • the amino acid sequence of such a homologue is, for example, an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of human Keap1, or human Keap1 1 to several amino acid sequences (e.g.
  • a molecule that “specifically binds to p62” means that the molecule has a substantially higher affinity for a particular amino acid sequence of p62 or a fragment thereof than its affinity for other amino acid sequences. It means to combine with.
  • substantially high affinity means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device.
  • K a a coupling constant Is at least 10 7 M -1 , Preferably at least 10 8 M -1 , More preferably 10 9 M -1 , Even more preferably, 10 10 M -1 10 11 M -1 10 12 M -1 Or higher, for example, up to 10 13 M -1 Or means a binding affinity that is higher.
  • Examples of “molecules that specifically bind to p62” include the following i) and ii). i) Keap1 interacting region of p62 (Keapl Interacting Region) [SKEVDPSTGELQSLQ (SEQ ID NO: 5), for example, 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of SEQ ID NO: 5.
  • KIP1 KIP1 interacting region
  • the inhibitor or suppressor can typically be used as i) an inhibitor or suppressor of ubiquitin-positive inclusion body formation, ii) a therapeutic agent for liver disease, or iii) an anticancer agent.
  • the “molecule that specifically binds to p62” is LC3.
  • the “molecule that specifically binds to p62” is an antibody that specifically binds to p62 (anti-p62 antibody).
  • the “p62 expression inhibitor” refers to a substance that inhibits transcription of p62 gene into mRNA and / or translation of p62 gene (mRNA) into protein. Examples of “p62 expression inhibitor” include the following iii) and iv).
  • the p62 expression inhibitor is an antisense nucleic acid or siRNA against the nucleotide sequence encoding p62, Iv) A nucleic acid having a ribozyme activity that specifically cleaves the transcription product (mRNA) of the p62 gene.
  • nucleic acid means RNA or DNA.
  • the “nucleic acid” herein may contain not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to.
  • a nucleic acid having an action of inhibiting the expression of p62 gene by RNAi effect can be used as an active ingredient.
  • RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, expression of the introduced foreign gene and target endogenous gene are both inhibited.
  • RNA used here include double-stranded RNA that causes RNA interference of 19 to 30 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin RNA). .
  • dsRNA double strand RNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • RNA can be locally delivered to a desired site by a delivery system such as a liposome, and can be locally expressed using a vector capable of generating the double-stranded RNA.
  • Methods for preparing and using such double-stranded RNA are known from many literatures (Japanese translations of PCT publication No. 2002-516062; US Publication No.
  • the length of the double-stranded RNA exhibiting the RNAi effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, and most preferably 21 to 23 bases.
  • “antisense nucleic acid” or “antisense polynucleotide” has a polynucleotide complementary to at least a part of a DNA region of interest, and the polynucleotide is at least one of the region. It means a nucleic acid capable of hybridizing with a part.
  • the antisense nucleic acid or antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those that are resistant to degradation of polynucleotide amides and oligonucleotide amides.
  • the antisense nucleic acid used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 'side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method.
  • the sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the animal to be transformed, but it is not completely complementary as long as the gene expression can be effectively suppressed. May be.
  • Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more complement to the target gene transcript. Have sex.
  • the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40 bases), preferably about 15 bases or more, More preferably, it is about 100 bases or more, More preferably, it is about 500 bases or more.
  • Antisense nucleic acids can be designed with reference to known literature (for example, Hirashima and Inoue, Shinsei Kagaku Koza 2 Lecture and Expression of Nucleic Acid IV Gene, edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1993, p.319) -347), J. et al. Kawakami et al. , Pharm Tech Japan. Vol. 8, p. 247, 1992; Vol.
  • ribozyme activity refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene.
  • Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Protein Nucleic Acid Enzyme, 1990, 35, p. 2191).
  • ribozymes see, for example, FEBS Lett, 1988, 228, p. 228; FEBS Lett, 1988, 239, p. 285; protein nucleic acid enzyme, 1990, 35, p. 2191; Nucl Acids Res, 1989, 17, p. 7059 and the like can be referred to.
  • hairpin ribozymes see, for example, Nature, 1986, 323, p. 349; Nucl Acids Res, 1991, 19,. 6751; Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, p. 112 and the like can be referred to.
  • a method for treating liver disease and / or cancer comprising a step of administering a substance that inhibits or suppresses the interaction between p62 and Keap1 to a patient in need thereof. Provided. 2.
  • an inhibitor or inhibitor of binding between Keap1 and p62 is provided.
  • This agent typically contains a substance that inhibits or suppresses the interaction between p62 and Keap1 described above.
  • this agent I) a molecule that specifically binds to p62, or Ii) p62 expression inhibitor Containing.
  • the above agent of the present invention can be used as a preparation for treating liver disease or cancer.
  • Such a preparation can be produced by mixing a substance that inhibits or suppresses the interaction between p62 and Keap1 with a commonly used preparation carrier.
  • the pharmaceutical carrier may be used in appropriate combination depending on the dosage form.
  • excipients such as lactose; lubricants such as magnesium stearate; disintegrants such as carboxymethylcellulose; binders such as hydroxypropylmethylcellulose Surfactants such as macrogol; foaming agents such as sodium hydrogen carbonate; solubilizing agents such as cyclodextrin; sour agents such as citric acid; stabilizers such as sodium edetate; pH adjusters such as phosphate Is mentioned.
  • excipients such as lactose; lubricants such as magnesium stearate; disintegrants such as carboxymethylcellulose; binders such as hydroxypropylmethylcellulose Surfactants such as macrogol; foaming agents such as sodium hydrogen carbonate; solubilizing agents such as cyclodextrin; sour agents such as citric acid; stabilizers such as sodium edetate; pH adjusters such as phosphate Is mentioned.
  • a solid preparation for internal use for oral administration a liquid for internal use, and an injection, external preparation, suppository
  • Oral solid preparations for oral administration include tablets, pills, capsules, powders, granules and the like.
  • Capsules include hard capsules and soft capsules.
  • Tablets include sublingual tablets, buccal adhesive tablets, buccal quick disintegrating tablets and the like.
  • one or more active substances are left as they are, or excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, Mixed with magnesium metasilicate aluminate, etc.), disintegrating agents (such as calcium calcium glycolate), lubricants (such as magnesium stearate), stabilizers, solubilizing agents (such as glutamic acid, aspartic acid), etc. Used by formulating. If necessary, it may be coated with a coating agent (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.), or may be coated with two or more layers.
  • excipients lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.
  • binders hydroxypropylcellulose, polyvinylpyrrolidone, Mixed with magnesium metasilicate aluminate, etc.
  • disintegrating agents such
  • Oral liquids for oral administration include pharmaceutically acceptable solutions, suspensions / emulsions, syrups, elixirs and the like.
  • a solution one or more active substances are dissolved, suspended or emulsified in a commonly used diluent (purified water, ethanol or a mixture thereof).
  • this liquid agent may contain a wetting agent, a suspending agent, an emulsifier, a sweetening agent, a flavoring agent, a fragrance, a preservative, a buffering agent and the like.
  • External dosage forms for parenteral administration include, for example, ointments, gels, creams, poultices, patches, liniments, sprays, inhalants, sprays, eye drops, and nasal drops Etc. are included. These contain one or more active substances and are produced and prepared by known methods or commonly used formulations. Included as injections for parenteral administration are solutions, suspensions, emulsions and solid injections used by dissolving or suspending in a solvent at the time of use. An injection is used by dissolving, suspending or emulsifying one or more active substances in a solvent.
  • the solvent for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof are used. Further, this injection may contain a stabilizer, a solubilizing agent (such as glutamic acid, aspartic acid, polysorbate 80 (registered trademark)), a suspending agent, an emulsifier, a soothing agent, a buffering agent, a preservative and the like. . These are sterilized in the final process or manufactured and prepared by aseptic manipulation. In addition, a sterile solid preparation, for example, a lyophilized product, can be produced and used by dissolving it in sterilized or sterile distilled water for injection or other solvent before use.
  • a solubilizing agent such as glutamic acid, aspartic acid, polysorbate 80 (registered trademark)
  • a suspending agent such as glutamic acid, aspartic acid, polysorbate 80 (registered trademark)
  • the present invention provides a method for screening an agent that inhibits or suppresses the interaction between Keap1 and p62.
  • This method is typically I) measuring the binding amount of Keap1 and p62 in the presence and absence of the test substance, and ii) comparing the amount of binding between Keap1 and p62 in the presence and absence of the test substance, and the amount of binding in the presence of the test substance is greater than that in the absence of the test substance.
  • a substance that inhibits or suppresses the interaction between p62 and Keap1 can be applied to a liver disease therapeutic agent and an anticancer agent.
  • a novel drug for the treatment of liver disease can be screened.
  • the interaction can be detected by a pull-down method (eg, FIG. 3), a method using a mass spectrometer, a method for detecting protein-protein interaction as a fluorescent signal, or an imaging using a fluorescent label.
  • the step of measuring the amount of binding between Keap1 and p62 in the presence and absence of a test substance comprises the KIR sequence of p62 (eg, SKEVDPSTGELQSLQ (SEQ ID NO: 5), for example, an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 This includes measuring the amount of Keap1 binding to 1 to several (eg, amino acid sequences having 1, 2, 3, or more amino acid residue deletions, substitutions, insertions or additions).
  • the “substance that inhibits or suppresses the interaction between Keap1 and p62” is aspartic acid at position 349 in the amino acid sequence of mouse p62 (SEQ ID NO: 3), It specifically binds to proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354.
  • the screening method of the present invention Iii) administering a substance that inhibits or suppresses the interaction between Keap1 and p62 to a liver disease model animal and measuring the activity of Nrf2, and Iv) When the activity of Nrf2 decreases, the above-mentioned substance is selected as a therapeutic agent for liver disease Is included.
  • the substance obtained by the screening method of the present invention include LC3, anti-p62 antibody and the like, but are not limited thereto, low molecular weight compounds (natural or synthetic), proteins or peptides, polysaccharides, lipids , Nucleic acids and the like may be included.
  • a substance that inhibits or suppresses the interaction between p62 and Keap1, or a substance that decreases the amount of intracellular p62 can be applied to a therapeutic agent for liver disease and an anticancer agent. These substances inactivate (decompose) Nrf2 by reducing the amount of p62 that binds to Keap1 and increasing the binding between Keap1 and Nrf2.
  • a novel drug You can find Detection of the interaction between Keap1 and p62 was performed by transiently cultivating a plasmid in which Nrf2 transcriptional activity reporter ARE (antioxidant-respondive element) was connected to luciferase gene to cultured cells (Huh-1, JHH5, Hepa-1, HEK293T, etc.). By luciferase assay using a cell line introduced into or stably maintained (see FIGS.
  • Nrf2 transcriptional activity reporter assay using lactamase and fluorescence resonance energy transfer (FRET)
  • FRET fluorescence resonance energy transfer
  • the measurement of the binding amount of Keap1 and p62 in the presence and absence of a test substance is carried out using a pull-down method, mass spectrometry, protein-protein interaction as a fluorescence signal. This is performed using either a detection method or imaging using a fluorescent label.
  • a substance that inhibits or suppresses the interaction between p62 and Keap1 or a substance that reduces the amount of intracellular p62 may be applicable to a therapeutic agent for liver disease and an anticancer agent. These substances reduce the amount of intracellular ubiquitin positive inclusions in the liver lacking mouse autophagy.
  • a substance that inhibits or suppresses the interaction of Keap1 or a substance that decreases the amount of intracellular p62 is administered to mice, and the amount of ubiquitin positive inclusion bodies, the activity of Nrf2, and the level of liver damage are measured. Drug candidates can be screened. Detection of ubiquitin positive inclusion bodies can be performed by immunostaining mouse liver tissue (FIG. 9). In the Nrf2 activity measurement, the expression of the detoxification enzyme gene group which is a downstream factor of Nrf2 can be confirmed by real time PCR (FIG. 8). Liver damage level measurement includes liver weight measurement (FIG. 13a), hepatocyte hypertrophy and lobular structure collapse detection by HE staining of liver tissue (FIG.
  • liver function test (AST, ALT, ALP) (FIG. 14) Can be done by.
  • the substance obtained by the screening method of the present invention can be used as i) an inhibitor or suppressor of ubiquitin-positive inclusion body formation, ii) a therapeutic agent for liver disease, or iii) an anticancer agent. 4). Diagnostic method, diagnostic agent, and diagnostic kit for liver disease
  • the present invention provides a method for diagnosing liver disease.
  • a method for determining or diagnosing a patient to which the therapeutic agent for liver disease of the present invention is applied is provided.
  • This method is typically I) measuring the expression level of p62 or Nrf2 by subject's liver biopsy, or Ii) measuring the activity of Nrf2 by subject's liver biopsy, including. If it is found that the expression level of p62 in a liver biopsy of a subject with liver disease is excessive as compared with that of a normal subject, it is highly likely that the liver disease of the subject is caused by overexpression of p62. In such a case, the therapeutic agent for liver disease according to the present invention is applied to a patient who needs to treat the disease in order to inhibit or suppress the interaction between p62 and Keap1 or to inhibit the expression of p62. By doing so, it is considered that an advantageous effect can be obtained. Therefore, it is useful to determine or diagnose the patient to whom such treatment is applied.
  • the liver disease of the subject is due to abnormal activation of Nrf2.
  • the therapeutic agent for liver disease of the present invention is applied to a patient in need of treatment of the disease, the interaction between p62 and Keap1 is inhibited or suppressed, or the expression of p62 is inhibited.
  • the amount of p62 competing with the interaction between Nrf2 and Keap1 can be reduced, so that the amount of free Nrf2 is reduced and the activation of Nrf2 is suppressed.
  • Measurement of the expression level of p62 by liver biopsy of a subject can be performed by methods well known to those skilled in the art, such as imaging with a fluorescent label, immunoblotting, and mass spectrometry.
  • Measurement of the activity of Nrf2 by liver biopsy of a subject includes quantitative measurement of luciferase assay using a target promoter of Nrf2, measurement of the amount of Nrf2 in cell nuclei by immunoblotting, and enzymes induced by Nrf2 (such as Nqol and Gstm).
  • RNA sample can be performed by methods well known to those skilled in the art, such as expression analysis by PCR, immunoblotting or fluorescence imaging, and enzyme activity measurement.
  • measurement of Nrf2 activity and / or detection of ubiquitin positive inclusion bodies is included.
  • a further embodiment of the method for determining a patient to which the therapeutic agent for liver disease of the present invention is applied includes measuring the concentration of AST, ALT, ALP, and / or ⁇ -GTP in the blood of the subject.
  • ⁇ -GTP ( ⁇ -glutamyl transpeptidase) is an enzyme related to the detoxification of the liver, and when the cells of the liver and bile duct break down, ⁇ -GTP flows into the blood. It is said to be a “deviation enzyme” and is generally used as an indicator that the cells of the liver and bile ducts have been broken.
  • a diagnostic kit or diagnostic agent used in the diagnostic method of the present invention is provided. These diagnostic kits or diagnostic agents typically include an anti-p62 antibody or an anti-Nrf2 antibody. Further, these diagnostic kits or diagnostic agents may contain a labeling agent for labeling the antibody (for example, a fluorescent label) or a fluorescently labeled antibody.
  • the diagnostic kit may further include instructions for use.
  • the instructions for use may describe the method for using the antibody, the labeling agent, etc. included in the kit, the experimental procedure including immunoblotting, and the like.
  • the relationship between the amino acid sequence or nucleotide sequence appearing in this specification and the sequence number is as follows. [SEQ ID NO: 1] This sequence represents the amino acid sequence of human p62. [SEQ ID NO: 2] This sequence represents a nucleotide sequence encoding the amino acid sequence of human p62 (SEQ ID NO: 1). [SEQ ID NO: 3] This sequence represents the amino acid sequence of mouse p62.
  • [SEQ ID NO: 4] This sequence represents a nucleotide sequence encoding the amino acid sequence of mouse p62 (SEQ ID NO: 3).
  • [SEQ ID NO: 5] This sequence represents a partial sequence (KIR sequence) consisting of consecutive amino acid residues from the 345th position to the 359th position of the amino acid sequence of mouse p62 (SEQ ID NO: 3).
  • Example 1 In Example 1, the interaction between p62 and Keap1 and the region of Keap1 that binds to p62 were examined. Hek293T cells in which each mutant protein of mouse Keap1 and Keap1 in which Flag-tag was linked to the N terminus were expressed were lysed with lysis buffer to obtain a cell extract. Next, immunoprecipitation was performed using an anti-Flag antibody, and Keap1 to which Flag-tag was added or its deletion mutant and its interaction complex were recovered.
  • the recovered protein complex and cell extract are developed in a gel by SDS-PAGE (polyacrylamide gel electrophoresis) and immunized with anti-Flag antibody, anti-p62 antibody, anti-Nrf2 antibody, and anti-actin antibody. Blotting (IB) was performed. The results are shown in FIG. 1 shows the functional domain region of Keap1 and the names and schematic diagrams of Keap1 and each mutant with FLAG-tag attached, and I.I. B. And I. using cell extract. B. It is a photograph of. At the top of the schematic diagram, the domain region of Keap1 and its name are shown.
  • NTR N-terminal region
  • BTB Broadcomplex, Tramtrac, and Brick-a-Brac
  • IVR intervening region
  • DSG the double glyceine repeat crea- te
  • Example 2 In Example 2, the direct interaction between p62 and Keap1 and the region of p62 that binds to Keap1 were examined.
  • FIG. 2 is a photograph of mouse p62 with a functional domain region of p62 and MBP-tag, names and schematics of each mouse, and stained with SDS-PAGE and CBB.
  • the top part of the schematic diagram shows the domain region of p62 and its name.
  • PB1 Phox and Bemlp
  • Zinc Zinc finger
  • LRS LC3-recognition sequence
  • UBA ubiquitin-associated domain
  • the binding region of p62 to Keap1 is a region of 345-359 amino acids. I found out. Further, this region is highly conserved among species as shown in FIG. 2b, and this amino acid sequence region was named a KIR (Keap1 interacting region) (Keap1 interaction region) sequence.
  • Example 3 amino acids in the p62-KIR sequence important for Keap1 binding were examined.
  • a pull-down assay similar to that in Example 2 was performed using a protein in which each amino acid in the -KIR sequence of MBP-p62M80 was substituted with alanine and GST-Keap1-DC. The results are shown in FIG.
  • the amino acid substitution site for each sample number is shown in the upper left of the figure, the lower part shows a photograph of the gel after staining with SDS-PAGE and CBB, and the right side of the photograph shows the name of the protein indicated by the band.
  • Example 4 In Example 4, it was examined that p62 and Nrf2 were bound in the same pocket of Keap1. Protein crystallization three-dimensional structure analysis predicted amino acids important for binding of Keap1-DC to p62 or Nrf2. The Keap1-DC mutant and Keap1-DC proteins in which these amino acids were each replaced with alanine were FLAG-tagged and expressed in Hek293T cells.
  • Example 1 immunoprecipitation and I.V. B. Went. The results are shown in FIG. The left side of FIG. 4 shows each sample number and the amino acid substitution site for that number.
  • I. Immunoprecipitation of anti-FLAG antibody B. are shown in IP: FLAG.
  • I. B. These photographs are represented by Crude, which shows confirmation of the expression of Keap1, p62, and actin (loading control) in each cell.
  • the antibody used is shown on the right side of the photo. As shown in FIG.
  • Example 5 shows that p62 competitively inhibits Keap1-Nrf2 binding.
  • Hek293T cells expressing p62 and p62-T352A in which Flag-tag was linked to the N terminus were lysed with lysis buffer to obtain a cell extract. Next, immunoprecipitation was performed using an anti-Nrf2 antibody, and Nrf2 and its interaction complex were recovered.
  • Nrf2 lane 1 the detection level of ubiquitinated Nrf2 and Nrf2 itself is low, but is significantly higher when a proteasome inhibitor is added (lane 2). Become. This can be explained by the fact that Nrf2 usually binds to keap1 and is ubiquitinated and degraded by the proteasome. When p62 was overexpressed in this state, the amount of Nrf2 protein significantly increased. On the other hand, ubiquitinated Nrf2 decreased (IP: Nrf2 lanes 3 and 4).
  • Nrf2 is increased because p62 binds to Keap1 and the binding between Nrf2 and Keap1 decreases and Nrf2 is not ubiquitinated and degraded.
  • IP Nrf2 lanes 5 and 6
  • IP Nrf2 lanes 1 and 2
  • Nrf2, Keap1, and p62 wild-type or p62 mutants were introduced into Hepa1 cells derived from mouse hepatocellular carcinoma together with pNQO1-ARE reporter plasmid and pRL-TK (internal control of lipofection), and 36 hours later The luciferase activity was measured. The results are shown in FIG. The presence or absence of Nrf2, Keap1, p62-WT, p62-mutants introduction is indicated by +-, and the relative luciferase activity is shown with no expression vector introduced (column 1) as a standard.
  • Nrf2 When Nrf2 is overexpressed (column 2), the activity increases significantly, and in the case of double transfection of Nrf2 and Keap1 (column 3), Nrf2 is captured by Keap1 and degraded to the same level as column 1 Decreased.
  • p62 In the triple transfection of Nrf2, Keap1, and p62-WT, p62 binds to Keap1 and free Nrf2 increases, so the luciferase activity increased, but Nrf2, Keap1, p62-mutant triple transfection ( In column 5-10), p62 cannot bind to Keap1, so the activity was not different from column 4.
  • Example 7 shows that in mice lacking autophagy specifically in the liver, Keap1 accumulates and insolubilizes with p62, and insolubilization of Keap1 is dependent on p62. The results are shown in FIG. F / F: Mx1 is a conditional knockout mouse for the Atg7 gene.
  • each fraction is developed by SDS-PAGE and immunoblotted with anti-p62 antibody, anti-Keap1 antibody, anti-LC3 antibody, anti-Nqo1 antibody, and anti-actin antibody (loading control).
  • the left side of the figure shows the type of antibody used, the upper part shows the number of days after administration, or the genotype of the mouse.
  • Total shown in the lower part of the figure is the whole cell extract, Sol.
  • Is the soluble fraction, Insol. Indicates an insoluble fraction. 8-12 hours after autophagy is lost by PIPC administration, p62 is excessively expressed, but Keap1 expression is hardly changed. Insoluble p62 accumulates with overexpression of p62, and at the same time, insoluble Keap1 accumulates.
  • RNA is extracted from the mouse liver 12 hours after PIPC administration to each genotype mouse.
  • cDNA was synthesized from total RNA 1 microg, and LightCycler 480 Probes Masterin (Rochet Cycle Probe Master (Phc).
  • the signal was normalized with ⁇ -glucuronidase (GUS).
  • the results are shown in FIG.
  • the upper part of FIG. 8 shows the gene name, the left part shows the ratio of the amount of mRNA when F / F is 1, and the lower part shows each genotype.
  • both Nqo1 and Gstm1 have dramatically increased expression levels in autophagy-deficient mice (F / F: Mx1).
  • Nrf2 downstream gene is increased by autophagy deficiency.
  • mice lacking p62 p62 and F / F: Mx1: p62
  • F / F wild type mice
  • activation of Nqo1 and Gstm1 due to autophagy deficiency depends on p62. It was shown that [Example 9] In Example 9, it is shown in mouse hepatocytes that Keap1 is sequestered in inclusion bodies in a p62-dependent manner.
  • the liver obtained from a mouse 28 hours after PIPC administration was cut into the genotype mouse shown in Example 7, and the intracellular localization of p62 and Keap1 by double immunostaining using anti-p62 antibody and anti-Keap1 antibody Was observed with fluorescence.
  • the results are shown in FIG.
  • the upper row in FIG. 9 is p62, the middle row is Keap1, the lower row is a combination of p62 and Keap1 localization (Marge), and the respective genotypes are written in the upper left part of the nuclear staining diagram.
  • F / F An enlarged view of the area surrounded by the white square frame at the upper right part of Mx1 is shown in the lower left.
  • Keap1 Due to autophagy deficiency, Keap1 is insolubilized depending on the expression of p62, and the expression of downstream factors of Nrf2 is also increased. Therefore, in addition to the F / F and F / F: Mx1 shown in Example 7 used so far, Nrf2-deficient mice (Nrf2 ⁇ / ⁇ , F / F: Mx1: Nrf2 ⁇ / ⁇ ) were prepared. The insolubilization of Keap1 was examined by the same method as in Example 7. The expression of Nrf2 due to Nrf2 deletion and the expression of Nrf2 downstream genes (Nqo1 and Gstm1) were also confirmed at the same time. The results are shown in FIG.
  • Example 11 shows that the accumulation of p62, Keap1 positive inclusion bodies due to autophagy deficiency is dependent on Nrf2.
  • Example 12 shows that overexpression of Nrf2 downstream gene due to autophagy deficiency is dependent on Nrf2.
  • Nrf2 downstream gene (Nqo1, Gstm1, Cyp2a5) seen in autophagy deficiency (F / F: Mx1) is caused when Nrf2 is deficient (F / F: Mx1: Nrf2 ⁇ / -) Dramatically decreased.
  • F / F: Mx1 Nrf2 ⁇ / -
  • Example 13 In order to examine whether the liver damage due to autophagy deficiency is a result of lack of Nrf2-Keap1 pathway due to abnormal accumulation of p62, the genotype mouse of Example 10 was used to measure the weight of the mouse liver 28 days after pIpC administration. Furthermore, hepatocyte hypertrophy of each gene-deficient mouse was measured by HE staining. The results are shown in FIGS. 13a and 13b, respectively.
  • Example 14 In autophagy-deficient mice (F / F: Mx1), the weight of the liver is greatly increased and hepatic hypertrophy, whereas autophagy / Nrf2 double-deficient mice do not show an increase. Therefore, Nrf2-Kap1pathway due to abnormal accumulation of p62. It was found that the lack caused liver enlargement. [Example 14] In Example 14, in order to examine whether liver damage due to autophagy deficiency is a result of lack of Nrf2-Keap1 pathway due to abnormal accumulation of p62, as an indicator of liver damage, aspartate aminotransferase (AST), alanine aminotransferase (ALT), enzyme activity of alkaline phosphatase (ALP) was measured.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP enzyme activity of alkaline phosphatase
  • Example 16 shows the presence of the Nrf2 activity system in p62 accumulation in a cell line stably retaining the ARE-Luciferase gene of hepatoma-derived cells (Hepa-1), and for screening for p62-Keap1 binding inhibitors. Indicates that it can be used. The result of the experiment is shown in FIG.
  • Example 17 shows the molecular state of tumors, Keap1 and Nrf2 downstream factors in the liver of autophagy-deficient mice or autophagy-deficient p62 knockout mice. The results are shown in FIG. Upper left figure shows Apg7 at 4, 7, 9, 12, 14, 16 months f / f Mouse (control) and Apg7 f / f; It is a photograph of the liver of an Alb-Cre mouse (liver-specific autophagy-deficient mouse).
  • Apg7 f / f In Alb-Cre mice, small tumors (arrows) were confirmed after 7 months of age, and the number and size of tumors increased with age. Bottom left figure is 12 months old Apg7 f / f (Control) Mouse liver, p62 knockout liver, Apg7 f / f; Liver non-tumor part, liver tumor part and Apg7 of Alb-Cre mice f / f; It is the result of the Western blot analysis which used Alb-Cre; p62 knockout liver extract.
  • the present invention is useful as a therapeutic agent for liver diseases and an anticancer agent, a diagnostic agent or a screening method thereof based on a novel mechanism of action.

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Abstract

Provided is a method for controlling the interaction between Keap1 and p62 which control the formation of an ubiquitin-positive inclusion body.

Description

肝疾患治療薬Liver disease treatment
　本発明は、細胞内のユビキチン陽性封入体の形成を制御するｐ６２とＫｅａｐ１のタンパク質相互作用、前記相互作用を阻害又は抑制する物質を有する肝疾患治療薬および抗がん剤、前記相互作用を用いたスクリーニング方法などに関する。 The present invention relates to a protein interaction between p62 and Keap1 that controls the formation of ubiquitin-positive inclusion bodies in cells, a therapeutic agent for liver disease and an anticancer agent having a substance that inhibits or suppresses the interaction, and uses the interaction Related to screening methods.
　肝臓はヒトにおける最大の代謝器官であり、生命維持に必須の臓器である。この臓器は、高カロリー・高脂肪の飲食、ストレスなどにより肝肥大、脂肪肝などを引き起こし、機能低下を招く。またウイルス感染やアルコール摂取などにより、ウイルス性肝炎、アルコール性肝炎を引き起こし、これらの障害が長期にわたると肝硬変や肝がんに至る。
　しかしながら、これらの肝疾患に対する有効な薬剤が開発されていないのが現状である。そのため、様々な肝疾患に対して有効な薬剤の開発が望まれている。その中で、本発明者らのオートファジーの研究から有望な肝疾患治療薬および抗がん剤を提供する。
　オートファジーは細胞成分の代謝回転、無用なオルガネラの除去、凝集易発性の変異タンパク質の除去などに関与する細胞内大規模分解経路である。オートファジーはまず、隔離膜が伸長し、細胞質構成成分を取り囲んだ脂質二重膜構造体（オートファゴソーム）を形成する。このオートファゴソームがリソソームと融合し、リソソームの加水分解酵素により囲い込まれた細胞質成分を分解する。酵母遺伝学の研究により、数多くのＡＴＧ遺伝子がオートファジーに必須の役割を担っていることが知られるようになった。オートファゴソームに局在するＬＣ３は酵母Ａｔｇ８のヒトホモログとして同定され、隔離膜の伸長に必須の役割を担っている。そのＬＣ３との結合因子としてｐ６２が取得された（非特許文献１：Ｋｏｍａｔｓｕ　ｅｔ　ａｌ．２００７　Ｃｅｌｌ）。
　ｐ６２はシグナル伝達を担う多彩な分子群と相互作用する。またＣ末端にユビキチン会合ドメインを有しており、ユビキチン化タンパク質のプロテアソームへのシャトル分子として知られている（非特許文献２：Ｓｅｉｂｅｎｈｅｎｅｒ　ｅｔ　ａｌ．２００４　ＭＣＢ）。またｐ６２はＬＣ３との相互作用を介してオートファジー・リソソーム系で分解されることから、オートファジーに対する選択的基質とされている（非特許文献３：Ｂｊｏｋｏｙ　ｅｔ　ａｌ．２００５　ＪＣＢ，非特許文献４：Ｐａｎｋｉｖ　ｅｔ　ａｌ．２００７　ＪＢＣ，非特許文献５：Ｉｃｈｉｍｕｒａ　ｅｔ　ａｌ．２００８　ＪＢＣ）。実際にｐ６２のノックアウトマウスにおいて正常なオートファゴソーム形成およびリソソームによるタンパク質分解が確認されている。またｐ６２ノックアウトマウスは２型糖尿病を発症するが、オートファジー欠損マウスにおいて観察される肝障害や神経変性は確認されていない（非特許文献６：Ｒｏｄｒｉｇｕｅｚ　Ａ　２００６　Ｃｅｌｌ　Ｍｅｔａｂ）。
　肝臓もしくは脳特異的Ａｔｇ７欠損マウスの肝臓もしくは脳において、ｐ６２が蓄積、不溶化し、最終的にｐ６２陽性の封入体（ｉｎｃｌｕｓｉｏｎ　ｂｏｄｉｅｓ）が確認される。またＡｔｇ７欠失肝臓、脳において、ユビキチン化タンパク質とｐ６２は比例的に蓄積し、ほぼ全ての封入体がユビキチン・ｐ６２陽性である（非特許文献７：Ｋｏｍａｔｓｕ　ｅｔ　ａｌ．２００５　ＪＣＢ，非特許文献１：Ｋｏｍａｔｓｕ　ｅｔ　ａｌ．２００７　Ｃｅｌｌ）。封入体の形成過程は、変異タンパク質の折りたたみ異常、可溶性オリゴマー形成、そして可溶性オリゴマーの不溶化・封入体として蓄積の３段階よりなる。この封入体の形成は、様々な病態（アルコール性肝炎やパーキンソン病、筋萎縮性側索硬化症、ＡＬＳなどの神経変性疾患）において観察されており、オートファジー欠損マウスでは、細胞内封入体形成を伴った肝細胞肥大、肝肥大を引き起こし、肝障害を発症する。Ａｔｇ７とｐ６２を同時に欠損させると、封入体の形成はほぼ完全に抑制され、またオートファジー不全で確認される肝肥大・肝障害を強く抑制した。このことは過度なｐ６２の蓄積が肝障害の主原因であることを示している（非特許文献１：Ｋｏｍａｔｓｕ　ｅｔ　ａｌ．２００７　Ｃｅｌｌ）。重要なことに、ユビキチン・ｐ６２陽性封入体の蓄積は、アルツハイマー病、筋萎縮性側索硬化症，星状細胞腫などの神経性疾患や、アルコール性肝炎、脂肪肝、肝がん患者の肝臓組織においても同定されている（非特許文献８：Ｓｔｕｍｐｔｎｅｒ　ｅｔ　ａｌ．，２００７　Ｈｐａｔｏｌｏｇｙ，非特許文献９：Ｚａｔｌｏｕｋａｌ　Ｋ　ｅｔ　ａｌ．ＡＭ　Ｊ　Ｐａｔｈｏｌ　２００２）。
　さらにオートファジー欠損肝臓においは、グルタチオンＳトランスフェラーゼ（ＧＳＴ），ＮＡＤ（Ｐ）Ｈ　Ｄｅｈｙｄｒｏｇｅｎａｓｅ，ＮＱＯ１（ｑｕｉｎｉｎｅ　１）など一連の解毒酵素群の転写が著しく誘導されている。これらの解毒酵素群の転写のほとんどは転写因子Ｎｒｆ２（ＮＦ−Ｅ２−ｒｅｌａｔｅｄ　ｆａｃｔｏｒ　２）によって制御されることが知られている。通常、Ｎｒｆ２はユビキチンリガーゼＫｅａｐ１（Ｋｅｌｃｈ−ｌｉｋｅ　ＥＣＨ−ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　１）に捕獲され、ユビキチン化を受けることによりプロテアソームによって分解されている。一方酸化ストレスないし親電子性物質に暴露されると、Ｋｅａｐ１の特定のシステイン残基が酸化されＮｒｆ２との結合を失う。その結果、分解を免れたＮｒｆ２は核内に移動し、ＧＳＴやＮＱＯ１などの解毒酵素群の転写を活性化することが明らかにされている（非特許文献１０：Ｔｏｎｇ　ＫＩ　ｅｔ　ａｌ．Ｍｏｌ　Ｃｅｌｌ　Ｂｉｏｌ　２００７）。またオートファジー欠損肝臓では、Ｎｒｆ２が核内に蓄積し、その蓄積はｐ６２の同時欠損で抑制される。これらの結果は，ｐ６２依存的なＮｒｆ２制御機構の存在を示唆する。 The liver is the largest metabolic organ in humans and an essential organ for life support. This organ causes liver enlargement, fatty liver, etc. due to high calorie / high fat eating and drinking, stress, etc., leading to functional deterioration. Moreover, viral hepatitis and alcoholic hepatitis are caused by viral infection and alcohol consumption, etc., and these disorders lead to cirrhosis and liver cancer over a long period of time.
However, the present condition is that the effective drug with respect to these liver diseases is not developed. Therefore, development of an effective drug for various liver diseases is desired. Among these, promising therapeutic agents for liver diseases and anticancer agents are provided from our autophagy research.
Autophagy is a large-scale intracellular degradation pathway involved in the turnover of cellular components, removal of unwanted organelles, removal of aggregation-prone mutant proteins, and the like. In autophagy, first, the isolation membrane extends to form a lipid bilayer structure (autophagosome) surrounding the cytoplasmic components. This autophagosome fuses with the lysosome and degrades the cytoplasmic component enclosed by the lysosomal hydrolase. Yeast genetics studies have revealed that many ATG genes play an essential role in autophagy. LC3 localized in the autophagosome has been identified as a human homologue of yeast Atg8 and plays an essential role in the elongation of the isolation membrane. P62 was obtained as a binding factor with LC3 (Non-patent Document 1: Komatsu et al. 2007 Cell).
p62 interacts with a diverse group of molecules responsible for signal transduction. Further, it has a ubiquitin-associated domain at the C-terminus, and is known as a shuttle molecule for ubiquitinated proteins to the proteasome (Non-Patent Document 2: Seibenhener et al. 2004 MCB). Moreover, since p62 is decomposed | disassembled by an autophagy lysosome system through interaction with LC3, it is made into the selective substrate with respect to an autophagy (nonpatent literature 3: Bjokoy et al.2005 JCB, nonpatent literature 4). : Pankiv et al. 2007 JBC, Non-Patent Document 5: Ichimura et al. 2008 JBC). In fact, normal autophagosome formation and lysosomal proteolysis have been confirmed in p62 knockout mice. In addition, p62 knockout mice develop type 2 diabetes, but hepatic damage and neurodegeneration observed in autophagy-deficient mice have not been confirmed (Non-patent Document 6: Rodriguez A 2006 Cell Metab).
In the liver or brain of the liver or brain-specific Atg7-deficient mouse, p62 accumulates and becomes insoluble, and finally p62-positive inclusion bodies are confirmed. In Atg7-deficient liver and brain, ubiquitinated protein and p62 accumulate proportionally, and almost all inclusion bodies are ubiquitin / p62 positive (Non-patent Document 7: Komatsu et al. 2005 JCB, Non-patent Document 1). : Komatsu et al. 2007 Cell). The formation process of the inclusion body is composed of three stages: abnormal folding of the mutant protein, formation of soluble oligomer, and insolubilization / accumulation of soluble oligomer. Formation of this inclusion body has been observed in various pathological conditions (neurodegenerative diseases such as alcoholic hepatitis, Parkinson's disease, amyotrophic lateral sclerosis, and ALS). In autophagy-deficient mice, intracellular inclusion formation Causes hepatocyte hypertrophy and hepatic hypertrophy accompanied by liver damage. When Atg7 and p62 were deleted at the same time, inclusion body formation was almost completely suppressed, and hepatic hypertrophy and liver damage confirmed by autophagy failure were strongly suppressed. This indicates that excessive p62 accumulation is the main cause of liver damage (Non-patent Document 1: Komatsu et al. 2007 Cell). Importantly, the accumulation of ubiquitin / p62-positive inclusions is associated with neurological diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, astrocytoma, alcoholic hepatitis, fatty liver, liver of liver cancer patients. It has also been identified in tissues (Non-patent document 8: Stumptner et al., 2007 Hpatology, Non-patent document 9: Zatloukal K et al. AM J Pathol 2002).
Furthermore, in the autophagy-deficient liver, transcription of a series of detoxification enzymes such as glutathione S-transferase (GST), NAD (P) H Dehydrogenase, NQO1 (quinine 1) is remarkably induced. It is known that most of the transcription of these detoxifying enzymes is controlled by the transcription factor Nrf2 (NF-E2-related factor 2). Normally, Nrf2 is captured by the ubiquitin ligase Keap1 (Kelch-like ECH-associated protein 1) and degraded by the proteasome by undergoing ubiquitination. On the other hand, when exposed to oxidative stress or an electrophilic substance, a specific cysteine residue of Keap1 is oxidized and loses binding to Nrf2. As a result, it has been clarified that Nrf2 that has escaped the degradation moves into the nucleus and activates transcription of detoxifying enzymes such as GST and NQO1 (Non-patent Document 10: Ton KI et al. Mol Cell Biol. 2007). In the autophagy-deficient liver, Nrf2 accumulates in the nucleus, and the accumulation is suppressed by the simultaneous deficiency of p62. These results suggest the existence of a p62-dependent Nrf2 control mechanism.
　上記の各知見により、過度のユビキチン陽性封入体形成が肝疾患（肝細胞がんや肝障害など）を引き起こし、またｐ６２が封入体の形成に深く関与することが示唆されている。
そこで本発明は、ｐ６２の封入体形成の制御メカニズムを同定し、そのメカニズムに基づく新規な肝疾患治療薬および抗がん剤、あるいは診断薬を提供することを主な目的とする。 Each of the above findings suggests that excessive ubiquitin-positive inclusion body formation causes liver diseases (such as hepatocellular carcinoma and liver injury), and that p62 is deeply involved in inclusion body formation.
Therefore, the main object of the present invention is to identify a control mechanism of p62 inclusion body formation and to provide a novel therapeutic agent for liver disease and anticancer agent or diagnostic agent based on the mechanism.
　本発明者らは、ｐ６２がＫｅａｐ１と結合することを初めて見出した。またＫｅａｐ１におけるｐ６２の結合部位は、Ｎｒｆ２の結合と同じポケットを使用していることも明らかにした。オートファジー欠損によってｐ６２が蓄積すると、Ｋｅａｐ１とｐ６２の結合が増加し、それに伴いフリーのＮｒｆ２が増加する。そして蓄積したＮｒｆ２が核内へ移行し、解毒酵素群の転写を上昇させている。
　また、本発明者らの実験によれば、オートファジー欠損の肝臓でＮｒｆ２も同時欠損させると、細胞内封入体の形成やそれに伴う肝肥大、肝障害を抑圧できた。これはＫｅａｐ１とｐ６２の結合増加が、直接Ｎｒｆ２の活性化、そして肝肥大や肝障害、そして肝臓がんを導くことを示す。したがって、ｐ６２とＫｅａｐ１の結合を阻害することでユビキチン−ｐ６２陽性封入体の形成を防ぎ、肝肥大、肝障害、肝臓がん治療あるいは、ユビキチン−ｐ６２陽性封入体形成が確認されている星状細胞腫の治療に繋がることが示唆される。
　したがって、本発明は、以下の剤、診断方法、診断キット、診断剤、およびスクリーニング方法を提供する。
（１）Ｋｅａｐ１とｐ６２との結合の阻害または抑制剤。
（２）上記（１）に記載の剤であって、
　ｉ）ｐ６２に特異的に結合する分子、または
　ｉｉ）ｐ６２の発現阻害物質
を含有する剤。
（３）ｉ）ユビキチン陽性封入体形成の阻害又は抑制剤、
　ｉｉ）肝疾患の治療剤、または
　ｉｉｉ）抗がん剤
として使用される、上記（１）または（２）に記載の剤。
（４）上記ｐ６２に特異的に結合する分子が、
　ｉ）ｐ６２のＫｅａｐ１相互作用領域に特異的に結合する物質、
　ｉｉ）マウスｐ６２のアミノ酸配列（配列番号３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、もしくは３５４位のグルタミン酸に特異的に結合する物質、または
　ｉｉｉ）上記ｐ６２の発現阻害物質が、ｐ６２をコードするヌクレオチド配列に対するアンチセンス核酸もしくはｓｉＲＮＡ
である、上記（２）または（３）に記載の剤。
（５）上記ｐ６２に特異的に結合する分子が、ＬＣ３または抗ｐ６２抗体である、上記（２）に記載の剤。
（６）上記（３）に記載の肝疾患の治療剤の適用患者を判定する方法であって、
　ｉ）被験者の肝生検によるｐ６２もしくはＮｒｆ２の発現量を測定する工程、または
　ｉｉ）被験者の肝生検によるＮｒｆ２の活性を測定する工程、
を含む、方法。
（７）被験者の血液中のＡＳＴ，ＡＬＴ，ＡＬＰ，またはγ−ＧＴＰの酵素活性を測定することをさらに含む、上記（６）に記載の方法。
（８）上記（６）に記載の方法に使用する診断キットまたは診断剤であって、
　抗ｐ６２抗体または抗Ｎｒｆ２抗体を含み、
　上記キットはさらに使用説明書
を含む、キットまたは剤。
（９）Ｋｅａｐ１とｐ６２との相互作用を阻害または抑制する物質（ａｇｅｎｔ）のスクリーニング方法であって、
　ｉ）試験物質の存在下および非存在下でＫｅａｐ１とｐ６２との結合量を測定する工程、および
　ｉｉ）上記試験物質の存在下と非存在下との間でＫｅａｐ１とｐ６２との上記結合量を比較し、上記試験物質の存在下での上記結合量が上記試験物質の非存在下での上記結合量よりも低い場合に、当該試験物質をＫｅａｐ１とｐ６２との相互作用を阻害または抑制する物質として選択する工程
を含む、方法。
（１０）上記結合量を測定する工程において、プルダウン法、質量分析法、タンパク質間相互作用を蛍光シグナルとして検出する方法、または蛍光標識を用いたイメージングのいずれかが用いられる、上記（９）に記載の方法。
（１１）ユビキチン陽性封入体形成を阻害または抑制する物質のスクリーニングのために使用する、上記（９）または（１０）に記載の方法。
（１２）肝疾患の治療剤のスクリーニングのために使用する、上記（９）~（１１）のいずれかに記載の方法。
（１３）上記結合量を測定する工程が、
　ｉ）ｐ６２のＫｅａｐ１相互作用領域、または
　ｉｉ）マウスｐ６２のアミノ酸配列（配列番号：３）中の３４５番から３５９番目のアミノ酸配列：ＳＫＥＶＤＰＳＴＧＥＬＱＳＬＱ（配列番号：５）
に対するＫｅａｐ１の結合量を測定することを含む、上記（９）~（１２）のいずれかに記載の方法。
（１４）上記物質が、マウスｐ６２のアミノ酸配列（配列番号：３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、または３５４位のグルタミン酸に特異的に結合する、上記（９）~（１３）のいずれかに記載の方法。
　本発明は、ユビキチン陽性封入体形成を制御するｐ６２とＫｅａｐ１の相互作用の制御方法を提供する。
　オートファジーが欠失することにより、細胞内のｐ６２タンパク質の蓄積と不溶化、ｐ６２−ユビキチン陽性封入体が蓄積、それに伴った肝障害が起こることが知られている。またこの封入体の蓄積がｐ６２の欠失により解消されることが知られている。
　本発明者らは今回、ｐ６２とＫｅａｐ１が直接結合すること、その結合部位はＫＩＲ（Ｋｅａｐ１　Ｉｎｔｅｒａｃｔｉｎｇ　Ｒｅｇｉｏｎ）配列（マウスｐ６２のアミノ酸配列（配列番号３）における３４５番目から３５９番目アミノ酸残基からなる領域に相当する）で、その中でも特に５つのアミノ酸（３４９番目アスパラギン酸、３５０番目プロリン、３５２番目トレオニン、３５３番目グリシン、３５４番目グルタミン酸）が結合に重要であることを見出した。
　またｐ６２とＫｅａｐ１の相互作用は、Ｋｅａｐ１の不溶化、肝細胞における解毒酵素群の転写因子であるＮｒｆ２の安定化と活性化を導き、解毒酵素群（抗酸化タンパク質等）の発現、細胞内封入体の蓄積、肝機能の低下を誘発することを新規に見出した。
　したがって、ｐ６２とＫｅａｐ１との相互作用（または結合）を制御（抑制または促進）することにより、Ｋｅａｐ１の不溶化、肝細胞における解毒酵素群の転写因子であるＮｒｆ２の安定化と活性化、解毒酵素群（抗酸化タンパク質等）の発現、細胞内封入体の蓄積、肝機能の低下等を制御できる。
　本発明は細胞保護酵素遺伝子の転写を制御するメカニズムにおける、選択的オートファジーの新たな役割を見出した点において、重要な意義を有する。
　続いて、本発明はｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する肝疾患の治療薬を提供する。
　上述の通り、オートファジーの減弱が、様々な疾患で確認されるユビキチン・ｐ６２陽性封入体を伴った肝細胞がんや肝障害を引き起こす。これはｐ６２とＫｅａｐ１の相互作用を抑制することで解消されるため、ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する物質は、肝疾患治療薬および抗がん剤に適用できる可能性が高い。
　Ｋｅａｐ１と結合するｐ６２の部位は、ｐ６２の他のタンパク質と相互作用する部位とは異なることから、この治療薬および抗がん剤によりオートファジーの異常が起きる可能性は低い。またＫｅａｐ１とｐ６２の相互作用がＮｒｆ２の安定化、活性化を導くが、ｐ６２−Ｋｅａｐ１の結合に伴うＮｒｆ２活性化システムは肝臓以外ではほとんど機能していないと考えられることから、ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する抗がん剤は、副作用が少ないと考える。
　さらに、本発明は細胞内のｐ６２の量を抑制することでＫｅａｐ１とｐ６２の相互作用を阻害又は抑制する肝疾患の治療薬および抗がん剤を提供する。
　上述の通り、オートファジーの減弱が、ｐ６２の蓄積を促し、肝細胞がんなどの肝疾患を引き起こす。この病変はｐ６２を欠損させることで大きく改善される。またｐ６２はＬＣ３と結合しオートファジーにより分解される。ｐ６２とＬＣ３が結合できない変異体を細胞に導入するのみで、ユビキチン陽性封入体が形成される。これらのことより、ｐ６２の発現そのものを抑制する物質や、ｐ６２を積極的分解に誘導できる物質は、肝疾患の治療薬に適用できる可能性が高い。 The present inventors have found for the first time that p62 binds to Keap1. It was also clarified that the binding site of p62 in Keap1 uses the same pocket as that of Nrf2. When p62 accumulates due to autophagy deficiency, the binding between Keap1 and p62 increases, and free Nrf2 increases accordingly. The accumulated Nrf2 moves into the nucleus and increases the transcription of the detoxification enzyme group.
Further, according to the experiments by the present inventors, when Nrf2 was also simultaneously deficient in the autophagy-deficient liver, formation of intracellular inclusion bodies, accompanying hepatic hypertrophy, and liver damage could be suppressed. This indicates that increased binding between Keap1 and p62 directly leads to activation of Nrf2, and liver hypertrophy, liver damage, and liver cancer. Therefore, the formation of ubiquitin-p62 positive inclusion bodies is prevented by inhibiting the binding between p62 and Keap1, and astrocytes that have been confirmed to be treated for hepatic hypertrophy, liver damage, liver cancer, or ubiquitin-p62 positive inclusion bodies. It is suggested that it leads to treatment of tumor.
Therefore, the present invention provides the following agents, diagnostic methods, diagnostic kits, diagnostic agents, and screening methods.
(1) An inhibitor or inhibitor of binding between Keap1 and p62.
(2) The agent according to (1) above,
i) a molecule that specifically binds to p62, or ii) an agent containing a substance that inhibits expression of p62.
(3) i) Inhibitor or suppressor of ubiquitin positive inclusion body formation,
The agent as described in said (1) or (2) used as a therapeutic agent of ii) liver disease, or iii) an anticancer agent.
(4) A molecule that specifically binds to p62 is
i) a substance that specifically binds to the Keap1 interaction region of p62;
ii) a substance that specifically binds to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3); or iii ) An antisense nucleic acid or siRNA against the p62-encoding nucleotide sequence, wherein the p62 expression inhibitor is
The agent according to (2) or (3) above.
(5) The agent according to (2) above, wherein the molecule that specifically binds to p62 is LC3 or an anti-p62 antibody.
(6) A method for determining an application patient of the therapeutic agent for liver disease described in (3) above,
i) measuring the expression level of p62 or Nrf2 by liver biopsy of the subject, or ii) measuring the activity of Nrf2 by liver biopsy of the subject,
Including a method.
(7) The method according to (6), further comprising measuring the enzyme activity of AST, ALT, ALP, or γ-GTP in the blood of the subject.
(8) A diagnostic kit or diagnostic agent used in the method according to (6) above,
An anti-p62 antibody or an anti-Nrf2 antibody,
The kit or kit further comprises instructions for use.
(9) A screening method for an agent that inhibits or suppresses the interaction between Keap1 and p62,
i) measuring the amount of Keap1 binding to p62 in the presence and absence of the test substance, and ii) determining the amount of Keap1 binding to p62 between the presence and absence of the test substance. In comparison, when the amount of binding in the presence of the test substance is lower than the amount of binding in the absence of the test substance, the test substance inhibits or suppresses the interaction between Keap1 and p62 A method comprising the step of selecting as
(10) In the step (9), in the step of measuring the binding amount, any one of a pull-down method, a mass spectrometry method, a method of detecting protein-protein interaction as a fluorescence signal, or imaging using a fluorescent label is used. The method described.
(11) The method according to (9) or (10) above, which is used for screening for a substance that inhibits or suppresses ubiquitin-positive inclusion body formation.
(12) The method according to any one of (9) to (11) above, which is used for screening a therapeutic agent for liver disease.
(13) The step of measuring the amount of binding comprises
i) Keap1 interaction region of p62, or ii) amino acid sequence from position 345 to position 359 in the amino acid sequence of mouse p62 (SEQ ID NO: 3): SKEVDPSTGELQSLQ (SEQ ID NO: 5)
The method according to any one of the above (9) to (12), which comprises measuring the amount of Keap1 bound to.
(14) The above substance is specific to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3). The method according to any one of (9) to (13), wherein the binding is performed.
The present invention provides a method for controlling the interaction between p62 and Keap1 that controls the formation of ubiquitin positive inclusion bodies.
It is known that deletion of autophagy causes accumulation and insolubilization of intracellular p62 protein, accumulation of p62-ubiquitin positive inclusion bodies, and accompanying liver damage. It is also known that this inclusion body accumulation is eliminated by deletion of p62.
The present inventors have now directly bound p62 and Keap1, and the binding site is a region consisting of amino acid residues 345 to 359 in the KIR (Keap1 Interacting Region) sequence (mouse p62 amino acid sequence (SEQ ID NO: 3)). In particular, it was found that five amino acids (349th aspartic acid, 350th proline, 352th threonine, 353rd glycine, 354th glutamic acid) are particularly important for binding.
The interaction between p62 and Keap1 leads to insolubilization of Keap1, stabilization and activation of Nrf2, a transcription factor of detoxification enzymes in hepatocytes, expression of detoxification enzymes (antioxidant proteins, etc.), intracellular inclusions Was found to induce the accumulation of liver and decrease in liver function.
Therefore, by controlling (suppressing or promoting) the interaction (or binding) between p62 and Keap1, insolubilization of Keap1, stabilization and activation of Nrf2 which is a transcription factor of detoxification enzymes in hepatocytes, detoxification enzymes It is possible to control the expression of (antioxidant proteins, etc.), accumulation of intracellular inclusions, decrease in liver function, and the like.
The present invention has an important significance in that a new role of selective autophagy has been found in a mechanism for controlling transcription of a cytoprotective enzyme gene.
Subsequently, the present invention provides a therapeutic agent for liver disease that inhibits or suppresses the interaction between p62 and Keap1.
As described above, attenuation of autophagy causes hepatocellular carcinoma and liver damage accompanied by ubiquitin / p62-positive inclusions confirmed in various diseases. Since this is eliminated by suppressing the interaction between p62 and Keap1, a substance that inhibits or suppresses the interaction between p62 and Keap1 is likely to be applicable to a therapeutic agent for liver disease and an anticancer agent.
Since the site of p62 that binds to Keap1 is different from the site that interacts with other proteins of p62, this therapeutic agent and anticancer agent are unlikely to cause autophagy abnormalities. The interaction between Keap1 and p62 leads to stabilization and activation of Nrf2. However, since the Nrf2 activation system associated with the binding of p62-Keap1 is considered to hardly function outside the liver, the interaction between p62 and Keap1 Anticancer agents that inhibit or suppress the action are considered to have few side effects.
Furthermore, this invention provides the therapeutic agent and anticancer agent of a liver disease which inhibit or suppress the interaction of Keap1 and p62 by suppressing the quantity of p62 in a cell.
As described above, attenuation of autophagy promotes the accumulation of p62 and causes liver diseases such as hepatocellular carcinoma. This lesion is greatly improved by deleting p62. P62 binds to LC3 and is decomposed by autophagy. A ubiquitin positive inclusion body is formed only by introducing into a cell a mutant that cannot bind p62 and LC3. From these things, the substance which suppresses the expression itself of p62, and the substance which can induce | guide | derive p62 to active decomposition | disassembly are highly likely to be applicable to the therapeutic agent of a liver disease.
　本発明は、新規な作用機序に基づく肝疾患治療薬および抗がん剤、診断薬またはそのスクリーニング方法などに応用できる。本発明の肝疾患治療剤は、副作用が少ないという利点を有する。 The present invention can be applied to liver disease therapeutic agents and anticancer agents, diagnostic agents or screening methods thereof based on a novel mechanism of action. The therapeutic agent for liver disease of the present invention has an advantage that there are few side effects.
Ｋｅａｐ１の欠失変異構築物（左パネル）の模式図とそれに対応する免疫沈降アッセイ（右パネル）の結果を示す図である。It is a figure which shows the result of the immunoprecipitation assay (right panel) corresponding to the schematic diagram of the deletion mutant construct (left panel) of Keap1. （ａ）ｐ６２の欠失変異構築物（左パネル）とそれに対応するインプット（右上パネル）およびプルダウンアッセイ（右下パネル）を示す図である。（ｂ）種々の種におけるｐ６２のホモログのＫｅａｐ１−ｉｎｔｅｒａｃｔｉｎｇ　ｒｅｇｉｏｎｓ（ＫＩＲ；赤線部）およびＬＣ３認識配列（ＬＲＳ；緑線部）のアラインメントを示す。黒およびグレーを付した部分は、それぞれ、完全保存および部分的保存の同一のアミノ酸残基を示す。(A) p62 deletion mutant construct (left panel) and corresponding input (upper right panel) and pull-down assay (lower right panel). (B) Alignment of Keap1-interacting regions (KIR; red line part) and LC3 recognition sequence (LRS; green line part) of the homologue of p62 in various species. Parts marked with black and gray indicate identical amino acid residues in full and partial conservation, respectively. ｐ６２の点変異構築物のリストおよびそれに対応するプルダウンアッセイを示す図である。ＧＳＴ−Ｋｅａｐ１−ＤＣ、ＭＢＰ−ｐ６２Ｍ８０およびその点変異、ならびにＭＢＰ−ｐ６２Ｍ８３が示されている。FIG. 6 shows a list of p62 point mutation constructs and the corresponding pull-down assay. GST-Keap1-DC, MBP-p62M80 and its point mutations, and MBP-p62M83 are shown. 免疫沈降アッセイの結果を示す図である。各ＦＬＡＧタグを付けたＫｅａｐ１およびリストされている点変異をＨｅｋ２９３Ｔ細胞において発現した。細胞溶解物を抗ＦＬＡＧ抗体により免疫沈降し、得られた免疫沈降物をＳＤＳ−ＰＡＧＥに供し、抗ＦＬＡＧ抗体および抗ｐ６２抗体で免疫ブロットにより分析した。ＦＬＡＧ−Ｋｅａｐ１、内因性のｐ６２、Ｎｒｆ２、およびアクチンが示されている。３つの独立した実験からの結果を示す。It is a figure which shows the result of an immunoprecipitation assay. Each FLAG-tagged Keap1 and the listed point mutations were expressed in Hek293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibody, and the resulting immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibody and anti-p62 antibody. FLAG-Keap1, endogenous p62, Nrf2, and actin are shown. Results from three independent experiments are shown. 免疫沈降アッセイの結果を示す図である。ＦＬＡＧタグの付いたｐ６２およびＫｅａｐ１との相互作用ができない変異体（ｐ６２　Ｔ３５２Ａ）を、アデノウイルスシステムを用いて野生型初代マウス肝細胞において過剰発現させた。感染の４８時間後に、細胞を１０μＭラクタシスチンの非存在下（レーン１，３，および５）または存在下（レーン２，４，および６）で４時間培養した。細胞溶解物を、抗Ｎｒｆ２抗体で免疫沈降した後、ＳＤＳ−ＰＡＧＥに供し、抗Ｎｒｆ２抗体および抗ユビキチン抗体で免疫沈降することにより分析した。Ｎｒｆ２、ユビキチン化Ｎｒｆ２、ＦＬＡＧ−ｐ６２、内因性ｐ６２、およびアクチンに対応するバンドが示されている。３つの独立した実験からの結果を示す。It is a figure which shows the result of an immunoprecipitation assay. A mutant (p62 T352A) that cannot interact with FLAG-tagged p62 and Keap1 was overexpressed in wild-type primary mouse hepatocytes using the adenovirus system. 48 hours after infection, cells were cultured for 4 hours in the absence ( lanes 1, 3, and 5) or presence ( lanes 2, 4, and 6) of 10 μM lactacystin. Cell lysates were analyzed by immunoprecipitation with anti-Nrf2 antibody, followed by SDS-PAGE and immunoprecipitation with anti-Nrf2 antibody and anti-ubiquitin antibody. Bands corresponding to Nrf2, ubiquitinated Nrf2, FLAG-p62, endogenous p62, and actin are shown. Results from three independent experiments are shown. Ｋｅａｐ１に対するｐ６２の競合的活性をルシフェラーゼアッセイによって測定した結果を示す図である。Ｎｒｆ２、Ｋｅａｐ１、およびｐ６２野生型またはその変異体についての発現プラスミドを、ｐＮｑｏ１−ＡＲＥレポータープラスミドおよび内部コントロールとしてのｐＲＬ−ＴＫっともにＨｅｐａ１細胞へトランスフェクトした。トランスフェクションの３６時間後に、製造業者の指示書に従ってルシフェラーゼ活性を測定した。アッセイは３連で２回行った。It is a figure which shows the result of having measured the competitive activity of p62 with respect to Keap1 by the luciferase assay. Expression plasmids for Nrf2, Keap1, and p62 wild type or mutants thereof were transfected into Hepa1 cells together with the pNqol-ARE reporter plasmid and pRL-TK as an internal control. Luciferase activity was measured 36 hours after transfection according to the manufacturer's instructions. The assay was performed twice in triplicate. （ａ）Ａｔｇ７欠損肝細胞におけるＫｅａｐ１の不溶化を示す図である。（ｂ）Ａｔｇ７欠損（Ｆ／Ｆ：Ｍｘ１）およびＡｔｇ７／ｐ６２欠損（Ｆ／Ｆ：Ｍｘ１：ｐ６２−／−）肝臓の免疫ブロット分析の結果を示す図である。(A) It is a figure which shows insolubilization of Keap1 in an Atg7 deficient hepatocyte. (B) It is a figure which shows the result of the immunoblot analysis of an Atg7 defect | deletion (F / F: Mx1) and an Atg7 / p62 defect | deletion (F / F: Mx1: p62 <-/-> ) liver. マウス肝臓におけるＮｑｏ１およびＧｓｔｍ１の定量的リアルタイムＰＣＲ分析の結果を示す図である。It is a figure which shows the result of the quantitative real-time PCR analysis of Nqo1 and Gstm1 in a mouse liver. ｐ６２およびＫｅａｐ１の細胞内局在の免疫蛍光分析の結果を示す図である。ｐＩｐＣ注入後２８日での図に示された遺伝子型のマウス由来の肝臓切片を抗ｐ６２抗体（上段パネル）および抗Ｋｅａｐ１抗体（中段パネル）で免疫染色した。下段パネルはＫｅａｐ１（グリーン）およびｐ６２（レッド）の画像を重ね合わせたものである。Ａｔｇ７欠損肝臓のパネルの中の各差し込み図は、四角で囲んだ領域の拡大図である。バーは２０μｍである。It is a figure which shows the result of the immunofluorescence analysis of the intracellular localization of p62 and Keap1. Liver sections from mice of the genotype shown in the figure 28 days after pIpC injection were immunostained with anti-p62 antibody (upper panel) and anti-Keap1 antibody (middle panel). The lower panel is an overlay of Keap1 (green) and p62 (red) images. Each inset in the panel of Atg7 deficient liver is an enlarged view of the area enclosed by a square. The bar is 20 μm. Ａｔｇ７欠損（Ｆ／Ｆ：Ｍｘ１）およびＡｔｇ７／Ｎｒｆ２欠損（Ｆ／Ｆ：Ｍｘ１：Ｎｒｆ２−／−）肝臓の免疫ブロッティングの結果を示す図である。It is a figure which shows the result of the immunoblotting of an Atg7 deficiency (F / F: Mx1) and an Atg7 / Nrf2 deficiency (F / F: Mx1: Nrf2 − / − ) liver. ｐ６２およびＫｅａｐ１の細胞内局在の免疫蛍光分析の結果を示す図である。It is a figure which shows the result of the immunofluorescence analysis of the intracellular localization of p62 and Keap1. マウス肝臓中のＮｑｏ１、Ｇｓｔｍ１、およびＣｙ２ａ５の定量的リアルタイムＰＣＲ分析の結果を示す図である。トータルＲＮＡを、ｐＩｐＣ注入後２８日目に図に示された遺伝子型の肝臓から調製した。値は、Ａｔｇ７Ｆ／Ｆ肝臓におけるｍＲＮＡ量に対して正規化したものである。実験は３回行った。パネル（ｄ）−（ｈ）：Ａｔｇ７Ｆ／Ｆ（コントロール）（パネル（ｄ））およびＡｔｇ７／Ｎｒｆ２欠損（Ａｔｇ７−／Ｎｒｆ２−）（パネル（ｅ）−（ｈ））肝臓の電子顕微鏡写真である。パネル（ｅ）および（ｇ）中の四角で囲んだ領域は拡大され、それぞれパネル（ｆ）および（ｈ）として示されている。コントロールの肝臓で容易に検出されるグリコーゲン領域（アスタリスク）は、大量の平滑ＥＲで占められ、多数のペルオキシソーム（そのいくつかはパネル（ｆ）およびパネル（ｇ）中、矢印で示されている）が周囲領域に蓄積していることに注意。パネル（ｇ）および（ｈ）はＡｔｇ７／Ｎｒｆ２欠損肝臓において観察される典型的な封入体を示す。バー：（ｄ）および（ｅ），５μｍ；（ｆ）および（ｇ），１μｍ；（ｈ），０．２μｍ。It is a figure which shows the result of the quantitative real-time PCR analysis of Nqo1, Gstm1, and Cy2a5 in a mouse liver. Total RNA was prepared from the liver of the genotype shown in the figure on day 28 after pIpC injection. Values are normalized to the amount of mRNA in Atg7 F / F liver. The experiment was performed three times. Panel (d)-(h): Atg7 F / F (control) (panel (d)) and Atg7 / Nrf2-deficient (Atg7- / Nrf2-) (panel (e)-(h)) is there. The squared regions in panels (e) and (g) are enlarged and shown as panels (f) and (h), respectively. The glycogen region (asterisk) that is easily detected in the control liver is occupied by a large amount of smooth ER and a large number of peroxisomes, some of which are indicated by arrows in panels (f) and (g) Note that is accumulated in the surrounding area. Panels (g) and (h) show typical inclusion bodies observed in Atg7 / Nrf2-deficient liver. Bars: (d) and (e), 5 μm; (f) and (g), 1 μm; (h), 0.2 μm. （ａ）ｐＩｐＣ注入後２８日目における異なる遺伝子型のマウス肝臓の重量の測定結果を表す図である。データは、各グループについて５匹のマウスの平均値±ＳＤを表す。＊＊＊Ｐ＜０．００１（Ｓｔｕｄｅｎｔ’ｓ　Ｔテスト）。（ｂ）図に示された遺伝子型のマウスの典型的な肝組織学を示す。ｐＩｐＣ注入後２８日目の肝臓を、Ｈ＆Ｅ染色で処理した。高次の拡大写真は、下側のパネルに示されている。ＣＶは中心静脈、Ｐは門脈である。バー：１００μｍ。(A) It is a figure showing the measurement result of the weight of the mouse liver of a different genotype in the 28th day after pIpC injection | pouring. Data represent the mean ± SD of 5 mice for each group. *** P <0.001 (Student's T test). (B) shows typical liver histology of mice of the genotype shown in the figure. The liver on day 28 after pIpC injection was treated with H & E staining. Higher-order magnified pictures are shown in the lower panel. CV is the central vein and P is the portal vein. Bar: 100 μm. 図１３で使用したマウスの肝機能テストの結果を示す図である。It is a figure which shows the result of the liver function test of the mouse | mouth used in FIG. ヒト肝細胞癌の二重免疫蛍光顕微鏡画像を示す図である。典型的な封入体を含有する肝細胞癌の３つの症例（組織学的段階を示した）について、抗Ｋｅａｐ１抗体および抗ｐ６２抗体で免疫染色した。Ｋｅａｐ１（グリーン）およびｐ６２（レッド）の画像を重ね合わせたものを右側のパネルに示した。バーは１０μｍである。It is a figure which shows the double immunofluorescence microscope image of human hepatocellular carcinoma. Three cases of hepatocellular carcinoma containing typical inclusion bodies (indicating histological stage) were immunostained with anti-Kap1 and anti-p62 antibodies. An overlay of Keap1 (green) and p62 (red) images is shown in the right panel. The bar is 10 μm. Ｎｒｆ２の標的プロモーターであるＡＲＥにＬｕｃｉｆｅｒａｓｅ遺伝子を繋げたプラスミドを安定に保持する細胞株にアデノウイルス感染系を用いてｐ６２−ＷＴ、ｐ６２−Ｔ３５２Ａ（Ｋｅａｐ１と結合できない変異体）、Ｋｅａｐ１、ＧＦＰ（コントロール）を過剰発現させて、ルシフェラーゼ活性を測定したアッセイの結果を示す図である。A cell line that stably holds a Luciferase gene linked to ARE, which is the target promoter of Nrf2, is p62-WT, p62-T352A (mutant that cannot bind to Keap1), Keap1, GFP (control) using an adenovirus infection system. ) Is overexpressed and the results of an assay in which luciferase activity was measured are shown. オートファジー欠損マウスの長期飼育により形成された腫瘍におけるＫｅａｐ１の不溶化およびＮｒｆ２の活性化は、ｐ６２依存的であることを示す図である。（Ａ）はＡｐｇ７ｆ／ｆマウス（コントロール）およびＡｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウス（肝特異的オートファジー欠損マウス）の各月齢における肝臓の写真を示す。（Ｂ）は各１２月齢マウスの肝臓から調節した抽出液の免疫ブロッティングの結果を示す図である。（Ｃ）は各１２月齢マウスの肝臓から調整したＲＮＡを用いた定量的ＰＣＲの結果を示す図である。It is a figure which shows that the insolubilization of Keap1 and the activation of Nrf2 in the tumor formed by the long-term breeding of the autophagy-deficient mouse are p62 dependent. (A) shows photographs of livers at each age of Apg7 f / f mice (control) and Apg7 f / f; Alb-Cre mice (liver-specific autophagy-deficient mice). (B) is a figure which shows the result of the immunoblotting of the extract adjusted from the liver of each 12-month-old mouse | mouth. (C) is a figure which shows the result of quantitative PCR using RNA adjusted from the liver of each 12-month-old mouse | mouth.
１．ｐ６２とＫｅａｐ１との相互作用を阻害または抑制する物質
　本発明は、１つの実施形態において、ｐ６２とＫｅａｐ１との相互作用を阻害または抑制する物質（ａｇｅｎｔ）を提供する。ｐ６２とＫｅａｐ１との相互作用を阻害又は抑制する物質は、肝疾患治療薬および抗がん剤として適用できる可能性がある。
　「ｐ６２とＫｅａｐ１との相互作用を阻害又は抑制する物質」としては、
　例えば
　（１）ｐ６２の立体構造中の、Ｋｅａｐ１との結合部位に対して特異的に結合する物質、
　（２）ｐ６２の立体構造中の、Ｋｅａｐ１との結合部位の近傍に結合することにより、前記相互作用を阻害または抑制する物質、
　（３）ｐ６２と結合して、ｐ６２の立体構造を変化させる物質、
　（４）ｐ６２の発現を阻害することにより、相互作用を阻害又は抑制する物質、
　（５）ｐ６２をオートファジーで分解することにより、相互作用を阻害又は抑制する物質、等が含まれる。
　典型的には、「ｐ６２とＫｅａｐ１との相互作用を阻害又は抑制する物質」としては、
　ｉ）ｐ６２に特異的に結合する分子、または
　ｉｉ）ｐ６２の発現阻害物質
が含まれる。
　「ｐ６２」は、ユビキチン結合タンパク質として知られている。ｐ６２のアミノ酸配列またはそれをコードするヌクレオチド配列の情報は、一般に利用可能な配列データベースから入手することができる。それらのデータベースのアクセッション番号のいくつかをここに記載する。［データベース名称：アクセッション番号（種名）］：ＮＣＢＩ：ＮＰ＿００３８９１．１（ヒト）、ＮＣＢＩ：ＮＭ＿００３９００．４（ヒト）、ＮＣＢＩ：ＮＰ＿０３５１４８．１（マウス）、ＮＣＢＩ：ＮＭ＿０１１０１８．２（マウス）Ｓｗｉｓｓ−Ｐｒｏｔ：００８６２３．１（ラット）、ＮＣＢＩ：ＮＰ＿７８７０３７．２（ラット）、ＮＣＢＩ：ＮＭ＿１７５８４３．３（ラット）、Ｓｗｉｓｓ−Ｐｒｏｔ：Ｑ５ＲＢＡ５．１（オランウータン）。本明細書中で「ｐ６２」という場合、ヒトｐ６２に限定されず、ｐ６２の他の種（例えば、他の哺乳動物種（例えば、サル、ウシ、ブタ、マウス、ラットなど）、は虫類、両生類、魚類など。）のホモログも含まれるものとする。そのようなホモログのアミノ酸配列は、例えば、ヒトｐ６２のアミノ酸配列（配列番号１）もしくはマウスｐ６２のアミノ酸配列（配列番号３）に対して、例えば、８０％以上、８５％以上、９０％以上、もしくは９５％以上の配列同一性を有するアミノ酸配列、またはヒトｐ６２のアミノ酸配列（配列番号１）もしくはマウスｐ６２のアミノ酸配列（配列番号３）において１~数個（例：２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０個、もしくはそれ以上）のアミノ酸残基の欠失、置換、挿入、もしくは付加を有するアミノ酸配列からなる。
　「Ｋｅａｐ１（Ｋｅｌｃｈ−ｌｉｋｅ　ＥＣＨ−ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　１）」は、ＮＦ−Ｅ２関連因子２（ＮＦ−Ｅ２−ｒｅｌａｔｅｄ　ｆａｃｔｏｒ　２（Ｎｒｆ２））とレドックス感受性な様式で相互作用し、細胞質でこのタンパク質が乖離するとＮｒｆ２は核へ移行することが知られている。この相互作用は、γグルタミルシステインシンセターゼの触媒サブユニットの発現を制御することが知られている。Ｋｅａｐ１のアミノ酸配列またはそれをコードするヌクレオチド配列の情報は、一般に利用可能な配列データベースから入手することができる。それらのデータベースにおけるアクセッション番号のいくつかをここに記載する。［データベース名称：アクセッション番号（種名）］：Ｓｗｉｓｓ−Ｐｒｏｔ：Ｑ１４１４５．２（ヒト）、Ｓｗｉｓｓ−Ｐｒｏｔ：Ｑ９Ｚ２Ｘ８．１（マウス）、ＧｅｎＢａｎｋ：ＢＡＡ３４６３９．１（マウス）、ＧｅｎＢａｎｋ：ＡＢ０２００６３．１（マウス）、Ｓｗｉｓｓ−Ｐｒｏｔ：Ｑ６８４Ｍ４．１（ブタ）。本明細書中で「Ｋｅａｐ１」という場合、ヒトＫｅａｐ１に限定されず、ｋｅａｐ１の他の種（例えば、他の哺乳動物種（例：サル、ウシ、ブタ、マウス、ラットなど）、は虫類、両生類、魚類など。）のホモログも含まれるものとする。そのようなホモログのアミノ酸配列は、例えば、ヒトＫｅａｐ１のアミノ酸配列に対して、例えば、８０％以上、８５％以上、９０％以上、もしくは９５％以上の配列同一性を有するアミノ酸配列、またはヒトＫｅａｐ１のアミノ酸配列において１~数個（例：２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０個、もしくはそれ以上）のアミノ酸残基の欠失、置換、挿入、もしくは付加を有するアミノ酸配列からなる。
　本明細書中、「ｐ６２に特異的に結合する」分子とは、その分子が他のアミノ酸配列に対するその親和性よりも、ｐ６２またはその断片の特定のアミノ酸配列に対して実質的に高い親和性で結合することを意味する。ここで、「実質的に高い親和性」とは、所望の測定装置によって、その特定のアミノ酸配列を他のアミノ酸配列から区別して検出することが可能な程度に高い親和性を意味し、典型的には、結合定数（Ｋａ）が少なくとも１０７Ｍ−１、好ましくは、少なくとも１０８Ｍ−１、より好ましくは、１０９Ｍ−１、さらにより好ましくは、１０１０Ｍ−１、１０１１Ｍ−１、１０１２Ｍ−１またはそれより高い、例えば、最高で１０１３Ｍ−１またはそれより高いものであるような結合親和性を意味する。
　「ｐ６２に特異的に結合する分子」の例には、下記のｉ）およびｉｉ）が含まれる。
　ｉ）ｐ６２のＫｅａｐ１相互作用領域（Ｋｅａｐ１　Ｉｎｔｅｒａｃｔｉｎｇ　Ｒｅｇｉｏｎ）〔ＳＫＥＶＤＰＳＴＧＥＬＱＳＬＱ（配列番号：５）、配列番号５のアミノ酸配列に対して、例えば、８０％以上、８５％以上、９０％以上、もしくは９５％以上の配列同一性を有するアミノ酸配列、または配列番号５のアミノ酸配列中に１~数個（例：１、２、３個、もしくはそれ以上）のアミノ酸残基の欠失、置換、挿入、もしくは付加を有するアミノ酸配列〕に特異的に結合する物質、
　ｉｉ）マウスｐ６２のアミノ酸配列（配列番号：３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、もしくは３５４位のグルタミン酸に特異的に結合する物質。
　本明細書中、「Ｋｅａｐ１相互作用領域（Ｋｅａｐ１　Ｉｎｔｅｒａｃｔｉｎｇ　Ｒｅｇｉｏｎ：ＫＩＰ）」とは、マウスｐ６２のアミノ酸配列中の３４５位~３５９位のアミノ酸残基からなる部分アミノ酸配列の領域、またはマウス以外の哺乳動物種（例：ヒト、サル、ウシ、ラット等）もしくはその他の種（例：は虫類、両生類、魚類等）のｐ６２のアミノ酸配列中の上記部分アミノ酸配列の領域に対応する高度に保存されたアミノ酸配列の領域であって、Ｋｅａｐ１と特異的に結合するｐ６２上の領域を指すものとする（図２ｂを参照）。
　ｐ６２はＫｅａｐ１と相互作用し、ｐ６２の過剰状態の際は、Ｋｅａｐ１と結合したｐ６２が増加し、ｐ６２−Ｋｅａｐ１複合体は不溶化状態となっている。Ｋｅａｐ１は通常ｐ６２と同じ領域でＮｒｆ２と結合しており、Ｎｒｆ２をユビキチン−プロテアソーム分解経路へと誘導する。しかしｐ６２が高発現すると、ｐ６２とＫｅａｐ１が結合・不溶化することでフリーのＮｒｆ２量が増大する。蓄積したＮｒｆ２は核内に移行し、下流因子の発現が誘導され、ｐ６２・ユビキチン陽性封入体が形成される。過剰な封入体形成は肝細胞がん、肝障害などの肝疾患を引き起こす。
　したがって、上記阻害または抑制剤は、典型的には、ｉ）ユビキチン陽性封入体形成の阻害又は抑制剤、ｉｉ）肝疾患の治療剤、またはｉｉｉ）抗がん剤として使用され得る。
　細胞内のｐ６２の量は、ＬＣ３との相互作用に対してオートファジーで規定されているので，この結合を増強させる物質の投与は細胞内でのｐ６２の量を減少させ、肝細胞がんや肝障害を抑える。
　したがって、本発明のさらなる実施形態では、上記「ｐ６２に特異的に結合する分子」がＬＣ３である。
　また、さらに別の実施形態では、上記「ｐ６２に特異的に結合する分子」は、ｐ６２に特異的に結合する抗体（抗ｐ６２抗体）である。
　本明細書中、「ｐ６２の発現阻害物質」とは、ｐ６２遺伝子のｍＲＮＡへの転写および／またはｐ６２遺伝子（ｍＲＮＡ）のタンパク質への翻訳を阻害する物質のことを指す。
　「ｐ６２の発現阻害物質」の例には、下記のｉｉｉ）およびｉｖ）が含まれる。
　ｉｉｉ）前記ｐ６２の発現阻害物質が、ｐ６２をコードするヌクレオチド配列に対するアンチセンス核酸もしくはｓｉＲＮＡ、
　ｉｖ）ｐ６２遺伝子の転写産物（ｍＲＮＡ）を特異的に切断するリボザイム活性を有する核酸。
　本明細書中、「核酸」とはＲＮＡまたはＤＮＡを意味する。ここでいう「核酸」は、プリンおよびピリミジン塩基を含有するのみでなく、修飾されたその他の複素環型塩基をもつようなものを含んでいてもよい。こうした修飾物は、メチル化されたプリンおよびピリミジン、アシル化されたプリンおよびピリミジン、アシル化されたプリンおよびピリミジン、あるいはその他の複素環を含むものであって良い。修飾されたヌクレオシドおよび修飾されたヌクレオチドはまた、糖部分が修飾されていて良く、例えば、１個以上の水酸基がハロゲンとか、脂肪族基などで置換されているか、あるいはエーテル、アミンなどの官能基に変換されていてよい。
　本発明の肝疾患の治療剤またはがん治療剤においては、ｐ６２遺伝子の発現をＲＮＡｉ効果により阻害する作用を有する核酸を有効成分として用いることができる。ＲＮＡｉとは、標的遺伝子配列と同一もしくは類似した配列を有する二重鎖ＲＮＡを細胞内に導入すると、導入した外来遺伝子および標的内在性遺伝子の発現がいずれも阻害される現象のことをいう。ここで用いられるＲＮＡとしては、例えば、１９~３０塩基長のＲＮＡ干渉を生ずる二重鎖ＲＮＡ、例えば、ｄｓＲＮＡ（ｄｏｕｂｌｅ　ｓｔｒａｎｄ　ＲＮＡ）、ｓｉＲＮＡ（ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｉｎｇ　ＲＮＡ）又はｓｈＲＮＡ（ｓｈｏｒｔ　ｈａｉｒｐｉｎ　ＲＮＡ）が挙げられる。このようなＲＮＡは、リポソームなどの送達システムにより所望の部位に局所送達させることも可能であり、また上記二重鎖ＲＮＡが生成されるようなベクターを用いてこれを局所発現させることができる。このような二重鎖ＲＮＡ（ｄｓＲＮＡ、ｓｉＲＮＡまたはｓｈＲＮＡ）の調製方法、使用方法などは、多くの文献から公知である（特表２００２−５１６０６２号；米国公開許第２００２／０８６３５６Ａ号；Ｎａｔｕｒｅ　Ｇｅｎｅｔｉｃｓ，２４（２），Ｆｅｂ．，１８０−１８３；Ｇｅｎｅｓｉｓ，２６（４），Ａｐｒｉｌ，２４０−２４４；Ｎａｔｕｒｅ，Ｓｐｅ．２１，４０７：６８０２，３１９−２０；Ｇｅｎｅｓ　＆　Ｄｅｖ．，Ｖｏｌ．１６，（８），Ａｐｒ．１６，９４８−９５８；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ．，９９（８），１６　Ａｐｒ．，５５１５−５５２０；Ｓｃｉｅｎｃｅ，２９６（５５６７），１９　Ａｐｒ．，５５０−５５３；Ｐｒｏｃ　Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，Ａｐｒ．３０，９９：９，６０４７−６０５２；Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｖｏｌ．２０（５），Ｍａｙ，４９７−５００；Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｖｏｌ．２０（５），Ｍａｙ，５００−５０８；Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，Ｍａｙ　１５など）。
　本発明で用いられるＲＮＡｉ効果を奏する二重鎖ＲＮＡの長さは、通常、１９~３０塩基、好ましくは２０~２７塩基、より好ましくは２１~２５塩基、最も好ましくは２１~２３塩基である。
　本明細書中、「アンチセンス核酸」、または「アンチセンスポリヌクレオチド」とは、ある対象となるＤＮＡ領域の少なくとも一部に相補的なポリヌクレオチドを有し、そのポリヌクレオチドが当該領域の少なくとも一部とハイブリダイズすることができる核酸のことをいう。本発明のアンチセンス核酸またはアンチセンスポリヌクレオチドは、ＲＮＡ、ＤＮＡ、あるいは修飾された核酸（ＲＮＡ、ＤＮＡ）である。それらは二本鎖ＤＮＡ、一本鎖ＤＮＡ、二本鎖ＲＮＡ、一本鎖ＲＮＡ、さらにＤＮＡ：ＲＮＡハイブリッドであってもよい。修飾された核酸の具体例としては、核酸の硫黄誘導体やチオホスフェート誘導体、さらにはポリヌクレオチドアミドやオリゴヌクレオチドアミドの分解に抵抗性を有するものなどが挙げられるが、それらに限定されるものではない。
　使用されるアンチセンス核酸は、適当なプロモーターの下流に連結され、好ましくは３’側に転写終結シグナルを含む配列が連結される。このようにして調製された核酸は、公知の方法を用いることで、所望の動物へ形質転換できる。アンチセンス核酸の配列は、形質転換される動物が持つ内在性遺伝子またはその一部と相補的な配列であることが好ましいが、遺伝子の発現を有効に抑制できる限りにおいて、完全に相補的でなくてもよい。
　遺伝子の翻訳阻害に効果的なアンチセンス核酸は、標的遺伝子の転写産物に対して約７０％以上、好ましくは約８０％以上、より好ましくは約９０％以上、最も好ましくは約９５％以上の相補性を有する。
　アンチセンス核酸を用いて標的遺伝子の発現を効果的に抑制するには、アンチセンス核酸の長さは少なくとも約１０塩基以上（例えば、１０~４０個程度）、好ましくは約１５塩基以上であり、より好ましくは約１００塩基以上であり、さらに好ましくは約５００塩基以上である。アンチセンス核酸は公知の文献を参照して設計することができる（例えば、平島および井上、新生化学実験講座２核酸ＩＶ遺伝子の複製と発現、日本生化学会編、東京化学同人、１９９３、ｐ．３１９−３４７）、Ｊ．Ｋａｗａｋａｍｉ　ｅｔ　ａｌ．，Ｐｈａｒｍ　Ｔｅｃｈ　Ｊａｐａｎ．Ｖｏｌ．８，ｐ．２４７，１９９２；Ｖｏｌ．８，ｐ．３９５，１９９２；Ｓ．Ｔ．Ｃｒｏｏｋｅ　ｅｔ　ａｌ．，ｅｄ．，Ａｎｔｉｓｅｎｓｅ　Ｒｅｓｅａｒｃｈ　ａｎｄ　Ａｐｐｌｉｃａｔｉｏｎｓ，ＣＲＣ　Ｐｒｅｓｓ，１９９３など参照）。
　本明細書中、「リボザイム活性」とは、ターゲットとする遺伝子の転写産物であるｍＲＮＡを部位特異的に切断する核酸のことをいう。リボザイムには、グループＩイントロン型やＲＮａｓｅＰに含まれるＭ１　ＲＮＡのように４００ヌクレオチド以上の大きさのものもあるが、ハンマーヘッド型やヘアピン型と呼ばれる４０ヌクレオチド程度の活性ドメインを有するものもある（タンパク質核酸酵素、１９９０、３５、ｐ．２１９１）。ハンマーヘッド型リボザイムについては、例えば、ＦＥＢＳ　Ｌｅｔｔ，１９８８，２２８，ｐ．２２８；ＦＥＢＳ　Ｌｅｔｔ，１９８８，２３９，ｐ．２８５；タンパク質核酸酵素，１９９０，３５，ｐ．２１９１；Ｎｕｃｌ　Ａｃｉｄｓ　Ｒｅｓ，１９８９，１７，ｐ．７０５９などを参照することができる。また、ヘアピン型リボザイムについては、例えば、Ｎａｔｕｒｅ，１９８６，３２３，ｐ．３４９；Ｎｕｃｌ　Ａｃｉｄｓ　Ｒｅｓ，１９９１，１９，．６７５１；菊池洋，化学と生物，１９９２，３０，ｐ．１１２などを参照することができる。このようなリボザイムを用いてｐ６２遺伝子の転写産物を特異的に切断することで、該遺伝子の発現を阻害することができる。
　これらの物質によりｐ６２の発現が抑制された場合、Ｋｅａｐ１とＮｒｆ２の結合が回復し、Ｎｒｆ２が不活性化することで、肝細胞がんや肝障害を抑えることができる。
　したがって、本発明のさらなる実施形態では、ｐ６２とＫｅａｐ１との相互作用を阻害または抑制する物質を、それを必要とする患者に投与する工程を包含する、肝疾患および／またはがんの治療方法が提供される。
２．ｐ６２とＫｅａｐ１との相互作用を阻害または抑制する物質を含有する剤
　本発明のさらなる（ｆｕｒｔｈｅｒ）実施形態では、Ｋｅａｐ１とｐ６２との結合の阻害または抑制剤が提供される。この剤（ｃｏｍｐｏｓｉｔｉｏｎ）は、典型的には、上記のｐ６２とＫｅａｐ１との相互作用を阻害又は抑制する物質を含有する。具体的には、この剤は、
　ｉ）ｐ６２に特異的に結合する分子、または
　ｉｉ）ｐ６２の発現阻害物質
を含有する。
　本発明の上記剤は、肝疾患またはがんの治療用製剤として使用され得る。このような製剤（ｐｈａｒｍａｃｅｕｔｉｃａｌ　ｃｏｍｐｏｓｉｔｉｏｎ）は、上記のｐ６２とＫｅａｐ１との相互作用を阻害又は抑制する物質を、慣用されている製剤担体とを混合することにより製造することができる。
　製剤担体は、投与形態に応じて、適宜、組み合わせて用いればよく、例えば、乳糖等の賦形剤；ステアリン酸マグネシウム等の滑沢剤；カルボキシメチルセルロース等の崩壊剤；ヒドロキシプロピルメチルセルロース等の結合剤；マクロゴール等の界面活性剤；炭酸水素ナトリウム等の発泡剤；シクロデキストリン等の溶解補助剤；クエン酸等の酸味剤；エデト酸ナトリウム等の安定化剤；リン酸塩等のｐＨ調整剤などが挙げられる。
　それを必要とする患者に投与する際には、経口投与のための内服用固形剤、内服用液剤および、非経口投与のための注射剤、外用剤、坐剤、点眼剤、吸入剤等として用いることもできる。
　経口投与のための内服用固形剤には、錠剤、丸剤、カプセル剤、散剤、顆粒剤等が含まれる。カプセル剤には、ハードカプセルおよびソフトカプセルが含まれる。また錠剤には舌下錠、口腔内貼付錠、口腔内速崩壊錠などが含まれる。
　このような内服用固形剤においては、ひとつまたはそれ以上の活性物質はそのままか、または賦形剤（ラクトース、マンニトール、グルコース、微結晶セルロース、デンプン等）、結合剤（ヒドロキシプロピルセルロース、ポリビニルピロリドン、メタケイ酸アルミン酸マグネシウム等）、崩壊剤（繊維素グリコール酸カルシウム等）、滑沢剤（ステアリン酸マグネシウム等）、安定剤、溶解補助剤（グルタミン酸、アスパラギン酸等）等と混合され、常法に従って製剤化して用いられる。また、必要によりコーティング剤（白糖、ゼラチン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロースフタレート等）で被覆していてもよいし、また２以上の層で被覆していてもよい。さらにゼラチンのような吸収されうる物質のカプセルも包含される。
　経口投与のための内服用液剤は、薬剤的に許容される水剤、懸濁剤・乳剤、シロップ剤、エリキシル剤等を含む。このような液剤においては、ひとつまたはそれ以上の活性物質が、一般的に用いられる希釈剤（精製水、エタノールまたはそれらの混液等）に溶解、懸濁または乳化される。さらにこの液剤は、湿潤剤、懸濁化剤、乳化剤、甘味剤、風味剤、芳香剤、保存剤、緩衝剤等を含有していてもよい。
　非経口投与のための外用剤の剤形には、例えば、軟膏剤、ゲル剤、クリーム剤、湿布剤、貼付剤、リニメント剤、噴霧剤、吸入剤、スプレー剤、点眼剤、および点鼻剤等が含まれる。これらはひとつまたはそれ以上の活性物質を含み、公知の方法または通常使用されている処方により製造、調製される。
　非経口投与のための注射剤としては、溶液、懸濁液、乳濁液および用時溶剤に溶解または懸濁して用いる固形の注射剤を包含する。注射剤は、ひとつまたはそれ以上の活性物質を溶剤に溶解、懸濁または乳化させて用いられる。溶剤として、例えば注射用蒸留水、生理食塩水、植物油、プロピレングリコール、ポリエチレングリコール、エタノールのようなアルコール類等およびそれらの組み合わせが用いられる。さらにこの注射剤は、安定剤、溶解補助剤（グルタミン酸、アスパラギン酸、ポリソルベート８０（登録商標）等）、懸濁化剤、乳化剤、無痛化剤、緩衝剤、保存剤等を含んでいてもよい。これらは最終工程において滅菌するか無菌操作法によって製造、調製される。また無菌の固形剤、例えば凍結乾燥品を製造し、その使用前に無菌化または無菌の注射用蒸留水または他の溶剤に溶解して使用することもできる。
３．本発明のスクリーニング方法
　本発明は、さらに別の実施形態において、Ｋｅａｐ１とｐ６２との相互作用を阻害または抑制する物質（ａｇｅｎｔ）のスクリーニング方法を提供する。この方法は、典型的には、
　ｉ）試験物質の存在下および非存在下でＫｅａｐ１とｐ６２との結合量を測定する工程、および
　ｉｉ）前記試験物質の存在下と非存在下との間でＫｅａｐ１とｐ６２との前記結合量を比較し、前記試験物質の存在下での前記結合量が前記試験物質の非存在下での前記結合量よりも低い場合に、当該試験物質をＫｅａｐ１とｐ６２との相互作用を阻害または抑制する物質として選択する工程、を包含する。
　ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する物質は、肝疾患治療薬および抗がん剤に適用し得る。ｐ６２とＫｅａｐ１のタンパク質間相互作用を阻害又は抑制する物質を探索することにより、肝疾患の治療のための新規薬剤をスクリーニングすることができる。
　相互作用の検出は、プルダウン法（例：図３）、質量分析計を用いた方法、タンパク質間相互作用を蛍光シグナルとして検出する方法、蛍光標識を用いたイメージング等の方法により行うことができる。
　本発明のスクリーニング方法のより具体的な実施形態では、「試験物質の存在下および非存在下でＫｅａｐ１とｐ６２との結合量を測定する工程」が、ｐ６２のＫＩＲ配列（例：ＳＫＥＶＤＰＳＴＧＥＬＱＳＬＱ（配列番号：５）、配列番号５のアミノ酸配列に対して、例えば、８０％以上、８５％以上、９０％以上、もしくは９５％以上の配列同一性を有するアミノ酸配列、または配列番号５のアミノ酸配列中に１~数個（例：１、２、３個、もしくはそれ以上）のアミノ酸残基の欠失、置換、挿入、もしくは付加を有するアミノ酸配列）に対するＫｅａｐ１の結合量を測定することを含む。
　本発明のスクリーニング方法のより具体的な実施形態では、「Ｋｅａｐ１とｐ６２との相互作用を阻害または抑制する物質」が、マウスｐ６２のアミノ酸配列（配列番号：３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、または３５４位のグルタミン酸に特異的に結合する。
　本発明のスクリーニング方法のより具体的な実施形態では、さらに、
　ｉｉｉ）Ｋｅａｐ１とｐ６２との相互作用を阻害または抑制する物質を肝疾患モデル動物に投与して、Ｎｒｆ２の活性を測定する工程、および
　ｉｖ）Ｎｒｆ２の活性が減少した場合に、上記物質を肝疾患の治療剤候補として選択する工程
を包含する。
　このような本発明のスクリーニング方法により得られる物質の例としては、ＬＣ３、抗ｐ６２抗体などが挙げられるが、これらに限定されず、低分子化合物（天然または合成）、タンパク質またはペプチド、多糖、脂質、核酸などが含まれ得る。
　ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する物質、あるいは細胞内のｐ６２の量を減少させる物質は肝疾患治療薬および抗がん剤に適用し得る。これらの物質はＫｅａｐ１と結合するｐ６２の量を低下させ、Ｋｅａｐ１とＮｒｆ２との結合を増大させることでＮｒｆ２を不活性化（分解）させる。ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する物質、あるいは細胞内のｐ６２の量を減少させる物質を肝疾患モデル動物（例：マウス）に投与して、Ｎｒｆ２の活性を測定することで、新規薬剤を見出し得る。
　Ｋｅａｐ１とｐ６２との相互作用の検出は、培養細胞（Ｈｕｈ−１，ＪＨＨ５，Ｈｅｐａ−１，ＨＥＫ２９３Ｔなど）へＮｒｆ２転写活性レポーターＡＲＥ（ａｎｔｉｏｘｉｄａｎｔ−ｒｅｓｐｏｎｓｉｖｅ　ｅｌｅｍｅｎｔ）をルシフェラーゼ遺伝子につなげたプラスミドを一過的に導入した、あるいは安定に保持する細胞株を用いたルシフェラーゼアッセイ（図６および図１６を参照）によって、Ｎｒｆ２転写活性レポーターとβ−ラクタマーゼ遺伝子を繋げたＡＲＥ−ｂｌａを組み込んだ培養細胞によるβ−ラクタマーゼと蛍光共鳴エネルギー転移（ＦＲＥＴ）を用いたＮｒｆ２転写活性レポーターアッセイによって、ｐ６２を過剰発現させた肝細胞を用いて、Ｎｒｆ２の下流因子の発現をｒｅａｌ　ｔｉｍｅ　ＰＣＲによって、免疫ブロット（Ｉｍｍｕｎｏｂｌｏｔ（以下Ｉ．Ｂ．））によって、またはＲＴ−ＰＣＲ等によって確認することができる。
　本発明のスクリーニング方法のより具体的な実施形態では、試験物質の存在下および非存在下でＫｅａｐ１とｐ６２との結合量の測定は、プルダウン法、質量分析法、タンパク質間相互作用を蛍光シグナルとして検出する方法、または蛍光標識を用いたイメージングのいずれかを用いて行われる。
　ｐ６２とＫｅａｐ１の相互作用を阻害又は抑制する物質、あるいは細胞内のｐ６２の量を減少させる物質は肝疾患治療薬および抗がん剤に適用できる可能性がある。これらの物質は、マウスのオートファジーを欠失させた肝臓における細胞内のユビキチン陽性封入体の量を減少させる。Ｋｅａｐ１の相互作用を阻害又は抑制する物質、あるいは細胞内のｐ６２の量を減少させる物質をマウスに投与して、ユビキチン陽性封入体の量、Ｎｒｆ２の活性、肝障害のレベルを測定することで新規薬剤候補をスクリーニングすることができる。
　ユビキチン陽性封入体の検出は、マウス肝臓組織の免疫染色を行うことができる（図９）。
　Ｎｒｆ２の活性測定は、Ｎｒｆ２の下流因子である解毒酵素遺伝子群の発現をｒｅａｌ　ｔｉｍｅ　ＰＣＲにより確認することができる（図８）。
　肝障害のレベル測定は、肝臓の重量測定（図１３ａ），肝臓組織のＨＥ染色による肝細胞肥大と小葉構造の崩壊検出（図１３ｂ），肝機能テスト（ＡＳＴ，ＡＬＴ，ＡＬＰ）（図１４）によって行うことができる。
　本発明のスクリーニング方法により得られた物質は、ｉ）ユビキチン陽性封入体形成の阻害又は抑制剤、ｉｉ）肝疾患の治療剤、またはｉｉｉ）抗がん剤として使用され得る。
４．肝疾患の診断方法、診断剤、及び診断用キット
　本発明は、さらなる実施形態において、肝疾患の診断方法を提供する。特に、上記の本発明の肝疾患の治療剤の適用患者を判定または診断する方法を提供する。この方法は、典型的には、
　ｉ）被験者の肝生検によるｐ６２もしくはＮｒｆ２の発現量を測定する工程、または
　ｉｉ）被験者の肝生検によるＮｒｆ２の活性を測定する工程、
を含む。
　肝疾患の被験者の肝生検によるｐ６２の発現量が正常者と比較して過剰であることが判明すれば、該被験者の肝疾患がｐ６２の過剰発現に起因している可能性が高い。そのような場合、上記の本発明の肝疾患の治療剤を、ｐ６２とＫｅａｐ１との相互作用を阻害もしくは抑制し、またはｐ６２の発現を阻害するために該疾患の治療を必要とする患者に適用することで有利な効果が得られると考えられる。したがって、このような治療の適用患者を判定または診断することは有用である。
　また、肝疾患の被験者の肝生検によるＮｒｆ２の活性が正常者と比較して高いことが判明すれば、該被験者の肝疾患がＮｒｆ２の異常活性化に起因している可能性が高い。そのような場合、上記の本発明の肝疾患の治療剤を該疾患の治療を必要とする患者に適用すれば、ｐ６２とＫｅａｐ１との相互作用を阻害もしくは抑制し、またはｐ６２の発現を阻害することによって、Ｎｒｆ２とＫｅａｐ１との相互作用と競合するｐ６２の量を減少させることができるため、遊離のＮｒｆ２の量が減少し、Ｎｒｆ２の活性化が抑制される。そのため、本発明の肝疾患の治療剤を該疾患の治療を必要とする患者に適用することで有利な効果が得られると考えられる。したがって、このような治療の適用患者を判定または診断することは有用である。
　被験者の肝生検によるｐ６２の発現量の測定は、蛍光標識によるイメージング、免疫ブロット法、質量分析法等の当業者に周知の方法によって行うことができる。
　被験者の肝生検によるＮｒｆ２の活性の測定は、Ｎｒｆ２の標的プロモーターを用いたルシフェラーゼアッセイ、免疫ブロットによる細胞核内のＮｒｆ２量測定、Ｎｒｆ２によって転写誘導される酵素（Ｎｑｏ１やＧｓｔｍなど）の、定量的ＰＣＲ、免疫ブロットや蛍光イメージングによる発現解析および酵素活性測定等の当業者に周知の方法によって行うことができる。
　本発明の肝疾患の治療剤の適用患者を判定する方法のさらなる実施形態では、Ｎｒｆ２の活性測定、および／またはユビキチン陽性封入体の検出を行うことを含む。
　本発明の肝疾患の治療剤の適用患者を判定する方法のさらなる実施形態では、被験者の血液中のＡＳＴ，ＡＬＴ，ＡＬＰ，および／またはγ−ＧＴＰの濃度を測定することを含む。なお、γ−ＧＴＰ（γグルタミルトランスペプチダーゼ）は、肝臓の解毒作用に関係する酵素であり、肝臓や胆管の細胞が壊れると血液中にγ−ＧＴＰが血液の中に流れ出てくることから、「逸脱酵素」といわれ、肝臓や胆管の細胞が壊れたことの指標として一般に利用されている。
　本発明のさらなる実施形態では、本発明の診断方法に用いられる診断用キットまたは診断剤が提供される。これらの診断用キットまたは診断剤は、典型的には、抗ｐ６２抗体または抗Ｎｒｆ２抗体を含む。さらに、これらの診断用キットまたは診断剤は、抗体を標識するための標識剤（例えば、蛍光標識）または蛍光標識された抗体を含んでいてもよい。診断用キットはさらに、使用説明書を含んでいてもよい。使用説明書には、キットに含まれる抗体、標識剤等の使用方法、免疫ブロッティングを含む実験の手順等が記載されていてもよい。
　なお、本明細書中に現れるアミノ酸配列またはヌクレオチド配列と配列番号との関係は以下のとおりである
［配列番号１］
　この配列は、ヒトｐ６２のアミノ酸配列を表す。
［配列番号２］
　この配列は、ヒトｐ６２のアミノ酸配列（配列番号１）をコードするヌクレオチド配列を表す。
［配列番号３］
　この配列は、マウスｐ６２のアミノ酸配列を表す。
［配列番号４］
　この配列は、マウスｐ６２のアミノ酸配列（配列番号３）をコードするヌクレオチド配列を表す。
［配列番号５］
　この配列は、マウスｐ６２のアミノ酸配列（配列番号３）の第３４５位~第３５９位までの連続するアミノ酸残基からなる部分配列（ＫＩＲ配列）を表す。
　以下、実施例を用いて本発明をより具体的に説明するが、本発明の範囲がこれらの実施例に限定されないことはいうまでもない。 1.Substances that inhibit or suppress the interaction between p62 and Keap1
In one embodiment, the present invention provides an agent that inhibits or suppresses the interaction between p62 and Keap1. A substance that inhibits or suppresses the interaction between p62 and Keap1 may be applicable as a therapeutic agent for liver disease and an anticancer agent.
“As substances that inhibit or suppress the interaction between p62 and Keap1”
For example
(1) A substance that specifically binds to a binding site with Keap1 in the three-dimensional structure of p62,
(2) a substance that inhibits or suppresses the interaction by binding in the vicinity of the binding site with Keap1 in the three-dimensional structure of p62;
(3) a substance that binds to p62 and changes the three-dimensional structure of p62,
(4) a substance that inhibits or suppresses interaction by inhibiting the expression of p62,
(5) Substances that inhibit or suppress the interaction by decomposing p62 by autophagy are included.
Typically, “a substance that inhibits or suppresses the interaction between p62 and Keap1”
I) a molecule that specifically binds to p62, or
Ii) p62 expression inhibitor
Is included.
“P62” is known as a ubiquitin-binding protein. Information on the amino acid sequence of p62 or the nucleotide sequence encoding it can be obtained from publicly available sequence databases. Some of these database accession numbers are listed here. [Database name: Accession number (species name)]: NCBI: NP_003891.1 (human), NCBI: NM_003900.4 (human), NCBI: NP_035148.1 (mouse), NCBI: NM_011018.2 (mouse) Swiss- Prot: 008623.1 (rat), NCBI: NP_78737.2 (rat), NCBI: NM_175843.3 (rat), Swiss-Prot: Q5RBA5.1 (orangutan). Reference herein to “p62” is not limited to human p62, but other species of p62 (eg, other mammalian species (eg, monkeys, cows, pigs, mice, rats, etc.), reptiles, amphibians, Homologs of fish, etc.). The amino acid sequence of such a homologue is, for example, 80% or more, 85% or more, 90% or more with respect to the amino acid sequence of human p62 (SEQ ID NO: 1) or the amino acid sequence of mouse p62 (SEQ ID NO: 3), Alternatively, one to several amino acids (for example: 2, 3, 4, 5) in the amino acid sequence having a sequence identity of 95% or more, the amino acid sequence of human p62 (SEQ ID NO: 1) or the amino acid sequence of mouse p62 (SEQ ID NO: 3) , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) deletion, substitution, insertion, or addition of amino acid residues It consists of an amino acid sequence having
“Keap1 (Kelch-like ECH-associated protein 1)” interacts with NF-E2-related factor 2 (NF-E2-related factor 2 (Nrf2)) in a redox-sensitive manner, and when this protein diverges in the cytoplasm. Nrf2 is known to migrate to the nucleus. This interaction is known to control the expression of the catalytic subunit of γ-glutamylcysteine synthetase. Information on the amino acid sequence of Keap1 or the nucleotide sequence encoding it can be obtained from publicly available sequence databases. Some of the accession numbers in those databases are listed here. [Database name: Accession number (species name)]: Swiss-Prot: Q14145.2 (human), Swiss-Prot: Q9Z2X8.1 (mouse), GenBank: BAA46339.1 (mouse), GenBank: AB020063.1 ( Mouse), Swiss-Prot: Q684M4.1 (pig). As used herein, the term “Keap1” is not limited to human Keap1, but other species of keap1 (eg, other mammalian species (eg, monkeys, cows, pigs, mice, rats, etc.), reptiles, amphibians, Homologs of fish, etc.). The amino acid sequence of such a homologue is, for example, an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of human Keap1, or human Keap1 1 to several amino acid sequences (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Or an amino acid sequence having deletion, substitution, insertion, or addition of amino acid residues.
As used herein, a molecule that “specifically binds to p62” means that the molecule has a substantially higher affinity for a particular amino acid sequence of p62 or a fragment thereof than its affinity for other amino acid sequences. It means to combine with. Here, “substantially high affinity” means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device. Includes a coupling constant (Ka) Is at least 107M-1, Preferably at least 108M-1, More preferably 109M-1, Even more preferably, 1010M-11011M-11012M-1Or higher, for example, up to 1013M-1Or means a binding affinity that is higher.
Examples of “molecules that specifically bind to p62” include the following i) and ii).
i) Keap1 interacting region of p62 (Keapl Interacting Region) [SKEVDPSTGELQSLQ (SEQ ID NO: 5), for example, 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of SEQ ID NO: 5. Deletion, substitution, insertion or addition of one to several amino acid residues (eg, 1, 2, 3 or more) in the amino acid sequence having the sequence identity of A substance that specifically binds to an amino acid sequence having
Ii) A substance that specifically binds to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3).
In this specification, “Keap1 interacting region (KIP1)” is a partial amino acid sequence region consisting of amino acid residues at positions 345 to 359 in the amino acid sequence of mouse p62, or a mammal other than mouse. Highly conserved amino acids corresponding to the above partial amino acid sequence region in the amino acid sequence of p62 of animal species (eg, human, monkey, cow, rat, etc.) or other species (eg, reptiles, amphibians, fish, etc.) It shall be the region of the sequence that refers to the region on p62 that specifically binds to Keap1 (see FIG. 2b).
P62 interacts with Keap1, and when p62 is in an excess state, p62 bound to Keap1 increases and the p62-Kap1 complex is insolubilized. Keap1 is normally bound to Nrf2 in the same region as p62, and induces Nrf2 into the ubiquitin-proteasome degradation pathway. However, when p62 is highly expressed, the amount of free Nrf2 is increased by binding and insolubilization of p62 and Keap1. The accumulated Nrf2 moves into the nucleus, the expression of downstream factors is induced, and a p62 / ubiquitin positive inclusion body is formed. Excessive inclusion body formation causes liver diseases such as hepatocellular carcinoma and liver damage.
Therefore, the inhibitor or suppressor can typically be used as i) an inhibitor or suppressor of ubiquitin-positive inclusion body formation, ii) a therapeutic agent for liver disease, or iii) an anticancer agent.
Since the amount of intracellular p62 is regulated by autophagy for the interaction with LC3, administration of a substance that enhances this binding decreases the amount of intracellular p62, which causes hepatocellular carcinoma and Reduce liver damage.
Therefore, in a further embodiment of the present invention, the “molecule that specifically binds to p62” is LC3.
In still another embodiment, the “molecule that specifically binds to p62” is an antibody that specifically binds to p62 (anti-p62 antibody).
In the present specification, the “p62 expression inhibitor” refers to a substance that inhibits transcription of p62 gene into mRNA and / or translation of p62 gene (mRNA) into protein.
Examples of “p62 expression inhibitor” include the following iii) and iv).
Iii) the p62 expression inhibitor is an antisense nucleic acid or siRNA against the nucleotide sequence encoding p62,
Iv) A nucleic acid having a ribozyme activity that specifically cleaves the transcription product (mRNA) of the p62 gene.
In this specification, “nucleic acid” means RNA or DNA. The “nucleic acid” herein may contain not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to.
In the therapeutic agent for liver disease or cancer therapeutic agent of the present invention, a nucleic acid having an action of inhibiting the expression of p62 gene by RNAi effect can be used as an active ingredient. RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, expression of the introduced foreign gene and target endogenous gene are both inhibited. Examples of RNA used here include double-stranded RNA that causes RNA interference of 19 to 30 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin RNA). . Such RNA can be locally delivered to a desired site by a delivery system such as a liposome, and can be locally expressed using a vector capable of generating the double-stranded RNA. Methods for preparing and using such double-stranded RNA (dsRNA, siRNA or shRNA) are known from many literatures (Japanese translations of PCT publication No. 2002-516062; US Publication No. 2002 / 086356A; Nature Genetics, 24 (2), Feb., 180-183; Genesis, 26 (4), April, 240-244; Nature, Spe.21, 407: 6802, 319-20; Genes & Dev., Vol.16, (8 ), Apr. 16, 948-958; Proc. Natl. Acad. Sci. USA., 99 (8), 16 Apr., 5515-5520; Science, 296 (5567), 19 Apr., 550-553; Natl.Acad.Sci.USA, Ap .30, 99: 9, 6047-6052; Nature Biotechnology, Vol. 20 (5), May, 497-500; Nature Biotechnology, Vol. 20 (5), May, 500-508; Nucleic Acids Res., May 15 Such).
The length of the double-stranded RNA exhibiting the RNAi effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, and most preferably 21 to 23 bases.
In this specification, “antisense nucleic acid” or “antisense polynucleotide” has a polynucleotide complementary to at least a part of a DNA region of interest, and the polynucleotide is at least one of the region. It means a nucleic acid capable of hybridizing with a part. The antisense nucleic acid or antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, or even a DNA: RNA hybrid. Specific examples of modified nucleic acids include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those that are resistant to degradation of polynucleotide amides and oligonucleotide amides. .
The antisense nucleic acid used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 'side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the animal to be transformed, but it is not completely complementary as long as the gene expression can be effectively suppressed. May be.
Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more complement to the target gene transcript. Have sex.
In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40 bases), preferably about 15 bases or more, More preferably, it is about 100 bases or more, More preferably, it is about 500 bases or more. Antisense nucleic acids can be designed with reference to known literature (for example, Hirashima and Inoue, Shinsei Kagaku Koza 2 Lecture and Expression of Nucleic Acid IV Gene, edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1993, p.319) -347), J. et al. Kawakami et al. , Pharm Tech Japan. Vol. 8, p. 247, 1992; Vol. 8, p. 395, 1992; T.A. Crooke et al. , Ed. , See Antisense Research and Applications, CRC Press, 1993, etc.).
In this specification, “ribozyme activity” refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Protein Nucleic Acid Enzyme, 1990, 35, p. 2191). For hammerhead ribozymes, see, for example, FEBS Lett, 1988, 228, p. 228; FEBS Lett, 1988, 239, p. 285; protein nucleic acid enzyme, 1990, 35, p. 2191; Nucl Acids Res, 1989, 17, p. 7059 and the like can be referred to. For hairpin ribozymes, see, for example, Nature, 1986, 323, p. 349; Nucl Acids Res, 1991, 19,. 6751; Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, p. 112 and the like can be referred to. By specifically cleaving the transcript of the p62 gene using such a ribozyme, the expression of the gene can be inhibited.
When the expression of p62 is suppressed by these substances, the binding between Keap1 and Nrf2 is restored, and Nrf2 is inactivated, so that hepatocellular carcinoma and liver damage can be suppressed.
Therefore, in a further embodiment of the present invention, there is provided a method for treating liver disease and / or cancer, comprising a step of administering a substance that inhibits or suppresses the interaction between p62 and Keap1 to a patient in need thereof. Provided.
2.Agents containing substances that inhibit or suppress the interaction between p62 and Keap1
In a further embodiment of the present invention, an inhibitor or inhibitor of binding between Keap1 and p62 is provided. This agent typically contains a substance that inhibits or suppresses the interaction between p62 and Keap1 described above. Specifically, this agent
I) a molecule that specifically binds to p62, or
Ii) p62 expression inhibitor
Containing.
The above agent of the present invention can be used as a preparation for treating liver disease or cancer. Such a preparation (pharmaceutical composition) can be produced by mixing a substance that inhibits or suppresses the interaction between p62 and Keap1 with a commonly used preparation carrier.
The pharmaceutical carrier may be used in appropriate combination depending on the dosage form. For example, excipients such as lactose; lubricants such as magnesium stearate; disintegrants such as carboxymethylcellulose; binders such as hydroxypropylmethylcellulose Surfactants such as macrogol; foaming agents such as sodium hydrogen carbonate; solubilizing agents such as cyclodextrin; sour agents such as citric acid; stabilizers such as sodium edetate; pH adjusters such as phosphate Is mentioned.
When administered to a patient in need thereof, as a solid preparation for internal use for oral administration, a liquid for internal use, and an injection, external preparation, suppository, eye drops, inhalant, etc. for parenteral administration It can also be used.
Oral solid preparations for oral administration include tablets, pills, capsules, powders, granules and the like. Capsules include hard capsules and soft capsules. Tablets include sublingual tablets, buccal adhesive tablets, buccal quick disintegrating tablets and the like.
In such solid preparations for internal use, one or more active substances are left as they are, or excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, Mixed with magnesium metasilicate aluminate, etc.), disintegrating agents (such as calcium calcium glycolate), lubricants (such as magnesium stearate), stabilizers, solubilizing agents (such as glutamic acid, aspartic acid), etc. Used by formulating. If necessary, it may be coated with a coating agent (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.), or may be coated with two or more layers. Also included are capsules of absorbable substances such as gelatin.
Oral liquids for oral administration include pharmaceutically acceptable solutions, suspensions / emulsions, syrups, elixirs and the like. In such a solution, one or more active substances are dissolved, suspended or emulsified in a commonly used diluent (purified water, ethanol or a mixture thereof). Furthermore, this liquid agent may contain a wetting agent, a suspending agent, an emulsifier, a sweetening agent, a flavoring agent, a fragrance, a preservative, a buffering agent and the like.
External dosage forms for parenteral administration include, for example, ointments, gels, creams, poultices, patches, liniments, sprays, inhalants, sprays, eye drops, and nasal drops Etc. are included. These contain one or more active substances and are produced and prepared by known methods or commonly used formulations.
Included as injections for parenteral administration are solutions, suspensions, emulsions and solid injections used by dissolving or suspending in a solvent at the time of use. An injection is used by dissolving, suspending or emulsifying one or more active substances in a solvent. As the solvent, for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof are used. Further, this injection may contain a stabilizer, a solubilizing agent (such as glutamic acid, aspartic acid, polysorbate 80 (registered trademark)), a suspending agent, an emulsifier, a soothing agent, a buffering agent, a preservative and the like. . These are sterilized in the final process or manufactured and prepared by aseptic manipulation. In addition, a sterile solid preparation, for example, a lyophilized product, can be produced and used by dissolving it in sterilized or sterile distilled water for injection or other solvent before use.
3.Screening method of the present invention
In yet another embodiment, the present invention provides a method for screening an agent that inhibits or suppresses the interaction between Keap1 and p62. This method is typically
I) measuring the binding amount of Keap1 and p62 in the presence and absence of the test substance, and
ii) comparing the amount of binding between Keap1 and p62 in the presence and absence of the test substance, and the amount of binding in the presence of the test substance is greater than that in the absence of the test substance. And a step of selecting the test substance as a substance that inhibits or suppresses the interaction between Keap1 and p62 when the amount is lower than the binding amount.
A substance that inhibits or suppresses the interaction between p62 and Keap1 can be applied to a liver disease therapeutic agent and an anticancer agent. By searching for a substance that inhibits or suppresses the protein-protein interaction between p62 and Keap1, a novel drug for the treatment of liver disease can be screened.
The interaction can be detected by a pull-down method (eg, FIG. 3), a method using a mass spectrometer, a method for detecting protein-protein interaction as a fluorescent signal, or an imaging using a fluorescent label.
In a more specific embodiment of the screening method of the present invention, “the step of measuring the amount of binding between Keap1 and p62 in the presence and absence of a test substance” comprises the KIR sequence of p62 (eg, SKEVDPSTGELQSLQ (SEQ ID NO: 5), for example, an amino acid sequence having sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 This includes measuring the amount of Keap1 binding to 1 to several (eg, amino acid sequences having 1, 2, 3, or more amino acid residue deletions, substitutions, insertions or additions).
In a more specific embodiment of the screening method of the present invention, the “substance that inhibits or suppresses the interaction between Keap1 and p62” is aspartic acid at position 349 in the amino acid sequence of mouse p62 (SEQ ID NO: 3), It specifically binds to proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354.
In a more specific embodiment of the screening method of the present invention,
Iii) administering a substance that inhibits or suppresses the interaction between Keap1 and p62 to a liver disease model animal and measuring the activity of Nrf2, and
Iv) When the activity of Nrf2 decreases, the above-mentioned substance is selected as a therapeutic agent for liver disease
Is included.
Examples of the substance obtained by the screening method of the present invention include LC3, anti-p62 antibody and the like, but are not limited thereto, low molecular weight compounds (natural or synthetic), proteins or peptides, polysaccharides, lipids , Nucleic acids and the like may be included.
A substance that inhibits or suppresses the interaction between p62 and Keap1, or a substance that decreases the amount of intracellular p62 can be applied to a therapeutic agent for liver disease and an anticancer agent. These substances inactivate (decompose) Nrf2 by reducing the amount of p62 that binds to Keap1 and increasing the binding between Keap1 and Nrf2. By administering a substance that inhibits or suppresses the interaction between p62 and Keap1 or a substance that decreases the amount of intracellular p62 to a liver disease model animal (eg, mouse) and measures the activity of Nrf2, a novel drug You can find
Detection of the interaction between Keap1 and p62 was performed by transiently cultivating a plasmid in which Nrf2 transcriptional activity reporter ARE (antioxidant-respondive element) was connected to luciferase gene to cultured cells (Huh-1, JHH5, Hepa-1, HEK293T, etc.). By luciferase assay using a cell line introduced into or stably maintained (see FIGS. 6 and 16) by β- by a cultured cell incorporating an ARE-bla linking the Nrf2 transcriptional activity reporter and the β-lactamase gene. By Nrf2 transcriptional activity reporter assay using lactamase and fluorescence resonance energy transfer (FRET), the expression of downstream factors of Nrf2 was immunized by real time PCR using hepatocytes overexpressing p62. It can be confirmed by blotting (Immunoblot (hereinafter referred to as IB)) or by RT-PCR or the like.
In a more specific embodiment of the screening method of the present invention, the measurement of the binding amount of Keap1 and p62 in the presence and absence of a test substance is carried out using a pull-down method, mass spectrometry, protein-protein interaction as a fluorescence signal. This is performed using either a detection method or imaging using a fluorescent label.
A substance that inhibits or suppresses the interaction between p62 and Keap1 or a substance that reduces the amount of intracellular p62 may be applicable to a therapeutic agent for liver disease and an anticancer agent. These substances reduce the amount of intracellular ubiquitin positive inclusions in the liver lacking mouse autophagy. A substance that inhibits or suppresses the interaction of Keap1 or a substance that decreases the amount of intracellular p62 is administered to mice, and the amount of ubiquitin positive inclusion bodies, the activity of Nrf2, and the level of liver damage are measured. Drug candidates can be screened.
Detection of ubiquitin positive inclusion bodies can be performed by immunostaining mouse liver tissue (FIG. 9).
In the Nrf2 activity measurement, the expression of the detoxification enzyme gene group which is a downstream factor of Nrf2 can be confirmed by real time PCR (FIG. 8).
Liver damage level measurement includes liver weight measurement (FIG. 13a), hepatocyte hypertrophy and lobular structure collapse detection by HE staining of liver tissue (FIG. 13b), liver function test (AST, ALT, ALP) (FIG. 14) Can be done by.
The substance obtained by the screening method of the present invention can be used as i) an inhibitor or suppressor of ubiquitin-positive inclusion body formation, ii) a therapeutic agent for liver disease, or iii) an anticancer agent.
4).Diagnostic method, diagnostic agent, and diagnostic kit for liver disease
In a further embodiment, the present invention provides a method for diagnosing liver disease. In particular, a method for determining or diagnosing a patient to which the therapeutic agent for liver disease of the present invention is applied is provided. This method is typically
I) measuring the expression level of p62 or Nrf2 by subject's liver biopsy, or
Ii) measuring the activity of Nrf2 by subject's liver biopsy,
including.
If it is found that the expression level of p62 in a liver biopsy of a subject with liver disease is excessive as compared with that of a normal subject, it is highly likely that the liver disease of the subject is caused by overexpression of p62. In such a case, the therapeutic agent for liver disease according to the present invention is applied to a patient who needs to treat the disease in order to inhibit or suppress the interaction between p62 and Keap1 or to inhibit the expression of p62. By doing so, it is considered that an advantageous effect can be obtained. Therefore, it is useful to determine or diagnose the patient to whom such treatment is applied.
In addition, if it is found that the activity of Nrf2 by liver biopsy of a subject with liver disease is higher than that of a normal subject, it is highly possible that the liver disease of the subject is due to abnormal activation of Nrf2. In such a case, if the therapeutic agent for liver disease of the present invention is applied to a patient in need of treatment of the disease, the interaction between p62 and Keap1 is inhibited or suppressed, or the expression of p62 is inhibited. As a result, the amount of p62 competing with the interaction between Nrf2 and Keap1 can be reduced, so that the amount of free Nrf2 is reduced and the activation of Nrf2 is suppressed. Therefore, it is considered that an advantageous effect can be obtained by applying the therapeutic agent for liver disease of the present invention to a patient in need of treatment for the disease. Therefore, it is useful to determine or diagnose the patient to whom such treatment is applied.
Measurement of the expression level of p62 by liver biopsy of a subject can be performed by methods well known to those skilled in the art, such as imaging with a fluorescent label, immunoblotting, and mass spectrometry.
Measurement of the activity of Nrf2 by liver biopsy of a subject includes quantitative measurement of luciferase assay using a target promoter of Nrf2, measurement of the amount of Nrf2 in cell nuclei by immunoblotting, and enzymes induced by Nrf2 (such as Nqol and Gstm). It can be performed by methods well known to those skilled in the art, such as expression analysis by PCR, immunoblotting or fluorescence imaging, and enzyme activity measurement.
In a further embodiment of the method for determining a patient to which the therapeutic agent for liver disease of the present invention is applied, measurement of Nrf2 activity and / or detection of ubiquitin positive inclusion bodies is included.
A further embodiment of the method for determining a patient to which the therapeutic agent for liver disease of the present invention is applied includes measuring the concentration of AST, ALT, ALP, and / or γ-GTP in the blood of the subject. Γ-GTP (γ-glutamyl transpeptidase) is an enzyme related to the detoxification of the liver, and when the cells of the liver and bile duct break down, γ-GTP flows into the blood. It is said to be a “deviation enzyme” and is generally used as an indicator that the cells of the liver and bile ducts have been broken.
In a further embodiment of the present invention, a diagnostic kit or diagnostic agent used in the diagnostic method of the present invention is provided. These diagnostic kits or diagnostic agents typically include an anti-p62 antibody or an anti-Nrf2 antibody. Further, these diagnostic kits or diagnostic agents may contain a labeling agent for labeling the antibody (for example, a fluorescent label) or a fluorescently labeled antibody. The diagnostic kit may further include instructions for use. The instructions for use may describe the method for using the antibody, the labeling agent, etc. included in the kit, the experimental procedure including immunoblotting, and the like.
The relationship between the amino acid sequence or nucleotide sequence appearing in this specification and the sequence number is as follows.
[SEQ ID NO: 1]
This sequence represents the amino acid sequence of human p62.
[SEQ ID NO: 2]
This sequence represents a nucleotide sequence encoding the amino acid sequence of human p62 (SEQ ID NO: 1).
[SEQ ID NO: 3]
This sequence represents the amino acid sequence of mouse p62.
[SEQ ID NO: 4]
This sequence represents a nucleotide sequence encoding the amino acid sequence of mouse p62 (SEQ ID NO: 3).
[SEQ ID NO: 5]
This sequence represents a partial sequence (KIR sequence) consisting of consecutive amino acid residues from the 345th position to the 359th position of the amino acid sequence of mouse p62 (SEQ ID NO: 3).
Hereinafter, the present invention will be described more specifically with reference to examples. However, it goes without saying that the scope of the present invention is not limited to these examples.
［実施例１］
　実施例１では、ｐ６２とＫｅａｐ１の相互作用およびｐ６２に結合するＫｅａｐ１の領域を調べた。
　Ｆｌａｇ−ｔａｇをＮ末に繋げたマウスＫｅａｐ１とＫｅａｐ１の各変異タンパク質を発現させたＨｅｋ２９３Ｔ細胞を、ｌｙｓｉｓ　ｂｕｆｆｅｒにより溶解し、細胞抽出液を得た。次に抗Ｆｌａｇ抗体を用いて免疫沈降を行い、Ｆｌａｇ−ｔａｇを付加したＫｅａｐ１あるいはそのｄｅｌｅｔｉｏｎ　ｍｕｔａｎｔ、およびその相互作用複合体を回収した。回収したタンパク質複合体と細胞抽出液をＳＤＳ−ＰＡＧＥ（ポリアクリルアミドゲル電気泳動）によりゲル内に展開し、抗Ｆｌａｇ抗体、抗ｐ６２抗体、抗Ｎｒｆ２抗体、抗ａｃｔｉｎ抗体の各抗体を用いて、免疫ブロッティング（Ｉ．Ｂ．）を行った。
　結果を図１に示す。図１はＫｅａｐ１の機能ドメイン領域およびＦＬＡＧ−ｔａｇをつけたＫｅａｐ１およびその各ｍｕｔａｎｔの名称および模式図、抗ＦＬＡＧ抗体を用いて免疫沈降を行った場合におけるＩ．Ｂ．の写真、および細胞抽出液を用いたＩ．Ｂ．の写真である。
　模式図最上部にはＫｅａｐ１のドメイン領域とその名称を示している。ＮＴＲ（Ｎ−ｔｅｒｍｉｎａｌ　ｒｅｇｉｏｎ），ＢＴＢ（Ｂｒｏａｄｃｏｍｐｌｅｘ，Ｔｒａｍｔｒａｃ，ａｎｄ　Ｂｒｉｃ−ａ−Ｂｒａｃ），ＩＶＲ（ｉｎｔｅｒｖｅｎｉｎｇ　ｒｅｇｉｏｎ），ＤＳＧ（ｔｈｅ　ｄｏｕｂｌｅ　ｇｌｙｃｉｎｅ　ｒｅｐｅａ　ｒｏｋｅｌｃｈ　ｒｅｐｅａｔ）ａｎｄ　ＣＴＲ（ｔｈｅ　Ｃ−ｔｅｒｍｉｎａｌ　ｒｅｇｉｏｎ）。ＦＬＡＧ−ｔａｇを付加したタンパクの模式図とその左に名称を記す。抗ＦＬＡＧ抗体の免疫沈降によるＩ．Ｂ．の写真をＩＰ：ＦＬＡＧで示す。また細胞抽出液によるＩ．Ｂ．の写真をＣｒｕｄｅで表し、これは各細胞でのＫｅａｐ１，ｐ６２，ａｃｔｉｎ（ｌｏａｄｉｎｇ　ｃｏｎｔｒｏｌ）の発現の確認を行う。Ｉ．Ｂ．の写真の左横には使用した抗体を示す。
　図１に示すように、Ｆｌａｇ−Ｋｅａｐ１を発現させた細胞で免疫沈降を行った場合（レーン２）、内在性ｐ６２が検出されたことから、ｋｅａｐ１とｐ６２は複合体を形成することが分かった。Ｋｅａｐ１の各ｍｕｔａｎｔを発現させた細胞で免疫沈降を行った場合（レーン３−９）では、レーン９でｐ６２が検出され、レーン６とレーン８では検出されないことから、ｐ６２と複合体を形成する為に必要なＫｅａｐ１の部位はＤＣ領域（ＤＧＲ＋ＣＴＲ）であることが分かった。
［実施例２］
　実施例２では、ｐ６２とＫｅａｐ１の直接の相互作用と、Ｋｅａｐ１に結合するｐ６２の領域を調べた。
　まず、大腸菌に実施例１で示したｐ６２と結合する領域Ｋｅａｐ１−ＤＣとのＧＳＴ（Ｇｌｕｔａｔｈｉｏｎｅ　Ｓ−ｔｒａｎｓｆｅｒａｓｅ）融合タンパク質、ＧＳＴタグのついたマウスＫｅａｐ１−ＤＣ（ＧＳＴ−Ｋｅａｐ１−ＤＣ）とマウスｐ６２あるいは各ｐ６２　ｍｕｔａｎｔのＭＢＰ（Ｍａｌｔｏｓｅ　Ｂｉｎｄｉｎｇ　Ｐｒｏｔｅｉｎ）融合タンパク質を産生させ、ｇｌｕｔａｔｈｉｏｎｅ−Ｓｅｐｈａｒｏｓｅ　４Ｂ　ｒｅｓｉｎとａｍｙｌｏｓｅ　ｒｅｓｉｎをそれぞれ用いたアフィニティークロマトグラフィーにより精製した。これらの精製タンパク質をＴＮＥ　ｂｕｆｆｅｒ中で混和させ、ａｍｙｌｏｓｅ　ｒｅｓｉｎで沈殿させる。Ａｍｙｌｏｓｅ　ｒｅｓｉｎ結合タンパク質を氷冷ＴＮＥで５回ｗａｓｈし、ＳＤＳ−ＰＡＧＥとＣＢＢ（Ｃｏｏｍａｓｓｉｅ　ｂｒｉｌｌｉａｎｔ　ｂｌｕｅ）染色を行った。
　結果を図２に示す。図２はｐ６２の機能ドメイン領域およびＭＢＰ−ｔａｇをつけたマウスｐ６２とその各ｍｕｔａｎｔの名称および模式図、ＳＤＳ−ＰＡＧＥ、ＣＢＢ染色後の写真である。
　模式図最上部にはｐ６２のドメイン領域とその名称を示している。ＰＢ１（Ｐｈｏｘ　ａｎｄ　Ｂｅｍｌｐ），Ｚｉｎｃ（Ｚｉｎｃ　ｆｉｎｇｅｒ），ＬＲＳ（ＬＣ３−ｒｅｃｏｇｎｉｔｉｏｎ　ｓｅｑｕｅｎｃｅ）ａｎｄ　ＵＢＡ（ｕｂｉｑｕｉｔｉｎ−ａｓｓｏｃｉａｔｅｄ　ｄｏｍａｉｎ）。ＭＢＰ−ｔａｇを付加したタンパクの模式図とその左に名称を記す。ＭＢＰ　ｉｍｍｏｂｉｌｉｚｅｄ　ＡＭ　ｒｅｓｉｎは各タンパク質の発現を表し、ＭＢＰ−Ｐｕｌｌ　ｄｏｗｎ　ａｓｓａｙでは各ｐ６２−ＭＢＰ融合タンパクへのＧＳＴ−Ｋｅａｐ１−ＤＣの結合を示す。
　図２ａに示すように、ＭＢＰ　ｉｍｍｏｂｉｌｉｚｅｄ　ＡＭ　ｒｅｓｉｎの図で各タンパク質の精製が全て確認された中で、ＭＢＰ−Ｐｕｌｌ　ｄｏｗｎ　ａｓｓａｙを行うと、ｐ６２とＫｅａｐ１の結合が確認されたことから、ｐ６２とＫｅａｐ１は直接結合することが示された。またレーン５，６，７，８，１１，１２，１４（１４については図３を参照）でｐ６２の結合が確認されたことから、Ｋｅａｐ１へのｐ６２の結合領域は３４５−３５９アミノ酸の領域であることが分かった。また、この領域は、図２ｂに示すように種間で高度に保存されており、このアミノ酸配列の領域をＫＩＲ（Ｋｅａｐ１　ｉｎｔｅｒａｃｔｉｎｇ　ｒｅｇｉｏｎ）（Ｋｅａｐ１相互作用領域）配列と名づけた。
［実施例３］
　実施例３では、Ｋｅａｐ１の結合に重要なｐ６２−ＫＩＲ配列中のアミノ酸を調べた。
　ＭＢＰ−ｐ６２Ｍ８０の−ＫＩＲ配列の各アミノ酸をそれぞれアラニンに置換したタンパク質と、ＧＳＴ−Ｋｅａｐ１−ＤＣを用いて実施例２と同様のプルダウンアッセイを行った。
　結果を図３に示す。図の左上には各サンプル番号に対するアミノ酸の置換部位を示し、下部ではＳＤＳ−ＰＡＧＥ，ＣＢＢ染色後のゲルの写真、写真の右側にはバンドが示すタンパク質の名称を示す。
　レーン６、７、９、１０、１１でＧＳＴ−Ｋｅａｐ１−ＤＣのバンドがほとんど見られず、結合が劇的に低下していることから、ｐ６２とＫｅａｐ１との結合にはｐ６２のＤ３４９，Ｐ３５０，Ｔ３５２，Ｇ３５３，Ｅ３５４の５アミノ酸が重要であることが分かった。
［実施例４］
　実施例４では、ｐ６２とＮｒｆ２はＫｅａｐ１の同じポケットで結合していることを調べた。
　タンパク質結晶化立体構造解析により、Ｋｅａｐ１−ＤＣのｐ６２あるいはＮｒｆ２との結合に重要なアミノ酸を予想した。これらのアミノ酸をそれぞれアラニンに置き換えたＫｅａｐ１−ＤＣ　ｍｕｔａｎｔおよびＫｅａｐ１−ＤＣタンパク質にＦＬＡＧタグを付けＨｅｋ２９３Ｔ細胞に発現させた。これらの細胞をｌｙｓｉｓ　ｂｕｆｆｅｒにより溶解し、細胞抽出液を得た。次に実施例１と同様に免疫沈降およびＩ．Ｂ．を行った。
　結果を図４に示す。図４の左には、各サンプル番号とその番号に対するアミノ酸置換部位を示す。抗ＦＬＡＧ抗体の免疫沈降によるＩ．Ｂ．の写真をＩＰ：ＦＬＡＧで示す。また細胞抽出液によるＩ．Ｂ．の写真をＣｒｕｄｅで表し、これは各細胞でのＫｅａｐ１，ｐ６２，ａｃｔｉｎ（ｌｏａｄｉｎｇ　ｃｏｎｔｒｏｌ）の発現の確認を示す。Ｉ．Ｂ．の写真の右横には使用した抗体を示す。
　図４で示すように、Ｒ４８３Ａ（レーン８）以外のＫｅａｐ１−ＤＣ　ｍｕｔａｎｔにおいてｐ６２との結合が劇的に低下しており、またＲ４８３Ａを含めた全てのｍｕｔａｎｔにおいてＮｒｆ２との結合も劇的に低下していることから、ｐ６２とＮｒｆ２は、Ｋｅａｐ１の同じポケットを使用して拮抗的に結合していることが示された。
［実施例５］
　実施例５では、Ｋｅａｐ１−Ｎｒｆ２の結合をｐ６２が拮抗的に阻害することを示す。
　Ｆｌａｇ−ｔａｇをＮ末に繋げたｐ６２とｐ６２−Ｔ３５２Ａを発現させたＨｅｋ２９３Ｔ細胞を、ｌｙｓｉｓ　ｂｕｆｆｅｒにより溶解し、細胞抽出液を得た。次に抗Ｎｒｆ２抗体を用いて免疫沈降を行い、Ｎｒｆ２およびその相互作用複合体を回収した。次にＳＤＳ−ＰＡＧＥ（ポリアクリルアミドゲル電気泳動）により、回収したタンパク質複合体と細胞抽出液をゲル内に展開し、抗ｕｂｉｑｕｉｔｉｎ抗体、抗Ｎｒｆ２抗体、抗ｐ６２抗体、抗ａｃｔｉｎ抗体の各抗体を用いて、Ｉ．Ｂ．を行った。
　結果を図５に示す。抗Ｎｒｆ２抗体を用いて免疫沈降を行った場合におけるＩ．Ｂ．の写真をＩＰ：Ｎｒｆ２で示す。また細胞抽出液を用いたＩ．Ｂ．の写真をＣｒｕｄｅで示す。写真上部にはＦＬＡＧ−ｐ６２あるいはＦＬＡＧ−ｐ６２−Ｔ３５２Ａの過剰発現，Ｌａｃｔａｃｙｓｔｉｎ（プロテアソーム阻害剤）の存在の有無を＋−で示す。また図の左右に、Ｉ．Ｂ．に使用した抗体名、検出されたタンパク名を示す。
　ｐ６２を過剰発現させていない細胞のＮｒｆ２免疫沈降（ＩＰ：Ｎｒｆ２のレーン１）では、ユビキチン化されたＮｒｆ２，Ｎｒｆ２そのものの検出レベルが低いが、プロテアソーム阻害剤を加える（レーン２）と顕著に高くなる。通常Ｎｒｆ２はｋｅａｐ１と結合しユビキチン化されプロテアソームにより分解されていることから説明できる。この状態でｐ６２を過剰発現させると、Ｎｒｆ２タンパク量が顕著に上昇した。一方、ユビキチン化されたＮｒｆ２が低下した（ＩＰ：Ｎｒｆ２のレーン３、４）。これはｐ６２がＫｅａｐ１と結合することにより、Ｎｒｆ２とＫｅａｐ１の結合が低下し、Ｎｒｆ２がユビキチン化および分解されないことから、Ｎｒｆ２が増加することを示す。またＫｅａｐ１と結合能が顕著に低下しているｐ６２−Ｔ３５２Ａを入れると（ＩＰ：Ｎｒｆ２のレーン５、６）ｐ６２を発現させない時と同様の状態（ＩＰ：Ｎｒｆ２のレーン１、２）を示すことから、Ｋｅａｐ１とＮｒｆ２の結合をｐ６２が拮抗的に阻害していることを示す。
［実施例６］
　実施例６では、Ｋｅａｐ１とｐ６２の結合がＮｒｆ２下流因子の発現活性を増大させることを示す。
　Ｎｒｆ２，Ｋｅａｐ１，そしてｐ６２　ｗｉｌｄ−ｔｙｐｅあるいはｐ６２　ｍｕｔａｎｔｓを発現するプラスミドをｐＮＱＯ１−ＡＲＥ　ｒｅｐｏｒｔｅｒ　ｐｌａｓｍｉｄとｐＲＬ−ＴＫ（リポフェクションの内部コントロール）と共にマウス肝細胞癌由来のＨｅｐａ１細胞に導入して、３６時時間後に、ルシフェラーゼ活性を測定した。
　結果を図６に示す。Ｎｒｆ２，Ｋｅａｐ１，ｐ６２−ＷＴ，ｐ６２−ｍｕｔａｎｔｓ導入の有無を＋−で示し、発現ベクターを導入していない（カラム１）をスタンダードとして相対的なルシフェラーゼの活性を示す。
　Ｎｒｆ２を過剰発現させると（カラム２）、活性が大幅に上昇し、Ｎｒｆ２とＫｅａｐ１の２重トランスフェクション（カラム３）ではＮｒｆ２がＫｅａｐ１に捕まえられプロテアソームにより分解される為カラム１と同程度まで活性が低下した。Ｎｒｆ２，Ｋｅａｐ１，ｐ６２−ＷＴの３重トランスフェクションでは、ｐ６２がＫｅａｐ１と結合することでフリーのＮｒｆ２が増加する為、ルシフェラーゼ活性が上昇したが、Ｎｒｆ２，Ｋｅａｐ１，ｐ６２−ｍｕｔａｎｔの３重トランスフェクション（カラム５−１０）では、ｐ６２はＫｅａｐ１と結合できない為、活性はカラム４と変わらなかった。またｐ６２−ＷＴ単独のトランスフェクションでも活性の上昇が見られ、ｐ６２−ｍｕｔａｎｔでは見られないことから、内在性のＫｅａｐ１に結合することで内在性Ｎｒｆ２が活性化したものと考えられる。以上の結果より、ｐ６２の過剰発現によりＫｅａｐ１と結合するｐ６２が増加し、Ｎｒｆ２の活性が上昇することを示唆している。
　また、カラム４とカラム５−１０でルシフェラーゼ活性に大きな差が見られることから、カラム４での高活性状態において、ｐ６２とＫｅａｐ１の阻害剤を加えると確実に活性が低下すると予想される。また他の肝臓がん由来細胞等では、もともとｐ６２の発現量が高くＮｒｆ２の転写活性も高い細胞も存在することから、これらの細胞にｐＮＱＯ１−ＡＲＥ　ｒｅｐｏｒｔｅｒ　ｐｌａｓｍｉｄを安定に持たせた安定株を作成し、ルシフェラーゼアッセイを行うことで、ｐ６２とｋｅａｐ１の結合をモニターでき、また阻害剤が探索可能となることが示唆された。
［実施例７］
　実施例７では、肝臓特異的にオートファジーを欠損させたマウスにおいて、ｐ６２と共にＫｅａｐ１が蓄積・不溶化し、Ｋｅａｐ１の不溶化はｐ６２に依存していることを示す。
　結果を図７に示す。Ｆ／Ｆ：Ｍｘ１はＡｔｇ７遺伝子のコンディショナルノックアウトマウスで、マウスの腹腔内にピペラシリン（ＰＩＰＣ）を投与すると肝臓特異的にＡｔｇ７遺伝子が欠損し、オートファジーが機能しなくなるマウスである。またｐ６２−／−はｐ６２が欠損したマウスで、Ｆ／Ｆ：Ｍｘ１：ｐ６２−／−はｐ６２が欠損したＦ／Ｆ：Ｍｘ１マウス，Ｆ／ＦはＷＴマウスを指す。図７ａでは、ＰＩＰＣを投与後、肝臓をスクロースバッファー（０．２５Ｍ　ｓｕｃｒｏｓｅ，１０ｍＭ　ＨＥＰＥＳ　ｐＨ７．４，１ｍＭ　ＤＴＴ）に回収し、ホモジナイズする。破砕液を０．５％　ＴｒｉｔｏｎＸ−１００可溶性と不溶性に分画する。各分画をＳＤＳ−ＰＡＧＥで展開し、抗ｐ６２抗体、抗Ｋｅａｐ１抗体、抗ＬＣ３抗体、抗Ｎｑｏ１抗体、および抗ａｃｔｉｎ抗体（ｌｏａｄｉｎｇ　ｃｏｎｔｒｏｌ）で免疫ブロットを行う。
　図の左側には使用した抗体の種類、上部には投与後の日数、あるいはマウスの遺伝子型を示す。図の下部に示した、Ｔｏｔａｌは全細胞抽出液、Ｓｏｌ．は可溶性分画、Ｉｎｓｏｌ．は不溶性分画を示す。ＰＩＰＣ投与によりオートファジーが欠損されてから８−１２時間後には、ｐ６２が過剰に発現されるようになるが、Ｋｅａｐ１の発現はほとんど変化しない。またｐ６２の過剰発現に伴い不溶性のｐ６２が蓄積し、同時に不溶性Ｋｅａｐ１も蓄積する。また各遺伝子型マウスのＰＩＰＣ投与後１２時間では、Ｆ／Ｆ：Ｍｘ１ではｐ６２の過剰発現、および不溶性のｐ６２とＫｅａｐ１の増加がみられるが、Ｆ／Ｆ：Ｍｘ１：ｐ６２−／−では見られない（図７ｂ）。これらの結果からオートファジーが欠損したマウスの肝臓においてｐ６２の過剰発現、および不溶性のｐ６２，Ｋｅａｐ１の蓄積がみられる。また不溶性ｋｅａｐ１の蓄積はｐ６２依存的であることが示された。
［実施例８］
　実施例８では、実施例７で用いた遺伝子型マウスの肝臓でのＮｒｆ２の下流因子の発現状態をＲＴ−ＰＣＲにより調べた。
　各遺伝子型マウスにＰＩＰＣ投与後１２時間後のマウス肝臓から、Ｔｏｔａｌ　ＲＮＡを抽出する。Ｔｒａｎｓｃｒｉｐｔｏｒ　Ｆｉｒｓｔ　Ｓｔｒａｎｄ　ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　Ｋｉｔ（Ｒｏｃｈｅ　Ａｐｐｌｉｅｄ　Ｓｃｉｅｎｃｅ）を使用し、ｔｏｔａｌ　ＲＮＡ　１ｍｉｃｒｏｇからｃＤＮＡを合成し、そしてＬｉｇｈｔＣｙｃｌｅｒ　４８０　Ｐｒｏｂｅｓ　Ｍａｓｔｅｒｉｎ（Ｒｏｃｈｅ　Ａｐｐｌｉｅｄ　Ｓｃｉｅｎｃｅ）を使いＬｉｇｈｔＣｙｃｌｅｒで定量的ＰＣＲを行った。シグナルはβ−ｇｌｕｃｕｒｏｎｉｄａｓｅ（ＧＵＳ）で標準化を行った。結果を図８に示す。
　図８の上部には、遺伝子名、左部にはＦ／Ｆを１とした時のｍＲＮＡ量の比率、下部には各遺伝子型を記す。
　示されたように、オートファジー欠損マウス（Ｆ／Ｆ：Ｍｘ１）ではＮｑｏ１，Ｇｓｔｍ１共に、発現量が劇的に上昇している。このことからオートファジー欠損によりＮｒｆ２下流遺伝子の発現が上昇することが示された。一方ｐ６２を欠損させたマウス（ｐ６２およびＦ／Ｆ：Ｍｘ１：ｐ６２）では野生型マウス（Ｆ／Ｆ）とほとんど変わらないことから、オートファジー欠損によるＮｑｏ１，Ｇｓｔｍ１の活性化はｐ６２に依存していることが示された。
［実施例９］
　実施例９では、Ｋｅａｐ１はｐ６２依存的に封入体へ隔離されることをマウスの肝細胞で示す。
　実施例７で示された遺伝子型マウスにＰＩＰＣ投与後２８時間のマウスから得た肝臓を切断し、抗ｐ６２抗体、および抗Ｋｅａｐ１抗体を用いた２重免疫染色によりｐ６２とＫｅａｐ１の細胞内局在を蛍光観察した。結果を図９に示す。
　図９の上段はｐ６２，中段はＫｅａｐ１、下段はｐ６２とＫｅａｐ１の局在を合わせたもの（Ｍａｒｇｅ）とし、核染色図の左上部にはそれぞれの遺伝子型を記す。Ｆ／Ｆ：Ｍｘ１右上部の白四角枠で囲った領域の拡大図を左下に示す。
　示されたように、オートファジー欠損マウス（Ｆ／Ｆ：Ｍｘ）では白で示したｐ６２とＫｅａｐ１は凝集し封入体を形成しており、共局在観察では黄色で示されたようにｐ６２とＫｅａｐ１の局在がほぼ一致していることから、ｐ６２とＫｅａｐ１が同じ封入体を形成しているものと考えられる。またオートファジー欠損マウスでｐ６２を欠失させると（Ｆ／Ｆ：Ｍｘ：ｐ６２−／−）、ｐ６２とＫｅａｐ１を含む封入体形成が見られなくなることから、Ｋｅａｐ１陽性封入体形成はｐ６２の発現に依存していることが示された。
　これまでの結果より、オートファジー欠損により、過剰発現したｐ６２がＫｅａｐ１と結合することで、Ｋｅａｐ１を不溶化、封入体を形成させ、またＮｒｆ２の下流因子の発現が上昇することが分かった。オートファジー欠損させたマウスの肝臓では、ｐ６２の発現上昇と不溶化に伴いＫｅａｐ１も不溶化し、Ｋｅａｐ１にトラップされないＮｒｆ２が増え、抗酸化タンパク質の発現を増加すると考えられる。
［実施例１０］
　実施例１０では、オートファジー欠損によるＫｅａｐ１の不溶化はＮｒｆ２に依存していることを示す。
　オートファジー欠損によりｐ６２の発現依存的にＫｅａｐ１が不溶化し、Ｎｒｆ２の下流因子の発現も上昇する。そこでこれまで用いた実施例７で示したＦ／Ｆ、Ｆ／Ｆ：Ｍｘ１に加えて、Ｎｒｆ２欠損マウス（Ｎｒｆ２−／−，Ｆ／Ｆ：Ｍｘ１：Ｎｒｆ２−／−）を作成し、実施例７と同様の方法でＫｅａｐ１の不溶化の検討を行った。
　Ｎｒｆ２欠失によるＮｒｆ２の発現、さらにＮｒｆ２下流遺伝子（Ｎｑｏ１とＧｓｔｍ１）の発現も同時に確認している。結果を図１０に示す。
　結果が示すように、オートファジー欠損マウス（Ｆ／Ｆ：Ｍｘ１）で見られたｐ６２の過剰発現および不溶タンパクの蓄積は、Ｎｒｆ２を２重で欠損させると劇的に低下した。またＮｒｆ２は通常Ｋｅａｐ１と結合することで分解されていることから、ｐ６２の蓄積はＫｅａｐ１を捕らえて不溶化し、Ｋｅａｐ１−Ｎｒｆ２のｐａｔｈｗａｙの欠如を導いていることが分かった。
［実施例１１］
　実施例１１では、オートファジー欠損によるｐ６２，Ｋｅａｐ１陽性封入体の蓄積はＮｒｆ２に依存していることを示す。
　実施例１０の遺伝子型マウスを使用し、実施例９と同様の方法で免疫染色を行った。結果を図１１に示す。
　結果が示すように、オートファジー欠損（Ｆ／Ｆ：Ｍｘ１）で見られたＫｅａｐ１陽性、ｐ６２陽性封入体の形成は、Ｎｒｆ２を欠損させると（Ｆ／Ｆ：Ｍｘ１：Ｎｒｆ２−／−）劇的に減少した。これはｐ６２陽性Ｋｅａｐ１陽性の封入体形成はＮｒｆ２に依存していることを示す。
［実施例１２］
　実施例１２では、オートファジー欠損によるＮｒｆ２下流遺伝子の過剰発現はＮｒｆ２に依存していることを示す。
　実施例１０の遺伝子型マウスを使用し、実施例９と同様の方法でＮｒｆ２下流遺伝子（Ｎｑｏ１，Ｇｓｔｍ１，Ｃｙｐ２ａ５）のＲＴ−ＰＣＲ解析を行った。結果を、図１２に示す。
　結果が示すように、オートファジー欠損（Ｆ／Ｆ：Ｍｘ１）で見られたＮｒｆ２下流遺伝子（Ｎｑｏ１，Ｇｓｔｍ１，Ｃｙｐ２ａ５）の過剰発現は、Ｎｒｆ２を欠損させると（Ｆ／Ｆ：Ｍｘ１：Ｎｒｆ２−／−）劇的に減少した。これはオートファジー欠損によるＮｒｆ２下流遺伝子の過剰発現はＮｒｆ２に依存していることを示す。
　実施例１０、１１、１２より、ｐ６２の異常蓄積によるＫｅａｐ１が不溶化は、ｐ６２陽性Ｋｅａｐ１陽性封入体を形成し、Ｎｒｆ２−Ｋｅａｐ１　ｐａｔｈｗａｙを欠如させることが示された。
　ｐ６２の過剰発現ではＫｅａｐ１と結合するＮｒｆ２の割合が減少することで、Ｎｒｆ２が蓄積し、Ｎｒｆ２の下流遺伝子の転写量が増大することが示唆される。
［実施例１３］
　オートファジー欠損による肝障害はｐ６２の異常蓄積によるＮｒｆ２−Ｋｅａｐ１ｐａｔｈｗａｙ欠如の結果であるかどうかを調べる為に、実施例１０の遺伝子型マウスを使用し、ｐＩｐＣ投与後２８日のマウス肝臓の重量を測定し、さらにＨＥ染色により、各遺伝子欠損マウスの肝細胞肥大を測定した。結果を、図１３ａおよび図１３ｂにそれぞれ示す。
　オートファジー欠損マウス（Ｆ／Ｆ：Ｍｘ１）では肝臓の重量が大きく増加し肝肥大を示すのに対し、オートファジー／Ｎｒｆ２ダブル欠損マウスでは上昇を示さないことから、ｐ６２の異常蓄積によるＮｒｆ２−Ｋｅａｐ１ｐａｔｈｗａｙ欠如により肝肥大を引き起こすことが分かった。
［実施例１４］
　実施例１４では、オートファジー欠損による肝障害はｐ６２の異常蓄積によるＮｒｆ２−Ｋｅａｐ１　ｐａｔｈｗａｙ欠如の結果であるかどうかを調べる為に、肝障害の指標として、マウス血中ａｓｐａｒａｔｅ　ａｍｉｎｏｔｒａｎｓｆｅｒａｓｅ（ＡＳＴ），ａｌａｎｉｎｅ　ａｍｉｎｏｔｒａｎｓｆｅｒａｓｅ（ＡＬＴ），ａｌｋａｌｉｎｅ　ｐｈｏｓｐｈａｔａｓｅ（ＡＬＰ）の酵素活性を測定した。結果を図１４に示す。
　オートファジー欠損マウス（Ｆ／Ｆ：Ｍｘ１）ではＡＳＴ，ＡＬＴ，ＡＬＰ全て野生型マウス（Ｆ／Ｆ）に比べて劇的な上昇を示すのに対し、オートファジー／Ｎｒｆ２ダブル欠損マウス（Ｆ／Ｆ：Ｍｘ１：Ｎｒｆ２）では上昇を示さない（野生型と同レベルである）ことから、ｐ６２の異常蓄積によるＮｒｆ２−Ｋｅａｐ１ｐａｔｈｗａｙ欠如により肝障害を引き起こすことが分かった。
［実施例１５］
　実施例１５では、ヒトの肝細胞がん腫においてｐ６２，Ｋｅａｐ１陽性封入体が観察されることを示す。抗ｐ６２抗体と抗Ｋｅａｐ１抗体を使ったヒトの肝細胞がん組織アレイ（ＢｉｏＣｈａｉｎ　Ｉｎｓｔｉｔｕｔｅ，Ｉｎｃから購入）の免疫組織化学染色を行った。結果を、図１５に示す。
　ヒト肝細胞がん腫２５サンプルのうち６サンプルについてｐ６２陽性の封入体が確認された。さらに重要なことにｐ６２陽性封入体が見られるサンプルにおいてＫｅａｐ１陽性封入体も確認された。さらにｐ６２，Ｋｅａｐ１　２重染色による蛍光顕微鏡では、ｐ６２とＫｅａｐ１の共局在が観察された。これらの結果、ヒトの肝細胞がん腫でもＮｒｆ２−Ｋｅａｐ１　ｐａｔｈｗａｙの異常調節が示唆された。
［実施例１６］
　実施例１６では、肝がん由来細胞（Ｈｅｐａ−１）のＡＲＥ−Ｌｕｃｉｆｅｒａｓｅ遺伝子を安定に保持するｃｅｌｌ　ｌｉｎｅにおいて、ｐ６２蓄積におけるＮｒｆ２活性システムの存在を示すと共に、ｐ６２−Ｋｅａｐ１結合阻害剤のスクリーニングに使用できることを示す。実験の結果を図１６に示す。
　Ｎｒｆ２の標的プロモーターであるＡＲＥにＬｕｃｉｆｅｒａｓｅ遺伝子を繋げたプラスミドを安定に保持する細胞株を作成し、この株にアデノウイルス感染系を用いてｐ６２−ＷＴ，ｐ６２−Ｔ３５２Ａ（Ｋｅａｐ１と結合できないｍｕｔａｎｔ），Ｋｅａｐ１，ＧＦＰ（ｃｏｎｔｒｏｌ）を過剰発現させて、ルシフェラーゼ活性を測定した。Ｈｅｐａ−１はもともとＮｒｆ２の活性が高いので、Ｋｅａｐ１を過剰発現させるとルシフェラーゼ活性が低下することが示された。Ｋｅａｐ１の過剰発現状態で、ｐ６２−ＷＴあるいはｐ６２−Ｔ３５２Ａを過剰発現させると、感染度を高くするとｐ６２−ＷＴではルシフェラーゼ活性が上昇し、ｐ６２−Ｔ３５２Ａでは上昇を示さなかった。またｐ６２−ＷＴ単独の発現ではルシフェラーゼ活性が劇的な上昇を示すが、ｐ６２−Ｔ３５２Ａ単独の過剰発現では少し低下することがわかった。これはｐ６２−ＷＴが過剰発現されると、ｐ６２と結合するＫｅａｐ１の割合が多くなりＮｒｆ２が活性化状態になり、またｐ６２−Ｔ３５２Ａの過剰発現ではドミナントネガティブ効果により若干活性が低下することを示す。
　このデータは、この細胞株でもｐ６２蓄積におけるＮｒｆ２活性システムの存在を示しており、またｐ６２−Ｋｅａｐ１の結合阻害剤を加えるとルシフェラーゼ活性低下を示すと予想されることから、ｐ６２−Ｋｅａｐ１結合阻害剤のスクリーニングに適している。
［実施例１７］
　実施例１７では、オートファジー欠損マウスまたはオートファジー欠損ｐ６２ノックアウトマウスの肝臓における、腫瘍やＫｅａｐ１やＮｒｆ２下流因子の分子状態を示す。結果を図１７に示す。
　左上図は、４、７、９、１２、１４、１６ヶ月齢のＡｐｇ７ｆ／ｆマウス（コントロール）およびＡｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウス（肝特異的オートファジー欠損マウス）の肝臓の写真である。Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスでは、７ヶ月齢以降に小さな腫瘍（矢印）が確認され、加齢と共に腫瘍の数および大きさが増大した。左下図は１２月齢のＡｐｇ７ｆ／ｆ（コントロール）マウス肝臓、ｐ６２ノックアウト肝臓、Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスの肝臓非腫瘍部、肝臓腫瘍部およびＡｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅ；ｐ６２ノックアウト肝臓抽出液を用いたウエスタンブロット解析の結果である。Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスの肝臓非腫瘍部、肝臓腫瘍部ともにｐ６２が過剰に蓄積・不溶化し、ｐ６２結合タンパク質Ｋｅａｐ１の不溶化も顕著であった。ｐ６２およびＫｅａｐ１の不溶化に伴い、Ｎｒｆ２の標的遺伝子産物であるＮｑｏ１のタンパク質量もＡｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスの肝臓非腫瘍部、肝臓腫瘍部で増加していた。上記Ｋｅａｐ１の不溶化およびＮｑｏ１の発現上昇は、Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅ；ｐ６２ノックアウト肝臓において抑制されていた。右下は１２月齢のＡｐｇ７ｆ／ｆ（コントロール）マウス肝臓、ｐ６２ノックアウト肝臓、Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスの肝臓非腫瘍部、肝臓腫瘍部およびＡｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅ；ｐ６２ノックアウト肝臓から調整したＲＮＡを用いた定量的ＰＣＲ解析の結果である。Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅマウスの肝臓非腫瘍部、肝臓腫瘍部ともに、Ｎｒｆ２の標的遺伝子であるＮｑｏ１やＧｓｔｍ１の遺伝子発現が増加していた。これらの発現上昇は、Ａｐｇ７ｆ／ｆ；Ａｌｂ−Ｃｒｅ；ｐ６２ノックアウト肝臓において抑制されていた。
　これらの結果より、オートファジー欠損マウスの長期飼育により形成された腫瘍におけるＫｅａｐ１の不溶化およびＮｒｆ２の活性化は、ｐ６２依存的であることが明らかとなった。このことから、ｉｎ　ｖｉｖｏ自然発症肝臓がんにおいても、ｐ６２−Ｋｅａｐ１のｐａｔｈｗａｙが機能していることが示唆される。 [Example 1]
In Example 1, the interaction between p62 and Keap1 and the region of Keap1 that binds to p62 were examined.
Hek293T cells in which each mutant protein of mouse Keap1 and Keap1 in which Flag-tag was linked to the N terminus were expressed were lysed with lysis buffer to obtain a cell extract. Next, immunoprecipitation was performed using an anti-Flag antibody, and Keap1 to which Flag-tag was added or its deletion mutant and its interaction complex were recovered. The recovered protein complex and cell extract are developed in a gel by SDS-PAGE (polyacrylamide gel electrophoresis) and immunized with anti-Flag antibody, anti-p62 antibody, anti-Nrf2 antibody, and anti-actin antibody. Blotting (IB) was performed.
The results are shown in FIG. 1 shows the functional domain region of Keap1 and the names and schematic diagrams of Keap1 and each mutant with FLAG-tag attached, and I.I. B. And I. using cell extract. B. It is a photograph of.
At the top of the schematic diagram, the domain region of Keap1 and its name are shown. NTR (N-terminal region), BTB (Broadcomplex, Tramtrac, and Brick-a-Brac), IVR (intervening region), DSG (the double glyceine repeat crea- te). A schematic diagram of a protein to which FLAG-tag has been added and the name are shown on the left. I. Immunoprecipitation of anti-FLAG antibody B. Are shown in IP: FLAG. In addition, I. B. This photograph is represented by Crude, which confirms the expression of Keap1, p62, and actin (loading control) in each cell. I. B. The antibody used is shown on the left side of the photo.
As shown in FIG. 1, when immunoprecipitation was performed on cells expressing Flag-Kap1 (lane 2), endogenous p62 was detected, and it was found that keap1 and p62 form a complex. . When immunoprecipitation was performed with cells expressing each mutant of Keap1 (lanes 3-9), p62 was detected in lane 9 and not detected in lanes 6 and 8, thus forming a complex with p62. It was found that the Keap1 site necessary for this purpose was the DC region (DGR + CTR).
[Example 2]
In Example 2, the direct interaction between p62 and Keap1 and the region of p62 that binds to Keap1 were examined.
First, GST (Glutathione S-transfer) fusion protein with the region Keap1-DC that binds to p62 shown in Example 1 in E. coli, GST-tagged mouse Keap1-DC (GST-Keap1-DC) and mouse p62 or Each p62 mutant MBP (Maltose Binding Protein) fusion protein was produced and purified by affinity chromatography using glutathione-Sepharose 4B resin and amylose resin, respectively. These purified proteins are mixed in TNE buffer and precipitated with amylose resin. The Amyrose resin binding protein was washed 5 times with ice-cold TNE, and stained with SDS-PAGE and CBB (Coomassie brilliant blue).
The results are shown in FIG. FIG. 2 is a photograph of mouse p62 with a functional domain region of p62 and MBP-tag, names and schematics of each mouse, and stained with SDS-PAGE and CBB.
The top part of the schematic diagram shows the domain region of p62 and its name. PB1 (Phox and Bemlp), Zinc (Zinc finger), LRS (LC3-recognition sequence) and UBA (ubiquitin-associated domain). A schematic diagram of the protein to which MBP-tag has been added and the name are shown on the left. MBP immobilized AM resin represents the expression of each protein, and MBP-Pull down assay indicates the binding of GST-Keap1-DC to each p62-MBP fusion protein.
As shown in FIG. 2a, when the purification of each protein was confirmed by the MBP immobilized AM resin diagram, when MBP-Pull down assay was performed, the binding between p62 and Keap1 was confirmed. Was shown to bind directly. In addition, since p62 binding was confirmed in lanes 5, 6, 7, 8, 11, 12, 14 (see FIG. 3 for 14), the binding region of p62 to Keap1 is a region of 345-359 amino acids. I found out. Further, this region is highly conserved among species as shown in FIG. 2b, and this amino acid sequence region was named a KIR (Keap1 interacting region) (Keap1 interaction region) sequence.
[Example 3]
In Example 3, amino acids in the p62-KIR sequence important for Keap1 binding were examined.
A pull-down assay similar to that in Example 2 was performed using a protein in which each amino acid in the -KIR sequence of MBP-p62M80 was substituted with alanine and GST-Keap1-DC.
The results are shown in FIG. The amino acid substitution site for each sample number is shown in the upper left of the figure, the lower part shows a photograph of the gel after staining with SDS-PAGE and CBB, and the right side of the photograph shows the name of the protein indicated by the band.
Since the bands of GST-Keap1-DC are hardly seen in lanes 6, 7, 9, 10, and 11 and the binding is drastically reduced, the binding of p62 to Keap1 is accompanied by D349, P350, It was found that 5 amino acids of T352, G353, and E354 are important.
[Example 4]
In Example 4, it was examined that p62 and Nrf2 were bound in the same pocket of Keap1.
Protein crystallization three-dimensional structure analysis predicted amino acids important for binding of Keap1-DC to p62 or Nrf2. The Keap1-DC mutant and Keap1-DC proteins in which these amino acids were each replaced with alanine were FLAG-tagged and expressed in Hek293T cells. These cells were lysed with a lysis buffer to obtain a cell extract. Next, as in Example 1, immunoprecipitation and I.V. B. Went.
The results are shown in FIG. The left side of FIG. 4 shows each sample number and the amino acid substitution site for that number. I. Immunoprecipitation of anti-FLAG antibody B. Are shown in IP: FLAG. In addition, I. B. These photographs are represented by Crude, which shows confirmation of the expression of Keap1, p62, and actin (loading control) in each cell. I. B. The antibody used is shown on the right side of the photo.
As shown in FIG. 4, binding to p62 is dramatically decreased in Keap1-DC mutants other than R483A (lane 8), and binding to Nrf2 is also dramatically decreased in all mutants including R483A. Thus, it was shown that p62 and Nrf2 are antagonistically bound using the same pocket of Keap1.
[Example 5]
Example 5 shows that p62 competitively inhibits Keap1-Nrf2 binding.
Hek293T cells expressing p62 and p62-T352A in which Flag-tag was linked to the N terminus were lysed with lysis buffer to obtain a cell extract. Next, immunoprecipitation was performed using an anti-Nrf2 antibody, and Nrf2 and its interaction complex were recovered. Next, the recovered protein complex and cell extract are developed in a gel by SDS-PAGE (polyacrylamide gel electrophoresis), and each of the anti-ubiquitin antibody, anti-Nrf2 antibody, anti-p62 antibody, and anti-actin antibody is used. I. B. Went.
The results are shown in FIG. In the case of immunoprecipitation using an anti-Nrf2 antibody, I.V. B. Is shown by IP: Nrf2. In addition, I. B. These photos are shown in Crude. In the upper part of the photograph, FLAG-p62 or FLAG-p62-T352A is overexpressed and the presence or absence of Lactacytin (proteasome inhibitor) is indicated by +-. Also, on the left and right of the figure, B. Shows the name of the antibody used and the name of the detected protein.
In the Nrf2 immunoprecipitation of cells not overexpressing p62 (IP: Nrf2 lane 1), the detection level of ubiquitinated Nrf2 and Nrf2 itself is low, but is significantly higher when a proteasome inhibitor is added (lane 2). Become. This can be explained by the fact that Nrf2 usually binds to keap1 and is ubiquitinated and degraded by the proteasome. When p62 was overexpressed in this state, the amount of Nrf2 protein significantly increased. On the other hand, ubiquitinated Nrf2 decreased (IP: Nrf2 lanes 3 and 4). This indicates that Nrf2 is increased because p62 binds to Keap1 and the binding between Nrf2 and Keap1 decreases and Nrf2 is not ubiquitinated and degraded. In addition, when p62-T352A whose binding ability to Keap1 is significantly reduced is added (IP: Nrf2 lanes 5 and 6), it shows the same state as when p62 is not expressed (IP: Nrf2 lanes 1 and 2). From these results, it is shown that p62 antagonistically inhibits the binding between Keap1 and Nrf2.
[Example 6]
Example 6 shows that binding of Keap1 and p62 increases the expression activity of Nrf2 downstream factor.
A plasmid expressing Nrf2, Keap1, and p62 wild-type or p62 mutants was introduced into Hepa1 cells derived from mouse hepatocellular carcinoma together with pNQO1-ARE reporter plasmid and pRL-TK (internal control of lipofection), and 36 hours later The luciferase activity was measured.
The results are shown in FIG. The presence or absence of Nrf2, Keap1, p62-WT, p62-mutants introduction is indicated by +-, and the relative luciferase activity is shown with no expression vector introduced (column 1) as a standard.
When Nrf2 is overexpressed (column 2), the activity increases significantly, and in the case of double transfection of Nrf2 and Keap1 (column 3), Nrf2 is captured by Keap1 and degraded to the same level as column 1 Decreased. In the triple transfection of Nrf2, Keap1, and p62-WT, p62 binds to Keap1 and free Nrf2 increases, so the luciferase activity increased, but Nrf2, Keap1, p62-mutant triple transfection ( In column 5-10), p62 cannot bind to Keap1, so the activity was not different from column 4. Moreover, since an increase in activity was observed even with transfection of p62-WT alone and not with p62-mutant, it is considered that endogenous Nrf2 was activated by binding to endogenous Keap1. The above results suggest that p62 binding to Keap1 increases due to overexpression of p62, and the activity of Nrf2 increases.
In addition, since there is a large difference in luciferase activity between column 4 and column 5-10, it is expected that the activity is surely reduced when inhibitors of p62 and Keap1 are added in the high activity state in column 4. In addition, since other liver cancer-derived cells originally have high expression levels of p62 and high transcription activity of Nrf2, stable strains in which these cells are stably provided with pNQO1-ARE reporter plasmid are prepared. It was suggested that by performing a luciferase assay, the binding between p62 and keap1 can be monitored, and inhibitors can be searched.
[Example 7]
Example 7 shows that in mice lacking autophagy specifically in the liver, Keap1 accumulates and insolubilizes with p62, and insolubilization of Keap1 is dependent on p62.
The results are shown in FIG. F / F: Mx1 is a conditional knockout mouse for the Atg7 gene. When piperacillin (PIPC) is administered into the abdominal cavity of a mouse, the Atg7 gene is deficient specifically in the liver and autophagy does not function. P62 − / − is a mouse deficient in p62, F / F: Mx1: p62 − / − is a F / F: Mx1 mouse deficient in p62, and F / F is a WT mouse. In FIG. 7a, after administration of PIPC, the liver is collected in sucrose buffer (0.25M sucrose, 10 mM HEPES pH 7.4, 1 mM DTT) and homogenized. The lysate is fractionated into 0.5% Triton X-100 soluble and insoluble. Each fraction is developed by SDS-PAGE and immunoblotted with anti-p62 antibody, anti-Keap1 antibody, anti-LC3 antibody, anti-Nqo1 antibody, and anti-actin antibody (loading control).
The left side of the figure shows the type of antibody used, the upper part shows the number of days after administration, or the genotype of the mouse. Total shown in the lower part of the figure is the whole cell extract, Sol. Is the soluble fraction, Insol. Indicates an insoluble fraction. 8-12 hours after autophagy is lost by PIPC administration, p62 is excessively expressed, but Keap1 expression is hardly changed. Insoluble p62 accumulates with overexpression of p62, and at the same time, insoluble Keap1 accumulates. In addition, 12 hours after PIPC administration of each genotype mouse, overexpression of p62 and increase of insoluble p62 and Keap1 were observed in F / F: Mx1, but it was observed in F / F: Mx1: p62 − / −. No (Figure 7b). From these results, overexpression of p62 and accumulation of insoluble p62 and Keap1 are observed in the liver of mice lacking autophagy. In addition, the accumulation of insoluble keap1 was shown to be p62-dependent.
[Example 8]
In Example 8, the expression state of the downstream factor of Nrf2 in the liver of the genotype mouse used in Example 7 was examined by RT-PCR.
Total RNA is extracted from the mouse liver 12 hours after PIPC administration to each genotype mouse. Using Transscriptor First Strand cDNA Synthesis Kit (Roche Applied Science), cDNA was synthesized from total RNA 1 microg, and LightCycler 480 Probes Masterin (Rochet Cycle Probe Master (Phc). The signal was normalized with β-glucuronidase (GUS). The results are shown in FIG.
The upper part of FIG. 8 shows the gene name, the left part shows the ratio of the amount of mRNA when F / F is 1, and the lower part shows each genotype.
As shown, both Nqo1 and Gstm1 have dramatically increased expression levels in autophagy-deficient mice (F / F: Mx1). From this, it was shown that expression of Nrf2 downstream gene is increased by autophagy deficiency. On the other hand, since mice lacking p62 (p62 and F / F: Mx1: p62) are almost the same as wild type mice (F / F), activation of Nqo1 and Gstm1 due to autophagy deficiency depends on p62. It was shown that
[Example 9]
In Example 9, it is shown in mouse hepatocytes that Keap1 is sequestered in inclusion bodies in a p62-dependent manner.
The liver obtained from a mouse 28 hours after PIPC administration was cut into the genotype mouse shown in Example 7, and the intracellular localization of p62 and Keap1 by double immunostaining using anti-p62 antibody and anti-Keap1 antibody Was observed with fluorescence. The results are shown in FIG.
The upper row in FIG. 9 is p62, the middle row is Keap1, the lower row is a combination of p62 and Keap1 localization (Marge), and the respective genotypes are written in the upper left part of the nuclear staining diagram. F / F: An enlarged view of the area surrounded by the white square frame at the upper right part of Mx1 is shown in the lower left.
As shown, in autophagy-deficient mice (F / F: Mx), p62 and Keap1 shown in white aggregate to form inclusion bodies, and in colocalization observation, p62 and Since the localization of Keap1 is almost the same, it is considered that p62 and Keap1 form the same inclusion body. In addition, when p62 is deleted in autophagy-deficient mice (F / F: Mx: p62 − / −), inclusion body formation including p62 and Keap1 is not observed, and Keap1 positive inclusion body formation contributes to p62 expression. It was shown that it depends.
From the results thus far, it was found that, due to autophagy deficiency, overexpressed p62 binds to Keap1, so that Keap1 is insolubilized and inclusion bodies are formed, and the expression of downstream factors of Nrf2 is increased. In the liver of autophagy-deficient mice, Keap1 is insolubilized as p62 is increased and insolubilized, and Nrf2 that is not trapped by Keap1 increases, which is thought to increase the expression of antioxidant proteins.
[Example 10]
Example 10 shows that Keap1 insolubilization due to autophagy deficiency depends on Nrf2.
Due to autophagy deficiency, Keap1 is insolubilized depending on the expression of p62, and the expression of downstream factors of Nrf2 is also increased. Therefore, in addition to the F / F and F / F: Mx1 shown in Example 7 used so far, Nrf2-deficient mice (Nrf2 − / −, F / F: Mx1: Nrf2 − / −) were prepared. The insolubilization of Keap1 was examined by the same method as in Example 7.
The expression of Nrf2 due to Nrf2 deletion and the expression of Nrf2 downstream genes (Nqo1 and Gstm1) were also confirmed at the same time. The results are shown in FIG.
As the results indicate, the overexpression of p62 and the accumulation of insoluble protein observed in autophagy-deficient mice (F / F: Mx1) were dramatically reduced when Nrf2 was deficient in duplicate. Moreover, since Nrf2 was normally decomposed | disassembled by couple | bonding with Keap1, it turned out that accumulation | storage of p62 capture | acquires and insolubilizes Keap1, leading to the lack of Pathway of Keap1-Nrf2.
[Example 11]
Example 11 shows that the accumulation of p62, Keap1 positive inclusion bodies due to autophagy deficiency is dependent on Nrf2.
Using the genotype mouse of Example 10, immunostaining was performed in the same manner as in Example 9. The results are shown in FIG.
As the results show, the formation of Keap1-positive and p62-positive inclusions seen with autophagy deficiency (F / F: Mx1) is dramatic when Nrf2 is lost (F / F: Mx1: Nrf2 − / −) Decreased. This indicates that p62-positive Keap1-positive inclusion body formation is dependent on Nrf2.
[Example 12]
Example 12 shows that overexpression of Nrf2 downstream gene due to autophagy deficiency is dependent on Nrf2.
Using the genotype mouse of Example 10, RT-PCR analysis of the Nrf2 downstream gene (Nqo1, Gstm1, Cyp2a5) was performed in the same manner as in Example 9. The results are shown in FIG.
As the results show, overexpression of the Nrf2 downstream gene (Nqo1, Gstm1, Cyp2a5) seen in autophagy deficiency (F / F: Mx1) is caused when Nrf2 is deficient (F / F: Mx1: Nrf2− / -) Dramatically decreased. This indicates that overexpression of the Nrf2 downstream gene due to autophagy deficiency is dependent on Nrf2.
From Examples 10, 11, and 12, it was shown that Keap1 insolubilization due to abnormal accumulation of p62 formed p62 positive Keap1 positive inclusion bodies and lacked Nrf2-Keap1 pathway.
When p62 is overexpressed, the ratio of Nrf2 binding to Keap1 decreases, suggesting that Nrf2 accumulates and that the amount of transcription of the downstream gene of Nrf2 increases.
[Example 13]
In order to examine whether the liver damage due to autophagy deficiency is a result of lack of Nrf2-Keap1 pathway due to abnormal accumulation of p62, the genotype mouse of Example 10 was used to measure the weight of the mouse liver 28 days after pIpC administration. Furthermore, hepatocyte hypertrophy of each gene-deficient mouse was measured by HE staining. The results are shown in FIGS. 13a and 13b, respectively.
In autophagy-deficient mice (F / F: Mx1), the weight of the liver is greatly increased and hepatic hypertrophy, whereas autophagy / Nrf2 double-deficient mice do not show an increase. Therefore, Nrf2-Kap1pathway due to abnormal accumulation of p62. It was found that the lack caused liver enlargement.
[Example 14]
In Example 14, in order to examine whether liver damage due to autophagy deficiency is a result of lack of Nrf2-Keap1 pathway due to abnormal accumulation of p62, as an indicator of liver damage, aspartate aminotransferase (AST), alanine aminotransferase (ALT), enzyme activity of alkaline phosphatase (ALP) was measured. The results are shown in FIG.
Autophagy-deficient mice (F / F: Mx1) show dramatic increases compared to wild-type mice (F / F) in all AST, ALT, and ALP, whereas autophagy / Nrf2 double-deficient mice (F / F) : Mx1: Nrf2) showed no increase (at the same level as the wild type), and it was found that lack of Nrf2-Kap1pathway due to abnormal accumulation of p62 caused liver damage.
[Example 15]
Example 15 shows that p62, Keap1 positive inclusion bodies are observed in human hepatocellular carcinoma. Immunohistochemical staining of human hepatocellular carcinoma tissue array (purchased from BioChain Institute, Inc.) using anti-p62 antibody and anti-Kap1 antibody was performed. The results are shown in FIG.
P62-positive inclusion bodies were confirmed in 6 out of 25 human hepatocellular carcinoma samples. More importantly, Keap1 positive inclusion bodies were also confirmed in samples in which p62 positive inclusion bodies were observed. Furthermore, co-localization of p62 and Keap1 was observed with a fluorescence microscope by p62, Keap1 double staining. These results suggest that abnormal regulation of Nrf2-Keap1 pathway is also observed in human hepatocellular carcinoma.
[Example 16]
Example 16 shows the presence of the Nrf2 activity system in p62 accumulation in a cell line stably retaining the ARE-Luciferase gene of hepatoma-derived cells (Hepa-1), and for screening for p62-Keap1 binding inhibitors. Indicates that it can be used. The result of the experiment is shown in FIG.
A cell line that stably holds a plasmid in which the Luciferase gene is linked to ARE, which is the target promoter of Nrf2, is prepared, and p62-WT, p62-T352A (mutant that cannot bind to Keap1) using this adenovirus infection system, Keap1, GFP (control) was overexpressed and luciferase activity was measured. Since Hepa-1 originally has a high activity of Nrf2, it was shown that luciferase activity decreases when Keap1 is overexpressed. When p62-WT or p62-T352A was overexpressed in an overexpressed state of Keap1, luciferase activity increased in p62-WT and no increase was observed in p62-T352A when the infectivity was increased. In addition, the expression of p62-WT alone showed a dramatic increase in luciferase activity, but it was found that the overexpression of p62-T352A alone slightly decreased. This shows that when p62-WT is overexpressed, the ratio of Keap1 binding to p62 increases and Nrf2 becomes activated, and overexpression of p62-T352A slightly decreases the activity due to the dominant negative effect. .
This data indicates the presence of the Nrf2 activity system in p62 accumulation in this cell line and is expected to show reduced luciferase activity when a p62-Keap1 binding inhibitor is added, so p62-Keap1 binding inhibitor Suitable for screening.
[Example 17]
Example 17 shows the molecular state of tumors, Keap1 and Nrf2 downstream factors in the liver of autophagy-deficient mice or autophagy-deficient p62 knockout mice. The results are shown in FIG.
Upper left figure shows Apg7 at 4, 7, 9, 12, 14, 16 months f / f Mouse (control) and Apg7 f / f; It is a photograph of the liver of an Alb-Cre mouse (liver-specific autophagy-deficient mouse). Apg7 f / f; In Alb-Cre mice, small tumors (arrows) were confirmed after 7 months of age, and the number and size of tumors increased with age. Bottom left figure is 12 months old Apg7 f / f (Control) Mouse liver, p62 knockout liver, Apg7 f / f; Liver non-tumor part, liver tumor part and Apg7 of Alb-Cre mice f / f; It is the result of the Western blot analysis which used Alb-Cre; p62 knockout liver extract. Apg7 f / f; P62 was excessively accumulated and insolubilized in the liver non-tumor part and liver tumor part of Alb-Cre mice, and insolubilization of p62-binding protein Keap1 was also remarkable. Along with the insolubilization of p62 and Keap1, the protein amount of Nqo1, the target gene product of Nrf2, was also increased to Apg7. f / f; It increased in the liver non-tumor part and liver tumor part of the Alb-Cre mouse. The insolubilization of Keap1 and the increase in Nqo1 expression were caused by f / f; Alb-Cre; was suppressed in p62 knockout liver. Bottom right is 12 months old Apg7 f / f (Control) Mouse liver, p62 knockout liver, Apg7 f / f; Liver non-tumor part, liver tumor part and Apg7 of Alb-Cre mice f / f; It is the result of quantitative PCR analysis using RNA adjusted from Alb-Cre; p62 knockout liver. Apg7 f / f; In both the liver non-tumor part and liver tumor part of Alb-Cre mice, gene expression of Nqo1 and Gstm1, which are target genes of Nrf2, was increased. These increases in expression are due to Apg7 f / f; Alb-Cre; was suppressed in p62 knockout liver.
These results revealed that Keap1 insolubilization and Nrf2 activation in tumors formed by long-term rearing of autophagy-deficient mice were p62-dependent. This suggests that the pathway of p62-Keap1 is functioning also in in vivo spontaneous liver cancer.
　本発明は、新規な作用機序に基づく肝疾患治療薬および抗がん剤、診断薬またはそのスクリーニング方法などとして有用である。
The present invention is useful as a therapeutic agent for liver diseases and an anticancer agent, a diagnostic agent or a screening method thereof based on a novel mechanism of action.

Claims (14)

  1. Ｋｅａｐ１とｐ６２との結合の阻害または抑制剤。 An inhibitor or inhibitor of binding between Keap1 and p62.
  2. 請求項１に記載の剤であって、
    　ｉ）ｐ６２に特異的に結合する分子、または
    　ｉｉ）ｐ６２の発現阻害物質
    を含有する剤。     The agent according to claim 1,
    i) a molecule that specifically binds to p62, or ii) an agent containing a substance that inhibits expression of p62.
  3. ｉ）ユビキチン陽性封入体形成の阻害又は抑制剤、
    　ｉｉ）肝疾患の治療剤、または
    　ｉｉｉ）抗がん剤
    として使用される、請求項１または２に記載の剤。     i) Inhibitor or inhibitor of ubiquitin positive inclusion body formation,
    The agent according to claim 1 or 2, which is used as a therapeutic agent for ii) liver disease, or iii) an anticancer agent.
  4. 前記ｐ６２に特異的に結合する分子が、
    　ｉ）ｐ６２のＫｅａｐ１相互作用領域に特異的に結合する物質、
    　ｉｉ）マウスｐ６２のアミノ酸配列（配列番号３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、もしくは３５４位のグルタミン酸に特異的に結合する物質、または
    　ｉｉｉ）前記ｐ６２の発現阻害物質が、ｐ６２をコードするヌクレオチド配列に対するアンチセンス核酸もしくはｓｉＲＮＡ
    である、請求項２または３に記載の剤。     A molecule that specifically binds to p62 is
    i) a substance that specifically binds to the Keap1 interaction region of p62;
    ii) a substance that specifically binds to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3); or iii ) An antisense nucleic acid or siRNA against the nucleotide sequence encoding p62 as the p62 expression inhibitor
    The agent according to claim 2 or 3, wherein
  5. 前記ｐ６２に特異的に結合する分子が、ＬＣ３または抗ｐ６２抗体である、請求項２に記載の剤。 The agent according to claim 2, wherein the molecule that specifically binds to p62 is LC3 or an anti-p62 antibody.
  6. 請求項３に記載の肝疾患の治療剤の適用患者を判定する方法であって、
    　ｉ）被験者の肝生検によるｐ６２もしくはＮｒｆ２の発現量を測定する工程、または
    　ｉｉ）被験者の肝生検によるＮｒｆ２の活性を測定する工程、
    を含む、方法。     A method for determining an application patient of the therapeutic agent for liver disease according to claim 3,
    i) measuring the expression level of p62 or Nrf2 by liver biopsy of the subject, or ii) measuring the activity of Nrf2 by liver biopsy of the subject,
    Including a method.
  7. 被験者の血液中のＡＳＴ，ＡＬＴ，ＡＬＰ，またはγ−ＧＴＰの酵素活性を測定することをさらに含む、請求項６に記載の方法。 The method according to claim 6, further comprising measuring enzyme activity of AST, ALT, ALP, or γ-GTP in the blood of the subject.
  8. 請求項６に記載の方法に使用する診断キットまたは診断剤であって、
    　抗ｐ６２抗体または抗Ｎｒｆ２抗体を含み、
    　前記キットはさらに使用説明書
    を含む、キットまたは剤。     A diagnostic kit or diagnostic agent for use in the method according to claim 6, comprising:
    An anti-p62 antibody or an anti-Nrf2 antibody,
    The kit further includes instructions for use.
  9. Ｋｅａｐ１とｐ６２との相互作用を阻害または抑制する物質（ａｇｅｎｔ）のスクリーニング方法であって、
    　ｉ）試験物質の存在下および非存在下でＫｅａｐ１とｐ６２との結合量を測定する工程、および
    　ｉｉ）前記試験物質の存在下と非存在下との間でＫｅａｐ１とｐ６２との前記結合量を比較し、前記試験物質の存在下での前記結合量が前記試験物質の非存在下での前記結合量よりも低い場合に、当該試験物質をＫｅａｐ１とｐ６２との相互作用を阻害または抑制する物質として選択する工程
    を含む、方法。     A method for screening an agent that inhibits or suppresses the interaction between Keap1 and p62,
    i) measuring the amount of Keap1 binding to p62 in the presence and absence of the test substance; and ii) determining the amount of Keap1 binding to p62 between the presence of the test substance and the absence of the test substance. In comparison, when the binding amount in the presence of the test substance is lower than the binding amount in the absence of the test substance, the test substance inhibits or suppresses the interaction between Keap1 and p62 A method comprising the step of selecting as
  10. 前記結合量を測定する工程において、プルダウン法、質量分析法、タンパク質間相互作用を蛍光シグナルとして検出する方法、または蛍光標識を用いたイメージングのいずれかが用いられる、請求項９に記載の方法。 The method according to claim 9, wherein in the step of measuring the amount of binding, any one of a pull-down method, mass spectrometry, a method of detecting protein-protein interaction as a fluorescence signal, or imaging using a fluorescent label is used.
  11. ユビキチン陽性封入体形成を阻害または抑制する物質のスクリーニングのために使用する、請求項９または１０に記載の方法。 The method of Claim 9 or 10 used for the screening of the substance which inhibits or suppresses ubiquitin positive inclusion body formation.
  12. 肝疾患の治療剤のスクリーニングのために使用する、請求項９~１１のいずれかに記載の方法。 The method according to any one of claims 9 to 11, which is used for screening for a therapeutic agent for liver disease.
  13. 前記結合量を測定する工程が、
    　ｉ）ｐ６２のＫｅａｐ１相互作用領域、または
    　ｉｉ）マウスｐ６２のアミノ酸配列（配列番号：３）中の３４５番から３５９番目のアミノ酸配列：ＳＫＥＶＤＰＳＴＧＥＬＱＳＬＱ（配列番号：５）
    に対するＫｅａｐ１の結合量を測定することを含む、請求項９~１２のいずれかに記載の方法。     The step of measuring the amount of binding,
    i) Keap1 interaction region of p62, or ii) amino acid sequence from position 345 to position 359 in the amino acid sequence of mouse p62 (SEQ ID NO: 3): SKEVDPSTGELQSLQ (SEQ ID NO: 5)
    The method according to any one of claims 9 to 12, which comprises measuring the amount of Keap1 bound to.
  14. 前記物質が、マウスｐ６２のアミノ酸配列（配列番号：３）中の３４９位のアスパラギン酸、３５０位のプロリン、３５２位のスレオニン、３５３位のグリシン、または３５４位のグルタミン酸に特異的に結合する、請求項９~１３のいずれかに記載の方法。 The substance specifically binds to aspartic acid at position 349, proline at position 350, threonine at position 352, glycine at position 353, or glutamic acid at position 354 in the amino acid sequence of mouse p62 (SEQ ID NO: 3). The method according to any one of claims 9 to 13.
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