WO2011083637A1 - Therapeutic agent for hepatic disorder - Google Patents

Abstract

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

Description

Liver disease treatment

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. 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.

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.

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.

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.

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 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. 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. 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. 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. 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) 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. It is a figure which shows the result of the quantitative real-time PCR analysis of Nqo1 and Gstm1 in a mouse liver. 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. 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. It is a figure which shows the result of the immunofluorescence analysis of the intracellular localization of p62 and Keap1. 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) 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. It is a figure which shows the result of the liver function test of the mouse | mouth used in FIG. 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. 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. 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.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 (K_a) 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.

[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.

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. An inhibitor or inhibitor of binding between Keap1 and p62.
2. 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) 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. 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. The agent according to claim 2, 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 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. 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. 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. 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. 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. The method of Claim 9 or 10 used for the screening of the substance which inhibits or suppresses ubiquitin positive inclusion body formation.
12. The method according to any one of claims 9 to 11, which is used for screening for a therapeutic agent for liver disease.
13. 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. 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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