JP7429413B2 - Protein that enhances activation of Wnt/β-catenin pathway - Google Patents

Description

The present invention relates to a mutant of the human IRAK1 protein that has the effect of enhancing activation of the Wnt/β-catenin pathway. The present invention also relates to a method of screening for a preventive or therapeutic agent for fibrosis or tumor in tissues or organs. More specifically, it relates to a method for screening for a preventive or therapeutic agent for tissue or organ fibrosis or tumor, which includes a step of selecting a substance that inhibits the binding of human IRAK1 to Axin or GSK3β, or a substance that binds to human IRAK1. .

Chronic inflammation increases the risk of tissue or organ fibrosis and tumorigenesis. In fact, it is known that interstitial pneumonia is frequently associated with alveolar fibrosis and lung tumors, and ulcerative colitis is also associated with a high incidence of colon tumors. However, at present, the molecular mechanisms of fibrosis and carcinogenesis induced by chronic inflammation are unknown, and treatments have not been established for most cases, and the identification of molecular targets, which are essential for preventive and therapeutic strategies, remains unclear. desired.

Chronic inflammation is involved in tumor development and is one of the various signaling pathways in tumor cells that trigger the expression of inflammatory molecules, including the pathway that activates Interleukin-1 receptor-associated kinase (IRAK) (TLR/ IL-1R pathway). The IRAK1 family consists of four family members (IRAK1, IRAK2, IRAK3, and IRAK4). Among them, IRAK1 has been reported to have enhanced phosphorylation and increased expression in various tumor types, and is reported to be a factor that promotes tumor growth, but its action is due to the TLR/IL-1R pathway, downstream factors NFκB, It was thought that this is an abnormality in signal transmission to JUN and p38 MAPK (eg, Non-Patent Documents 1 to 3).

Singer J.W. et al., Oncotarget, 9(70):33416-33439 (2018) Jain A. et al., Front Immunol, 5:553 (2014) Rhyasen G.W. and Starczynowski D.T., br j cancer, 112:232-237 (2015)

An object of the present invention is to provide an IRAK1 mutant that has an activation-enhancing effect on the Wnt/β-catenin pathway, and an animal model of tissue or organ fibrosis or tumor that has the mutant. The present invention also provides a method for screening a therapeutic drug, in which a test substance that inhibits activation of the Wnt/β-catenin pathway by human IRAK1 is used as a candidate drug for preventing or treating fibrosis or tumor in tissues or organs. The task is to do so.

In order to investigate the function of IRAK1 (Interleukin-1 receptor-associated kinase1) in vivo, the present inventors microinjected human IRAK1 mRNA into early Xenopus embryos and found that the activation of the Wnt/β-catenin pathway was detected. We found that the posteriorization of the early embryo (specific deletion of the head structure), which is noticeable during development, was observed (Figure 2). IRAK1 is a Toll-like receptor/IL-1 receptor (TLR/IL-1R) pathway factor, and is involved in abnormalities in signal transduction to NFκB, a downstream factor of the TLR/IL-1R pathway. The above findings were surprising because it is thought to enhance Wnt/β-catenin pathway and its relationship with the Wnt/β-catenin pathway was unknown. Therefore, in order to investigate the relationship between IRAK1 and the Wnt/β-catenin pathway in more detail, we established a variety of human cell lines that allow us to examine the presence or absence of activation of the Wnt/β-catenin pathway (Figure 3). By using these cell lines, it was confirmed that the activation of the Wnt/β-catenin pathway by IRAK1 observed in Xenopus was also observed in human cells (Figure 4). Further research revealed that compared to wild-type IRAK1, using a kinase activity-deficient strain of IRAK1 (KD mutant), activation of the Wnt/β-catenin pathway was significantly enhanced, while IRAK1 In the N-terminus-deficient strain, activation of the Wnt/β-catenin pathway disappears, and protein modification of the PPPSP motif within the N-terminus causes activation of the Wnt/β-catenin pathway and activation of the Wnt/β-catenin pathway. We have discovered that this is necessary for the interaction with Axin1 and GSK3β, which are inhibitory complex factors, and have completed the present invention.

That is, the present invention is as follows.
[1]
The following protein (a) or (b).
(a) Protein having a mutation in at least one amino acid selected from the group consisting of valine at position 412, lysine at position 239, threonine at position 209, glutamine at position 421, and glycine at position 289 of SEQ ID NO: 2 (b) In the protein of (a), the amino acid sequence has one or more amino acids deleted, substituted, inserted, and/or added (provided that the mutated amino acid residue of (a) is replaced with a wild-type amino acid residue) , and excluding the substitution of serine with another amino acid residue corresponding to position 173 of SEQ ID NO: 2), and has an activation-enhancing effect on the Wnt/β-catenin pathway [2]
A nucleic acid encoding the protein according to [1].
[3]
A cell containing the nucleic acid according to [2].
[4]
A non-human mammal or a part of its living body that expresses the nucleic acid according to [2] and exhibits a tissue or organ fibrosis or tumor phenotype.
[5]
The animal or part of its living body according to [4], wherein the mammal is a rodent.
[6]
(1) A step of contacting the test substance with the cells described in [3], or the animal or part of its living body described in [4] or [5]; and (2) the treatment of fibrosis or tumor in tissues or organs. A method for screening for a preventive or therapeutic drug for tissue or organ fibrosis or tumor, comprising the step of selecting a test substance with improved phenotype.
[7]
(1) A step in which the test substance is brought into contact with human IRAK1 and Axin or GSK3β; and (2) In step (1), the substance that inhibits the binding between human IRAK1 and Axin or GSK3β is applied to tissue or organ fibers. 1. A method for screening for a prophylactic or therapeutic agent for tissue or organ fibrosis or tumor, the method comprising the step of selecting as a candidate for a prophylactic or therapeutic agent for fibrosis or tumor.
[8]
(1) The step of bringing the test substance into contact with human IRAK1,
(2) Prevention of tissue or organ fibrosis or tumor, including a step in step (1) of selecting a test substance that binds to human IRAK1 as a candidate for a preventive or therapeutic agent for tissue or organ fibrosis or tumor. or screening methods for therapeutic drugs.
[9]
The method according to [7] or [8], wherein the tissue or organ is selected from lung, large intestine, brain, ovary, prostate and breast.

According to the present invention, there is provided a method for screening a therapeutic drug, which uses a test substance that inhibits activation of the Wnt/β-catenin pathway by human IRAK1 as a candidate drug for preventing or treating tissue or organ fibrosis or tumor. be done. Also provided are IRAK1 mutants that have the effect of enhancing activation of the Wnt/β-catenin pathway, and model animals that exhibit fibrosis or tumor phenotypes in tissues or organs, which contain the mutants.

Figure 1 shows a schematic diagram of the IL-1 receptor signaling pathway and the Wnt/β-catenin pathway. It is known that the IL-1 receptor signal pathway induces inflammatory and immune responses, and the Wnt/β-catenin pathway causes tissue or organ fibrosis and tumors. Figure 2 shows that when human IRAK1 mRNA was expressed in the expected head region of early Xenopus laevis embryos by microinjection, the axial posteriorization phenotype, which is characteristic when the Wnt/β-catenin pathway is activated, was observed. It shows. Figure 3 shows a schematic diagram of the TOPFLASH reporter assay using lentivirus. It has smaller experimental errors than transient assays using transfection methods and can be applied to a wider variety of human cells. Figure 4 shows the results of activation of the Wnt/β-catenin pathway in cultured cell lines by human IRAK1. Lung tumor cell line (H157 , H522 , H1299), colorectal tumor cell line (RKO), glioma cell line (U251 , U-87MG ), ovarian tumor cell line (Ovca420), prostate tumor cell line (Du145), breast tumor IRAK1 activates the reporter gene in the cell line (MDA-MB231) and mammary epithelial cell line (MCF10A), but the IRAK1 kinase activity-deficient strain (KD mutant) activates the reporter gene more strongly. The one into which GFP was introduced was used as a negative control, and the luciferase level in this control was set to 1. Figure 5 shows the structure and characteristics of human IRAK family proteins. The death domain is a domain required for multimerization between IRAKs, and is highly conserved among the IRAK family together with the kinase domain. IRAK4 phosphorylates and activates IRAK1. IRAK1 self-phosphorylates to control its activity and stability. The kinase domains in IRAK2 and IRAK3 have been suggested to be pseudokinases. * indicates the position of lysine, which is the center of conserved kinase activity. Figure 6 shows the results of activation of the Wnt/β-catenin pathway by the human IRAK family. Among the IRAK family, only IRAK1 activates TOPFLASH, 2) increases the expression of Axin2 mRNA, 3) increases the expression of Axin2 protein, and 4) increases the expression of active (non-phosphorylated) β-catenin. increase, cause. The one into which GFP was introduced was used as a negative control, and the luciferase activity and Axin2 mRNA level in this control were set to 1. FIG. 7 shows the results of an analysis using Kaplan-Meier plotter of the relationship between the expression level (low or high) of human IRAK family transcripts (mRNA) and overall survival rate. Among the IRAK family, only IRAK1 was shown to be a factor that worsens the prognosis of human lung glandular tumors. Figure 8 shows that human IRAK1 (hIRAK1) activates the Wnt/β-catenin pathway in the mouse NIH3T3 cell line, and mouse IRAK1 (mIRAK1) does not have similar activity in the human lung cancer H1299 cell line. FIG. 9 shows a schematic diagram of known post-translational modification sites and point mutation sites of human IRAK1. FIG. 10 shows the results of simultaneous analysis of Wnt/β-catenin activation by expression of a point mutant of human IRAK1 using Western blot and TOPFLASH reporter assay. The left panel shows the results of a cell line in which the mutant shown in Figure 7 was expressed in H1299T/R cells, and the middle panel shows the results obtained by introducing point mutations (W71D, R76D, S131A, S144A, S173A) into the KD mutant. Cell line results are shown. The figure on the right shows the results of comparing two active center mutants, K239A and K239Q. The one into which GFP was introduced was used as a negative control, and the luciferase activity in this control was set to 1. Figure 11 shows a summary of the expression of point mutants of human IRAK1 and Wnt/β-catenin pathway activity. FIG. 12 shows the results of comparing the Wnt/β-catenin pathway activity between the KD mutant and V412M mutant and wild-type IRAK1. Even when the KD mutant or V412M mutant, which has the same level of expression as wild-type IRAK1, is expressed in cells, the activity of TOPFLASH is higher than that of wild-type IRAK1, indicating that these mutants are hyperactive forms of β-catenin activation ( It is considered to be an activated (activated) IRAK1 mutant. FIG. 13 shows a schematic diagram of the binding between LPR5/6, a Wnt-coupled receptor, and β-catenin inhibitory complex factors. LPR5/6 has a PPPSP motif similar to human IRAK1 at its C-terminus, and is known to bind to β-catenin inhibitory complex factors Axin1 and GSK3β through phosphorylation of PPPSP that is dependent on Wnt ligand binding. It is being Figure 14 shows the binding ability of human IRAK1 to Axin1 and GSK3β. The KD mutant binds to Axin1 and GSK3β, which are components of the β-catenin repressive complex. Death domain deletion (ΔD), P-S/T domain deletion (ΔPS/T), and/or S173A point mutation within the PPPSP motif reduce or eliminate binding ability. It is known that ΔD and ΔPS/T do not have the ability to activate β-catenin. Figure 15 shows the results of activation of the Wnt/β-catenin pathway by cytokines (IL-1β or Wnt3A) in cell lines in which human IRAK1 was knocked down. IRAK1 is not required for TOPFLASH activation by Wnt3A. IRAK1 is required for activation of TOPFLASH by IL1β, although its protein amount is decreased by IL1β. A negative control was prepared by introducing siRNA (Csi) that does not target IRAK1, and the luciferase activity in this control was set to 1. FIG. 16 shows a hypothesis of disease progression due to activation of the IRAK1/β-catenin pathway. According to this hypothesis, (1) normal production and stimulation of IL-1 occurs due to infiltration of inflammatory cells, and (2) constitutive activation of the Wnt/β-catenin pathway occurs due to high expression or mutation of IRAK1. (3) EMT, fibrosis, promotion of proliferation, accumulation of genetic mutations, and promotion of tumorigenesis. FIG. 17 shows an outline of an example of producing a transgenic mouse expressing human active IRAK1.

1. Method for screening drugs for preventing or treating tissue or organ fibrosis or tumor As shown in the Examples below, human IRAK1 activates the Wnt/β-catenin pathway, and this activation involves the use of the PPPSP motif of IRAK1. was shown to require interaction with Axin1 and/or GSK3β, which are inhibitory complex factors of the Wnt/β-catenin pathway, upon modification of this motif. Therefore, if a test substance inhibits the activation of the Wnt/β-catenin pathway by IRAK1, it will suppress the activation of the Wnt/β-catenin pathway and thereby inhibit tissue or organ fibrosis or tumors caused by the activation. Can be prevented or treated. Therefore, the present invention provides a method for treating tissue or organ fibrosis, which includes the step of selecting a test substance that inhibits activation of the Wnt/β-catenin pathway by IRAK1 as a candidate for a preventive or therapeutic drug for tissue or organ fibrosis or tumor. The present invention provides a screening method for a prophylactic or therapeutic agent for a disease or tumor (hereinafter sometimes referred to as "the screening method of the present invention"). As used herein, a prophylactic or therapeutic agent for tissue or organ fibrosis or tumor refers to a substance that has a prophylactic or therapeutic activity for tissue or organ fibrosis or tumor.

In one embodiment, the screening method of the present invention includes (1) contacting a test substance, IRAK1, and Axin or GSK3β, and (2) inhibiting the binding between IRAK1 and Axin or GSK3β in step (1). The method may include the step of selecting the substance as a candidate for a prophylactic or therapeutic drug for tissue or organ fibrosis or tumor. In addition, a substance that binds to IRAK1, preferably a substance that binds to the PPPSP motif, can inhibit the binding of IRAK1 to Axin and/or GSK3β. ') A step of bringing the test substance into contact with IRAK1, and (2') In step (1'), the test substance that binds to IRAK1 is selected as a candidate for a prophylactic or therapeutic drug for tissue or organ fibrosis or tumor. The method may include the step of: Whether or not it binds to the PPPSP motif is determined, for example, by IRAK1 in which part (particularly, the serine residue) or the entire motif is deleted or substituted with other amino acid residues (e.g., alanine residue or glycine residue). This can be confirmed through comparative experiments.

It may be verified whether the substances selected by the screening method of the present invention inhibit the activation of the Wnt/β-catenin pathway. Methods known per se or assays in which reporter expression increases in response to Wnt/β-catenin activation (e.g., TOPFlash/FOPFlash luciferase assay system) (e.g., Li, X. et al., Chem. . Asian J. 4:540-547 (2009), etc.).

As used herein, "tissue or organ fibrosis" refers to a state in which extracellular matrix such as fibronectin or collagen fibers is significantly deposited in the tissue or organ, or where collagen fibers are significantly increased and cause scar tissue. This refers to a state in which fibrous connective tissue increases. Tissues with fibrosis or tumors include, for example, the brain, tongue, pharynx, skin, esophagus, lung, breast, stomach, pancreas, liver, gallbladder, bile duct, small intestine, large intestine, kidney, bladder, prostate, uterus, ovary, Examples include blood vessels and bone marrow, but lungs, large intestines, brains, ovaries, prostates, and breasts are preferred, and lungs, brains, ovaries, and prostates are more preferred. Therefore, examples of tissue or organ fibrosis that can be prevented or treated with drugs for prevention or treatment of tissue or organ fibrosis include fibrosis in endometriosis or uterine adenomyosis, chronic obstructive pulmonary disease ( COPD), cystic fibrosis, psoriasis, and liver fibrosis (e.g., viral hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease (NAFLD), and primary biliary cirrhosis (PBC)). liver cirrhosis, etc.), renal fibrosis (e.g., chronic kidney disease caused by chronic inflammation such as chronic nephritis or diabetes), pulmonary fibrosis (e.g., chronic obstructive pulmonary disease (COPD), pulmonary fibrosis accompanied by emphysema) (CPFE), idiopathic interstitial pneumonia (IIPs), idiopathic pulmonary fibrosis, etc.).

Furthermore, in this specification, the term "tumor" includes benign tumors, malignant tumors (also referred to as "cancer"), and tumors that can be diagnosed or determined to be benign or malignant. More specifically, tumors targeted for prevention or treatment with tumor prevention or treatment drugs include, for example, liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., ovarian clear cell adenocarcinoma), and childhood cancer. , lung cancer (e.g. squamous cell carcinoma, small cell lung cancer), testicular cancer (e.g. non-seminoma germ cell tumor), soft tissue tumors (e.g. liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g. cervical melanoma, adrenal tumors (e.g. adrenal adenoma), neurological tumors (e.g. Schwannoma), gastric cancer (e.g. adenocarcinoma of the stomach), renal cancer (e.g. Witz tumor), breast cancer (e.g. invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g. medullary carcinoma), laryngeal cancer (e.g. squamous cell carcinoma), bladder cancer (e.g. invasive transitional cell carcinoma) Examples include, but are not limited to, the following.

As used herein, a preventive or therapeutic agent for tissue or organ fibrosis refers to an agent capable of suppressing the accumulation of fibers in tissues and/or an increase in already accumulated fibers, and preferably inhibits the accumulation of fibers and and/or agents capable of reducing accumulated fibers. In addition, a tumor preventive or therapeutic drug means a drug that can suppress tumor formation and/or growth of an already formed tumor, and preferably a drug that has tumor formation inhibiting and/or tumor regression effects. be.

As used herein, human IRAK1 is intended to include fragments thereof. Such human IRAK1 includes, for example, a protein consisting of the amino acid sequence shown by SEQ ID NO: 2, and fragments of IRAK1 are not particularly limited as long as they can bind to Axin and/or GSK3β, but PPPSP Examples include fragments of IRAK1 containing motifs. Further, as a nucleic acid encoding human IRAK1, for example, a nucleic acid consisting of the nucleotide sequence shown by SEQ ID NO: 1 can be mentioned. Such a sequence has been published as Genebank Accession No: NM_001569. As used herein, the PPPSP motif of IRAK1 means a motif consisting of proline-proline-proline-serine-proline at positions 170 to 174 of the amino acid sequence shown in SEQ ID NO: 2, and is shown in the examples below. As shown, human IRAK1 forms a complex with Axin1 and GSK3β via this motif. Note that in this specification, the amino acid positions of IRAK1 are expressed based on the amino acid sequence shown in SEQ ID NO: 2. Furthermore, as described below, instead of IRAK1, mutant IRAK1, which was newly discovered by the present inventors and has an effect of enhancing activation of the Wnt/β-catenin pathway, may be used. For specific examples of such mutant IRAK1, see 2. below. As described in . Therefore, as used herein, human IRAK1 is intended to include mutant IRAK1 and fragments thereof.

In this specification, Axin and GSK3β include fragments thereof. Examples of such Axin or GSK3β and the nucleic acids encoding them include sequences published as Genebank Accession Nos: NM_003502 and NM_002093. The fragment of Axin or GSK3β is not particularly limited as long as it is a fragment that can bind to IRAK1.

The length of the above protein fragment is not particularly limited, but examples include lengths of 20 amino acids or more (e.g., 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000 amino acids or more). .

The above IRAK1, Axin, and GSK3β can be produced by known methods. Examples of such production methods include solid phase synthesis methods such as Fmoc synthesis method and Boc synthesis method, and liquid phase synthesis methods such as fragment condensation method.However, from the viewpoint of ease of operation, solid phase synthesis method law is preferred. Examples of the solid phase synthesis method include, but are not limited to, the method of R.B. Merrifield (J. Am. Chem. Soc., 1963, 85, p.2149-2154). Protein synthesis may be performed automatically or manually using commercially available protein synthesis equipment. It may be isolated from cells or tissues expressing IRAK1 by a method known per se. Alternatively, it may be produced using genetic engineering techniques. Such genetic engineering techniques include, for example, introducing an expression vector incorporating a nucleic acid encoding a peptide containing the above amino acid sequence into an appropriate host cell (e.g., mammalian cell, insect cell, Escherichia coli, etc.); Examples include a method in which the peptide is expressed in a host cell, then extracted and purified to obtain the peptide, and a method in which the protein is synthesized in a cell-free system. From the viewpoint of preserving protein modification, methods using host cells are desirable. In addition, proteins or parts thereof (e.g., PPPSP motif, known modification sites (e.g., W71, R76, S131, S144, S173, T209, K239), etc.) synthesized by the above synthesis method or cell-free system, etc. Appropriate modifications (e.g., phosphorylation, acylation, acetylation, alkylation, amidation, glycosylation, SUMOylation, ubiquitination, cis/trans isomerization conversion in proline, etc.) may be performed by known methods. .

The inhibitory effect of the test substance on the binding between human IRAK1 and Axin or GSK3β can be evaluated by methods known per se, including test tube-based methods and cell-based (in vitro) methods. Good too. In the case of using a test tube-based method, for example, immunoprecipitation method, Alpha (Amplified Luminescent Proximity Homogeneous Assay) method, Fluoppi system, FRET method, etc. can be used. For example, the immunoprecipitation method uses a carrier (e.g., beads) carrying an anti-IRAK1 antibody (if a tag (e.g., His tag, GST tag, FLAG (trademark) tag, etc.) is added to IRAK1, an antibody against the tag). etc.), IRAK1, Axin and/or GSK3β, and the test substance are mixed in the same solution, and after incubation, the amount of Axin or GSK3β obtained by the step of collecting the protein captured on the carrier is Decreased compared to the amount of protein obtained without contacting the substance or the amount of Axin or GSK3β obtained by contacting with a test substance that does not have an inhibitory effect on the binding of human IRAK1 to Axin or GSK3β. In this case, the test substance can be evaluated as having an inhibitory effect on the binding between human IRAK1 and Axin or GSK3β. Instead of a carrier carrying an anti-IRAK1 antibody or an antibody against a tag, a carrier carrying an anti-IRAK1 antibody or an antibody against a tag that is not supported on a carrier, and a secondary antibody against the antibody or Protein A or Protein G is used. Good too. The Alpha method and the Fluoppi system can be carried out using kits provided by, for example, PerkinElmer and according to the manufacturer's instructions. In this case, a test substance whose binding property between human IRAK1 and Axin or GSK3β is reduced upon contact can be evaluated as a substance that inhibits the binding.

Alternatively, evaluation may be made based on the 50% inhibitory concentration (IC50) of the binding between human IRAK1 and Axin or GSK3. IC50 can be determined by a method known per se (eg, functional antagonist test, competitive binding test, etc.). The above method may be performed using a kit (eg, AlphaLISA immunoassay kit, etc.). In a preferred embodiment, when the IC50 in a competitive binding test is 10 μM or less, it can be evaluated as having the above-mentioned inhibitory effect, and the lower the IC50 value, the higher the inhibitory effect. The IC50 of the test substance selected by the screening method of the present invention is preferably 10 μM or less, more preferably 5.0 μM or less, even more preferably 1.0 μM or less, even more preferably 0.5 μM or less, and especially 0.3 μM or less. Preferably, 0.2 μM or less is most preferable.

In addition, when performing in vitro, in addition to the above-mentioned Alpha (Amplified Luminescent Proximity Homogeneous Assay) method, Fluoppi system, and FRET method applied to cells, the binding of human IRAK1 and Axin or GSK3β in cells A test substance may be evaluated using a system (eg, yeast two-hybrid method) in which the expression level of the reporter gene changes accordingly, using the expression level of the reporter gene as an index.

As used herein, "binding" (also referred to as "having binding property") between IRAK1 and a test substance refers to the extent to which interaction can occur between the two substances when they are brought into contact. means to be in close proximity to each other or to be in a state of association. Examples of such interactions include covalent bonds, coordinate bonds, ionic bonds, hydrogen bonds, van der Waals bonds, and hydrophobic interactions.

The presence or absence of binding between IRAK1 and a test substance can be determined by measuring binding activity or affinity indicators. Examples of the binding affinity index include a dissociation constant (K _D ) and a binding constant (also referred to as an association constant), which is the reciprocal thereof. The dissociation constant can be measured by a method known per se, such as a method using surface plasmon resonance, an isothermal titration calorimetry method, and the like.

Examples of systems for measuring and calculating surface plasmon resonance include, but are not limited to, the Biacore system (GE Healthcare). When using the Biacore system, for example, K _D can be measured by the following procedure. (1) Immobilizing IRAK1 on the sensor chip of the Biacore system by amine coupling, etc.; (2) bringing IRAK1 into contact with the test substance in a solution containing the test substance prepared at multiple concentrations; (3) ) detecting the interaction between the two by surface plasmon resonance; (4) depicting a series of binding and dissociation reactions as a sensorgram with time on the horizontal axis and binding amount (RU) on the vertical axis; (5) each Using software (e.g. BIAevaluation software (GE Healthcare), etc.), fit the sensorgram created at the concentration to a 1:1 Langmuir model to calculate various rate parameters; (6) Dissociation from the various rate parameters. Calculate the constant.

Examples of systems used in the isothermal titration calorimetry method include, but are not limited to, the MicroCal system (GE Healthcare). When using the MicroCal system, for example, K _D can be measured by the following procedure. (1) A solution containing the test substance is titrated and stirred into a sample cell containing IRAK1, which is kept at a constant temperature. (2) Heat generation or absorption occurs in direct proportion to the amount of binding due to intermolecular interactions, and the sample cell The temperature of the solution inside changes. (3) The cell feedback network (CFB) senses the temperature difference (ΔT) with the reference cell. (4) The reference cell or sample cell is heated so that ΔT becomes 0. (5) Obtain the amount of heat generated or absorbed by the interaction by measuring the feedback power required to maintain ΔT=0. (6) Calculate the molar ratio of IRAK1 and the test substance using the amount of heat generated as the vertical axis. The dissociation constant is calculated from the binding isotherm on the horizontal axis.

In this specification, when measured using surface plasmon resonance, if the K _D is 1500 nM or less, it can be evaluated that it has binding property to IRAK1, and the lower the K _D value, the higher the binding property to IRAK1. It can be evaluated that the bonding property is high. K _D is preferably 1000 nM or less, more preferably 500 nM or less, even more preferably 300 nM or less, and particularly preferably 100 nM or less.

Alternatively, interactions between substances can be verified using the above-mentioned immunoprecipitation method, Alpha (Amplified Luminescent Proximity Homogeneous Assay) method, Fluoppi system, FRET method, methods using reporter genes, etc. The binding property between the test substance and IRAK1 may be evaluated using this method.

Test substances used in the screening method of the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified proteins or crude proteins, peptides, non-peptide compounds, Examples include synthetic low-molecular compounds and natural compounds.

The test substance can also be used in (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography selection. Biological library methods using affinity chromatography selection are limited to peptide libraries, but the other four approaches can be applied to peptides, non-peptide oligomers, or small molecule libraries of compounds (Lam (1997) ) Anticancer Drug Des. 12:145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem . 37:1233-51). Compound libraries can be prepared in solution (see Houghten (1992) Bio/Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4) or chips (Fodor (1993) Nature 364:555- 6), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA Natl. Acad. Sci. USA 87 :6378-82; Felici (1991) J. Mol. Biol. 222:301-10; US Patent Application No. 2002103360).

2. A protein that has an activation-enhancing effect on the Wnt/β-catenin pathway The present invention provides a novel protein that has an activation-enhancing effect on the Wnt/β-catenin pathway (hereinafter also referred to as "mutant IRAK1 of the present invention"), and A nucleic acid encoding a protein (hereinafter also referred to as "mutant IRAK1 gene of the present invention") is provided. The mutant IRAK1 of the present invention is the following (a) or (b).
(a) Having a mutation in at least one amino acid selected from the group consisting of valine at position 412 of SEQ ID NO: 2, lysine at position 239 of SEQ ID NO: 2, threonine at position 209, glutamine at position 421, and glycine at position 289 Protein (b) An amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and/or added in the protein (a) (however, wild-type amino acid residues of the mutated amino acid residues in (a)) (excluding substitution of serine corresponding to position 173 of SEQ ID NO: 2 with other amino acid residues) and having an activation-enhancing effect on the Wnt/β-catenin pathway.

As used herein, "Wnt/β-catenin pathway" means the following pathway. When Wnt binds to receptors (Frizzled) and coupled receptors (LRP5/6) on the cell membrane, it further binds to Axin and GSK-3β (Glycogen synthase kinase-3β), which are β-catenin degrading complexes, and -Suppresses catenin phosphorylation. Unphosphorylated β-catenin that has escaped degradation translocates into the nucleus, where it cooperates with the transcription factor TCF protein to induce the expression of target genes and promote cell proliferation and differentiation. In the absence of Wnt, β-catenin in the cytoplasm binds to Axin together with APC (adenomatous polyposis coli) and GSK-3β, and in the β-catenin degradation complex (Axin complex), CKI-1α (casein kinase Iα) ) and GSK-3β, β-catenin is further ubiquitinated and degraded by the proteasome, so the amount of β-catenin in cells is maintained at a low level. Furthermore, activation of the Wnt/β-catenin pathway means that β-catenin translocates into the nucleus and causes target genes to be expressed.

As used herein, the Wnt/β-catenin pathway activation-enhancing effect means that the Wnt/β-catenin pathway is activated at a level higher than that activated by wild-type IRAK1. In addition, the activation-enhancing effect of the Wnt/β-catenin pathway promotes fibrosis or tumorigenesis of tissues or organs. It may be read as "accelerating effect of organ fibrosis or tumorigenesis" as appropriate.

The mutants that enhanced activation of the Wnt/β-catenin pathway in the examples below were all mutations in the kinase domain, which strongly suggests that the function of the kinase domain suppresses β-catenin activation. be done. Therefore, regarding (a) above, substitutions to any amino acid residue are not limited to mutations and are not limited to substitutions to specific amino acid residues, as long as they reduce or eliminate the kinase activity compared to the wild type. Substitution of a specific amino acid residue in the mutant IRAK1 of the present invention may be a substitution with any amino acid residue, since the kinase activity may be reduced or eliminated by substitution. In fact, as shown in the Examples below (FIG. 10), the K239Q mutant, which mimics acetylation, also had the same activation-enhancing effect on the Wnt/β-catenin pathway as the K239A mutant. In a preferred embodiment, substitution of valine at position 412 of SEQ ID NO: 2 with methionine, substitution of lysine at position 239 of SEQ ID NO: 2 with alanine or glutamine, substitution of threonine at position 209 with alanine, substitution of glutamine at position 421 with histidine. Examples include, but are not limited to, variants in which a substitution has been introduced, and/or a substitution of glycine at position 289 with valine. Without wishing to be bound by any theory, the present inventors' idea regarding β-catenin activation is that amino acid mutations that cause decreased activity of IRAK1 kinase or structural changes in the kinase domain may cause activation of the kinase. It is speculated that when the function of IRAK1 is impaired, mutant IRAK1 is stabilized and its expression level increases, leading to activation of β-catenin.

In addition, mutants with multiple mutations in the kinase domain were shown to enhance activation of the Wnt/β-catenin pathway compared to mutants with only one mutation. Therefore, from the viewpoint of activation of the Wnt/β-catenin pathway, the mutant IRAK1 of the present invention is preferably a mutant having multiple mutations in the kinase domain. A group in which lysine is substituted with another amino acid residue (e.g., alanine, glycine, glutamine, etc.) and consisting of valine at position 412, threonine at position 209, glutamine at position 421, and glycine at position 289 of SEQ ID NO: 2. A protein having a mutation in at least one amino acid selected from the following is preferred, and among these, a mutant in which lysine at position 239 and valine at position 412 of SEQ ID NO: 2 are substituted with other amino acid residues is more preferred.

Regarding (b) above, more specifically, 1 to 70 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 30 amino acids, even more preferably 1 to 30 amino acids in the amino acid sequence of the protein of (i)(a). (ii) an amino acid sequence in the protein of (a), in which 20, and even more preferably one to several (10, 9, 8, 7, 6, 5, 4, 3 or 2) amino acids are deleted; 1 to 70 pieces, preferably 1 to 50 pieces, more preferably 1 to 30 pieces, even more preferably 1 to 20 pieces, even more preferably 1 to several (10, 9, 8, 7, 6, 5, 4 , 3 or 2) amino acid sequence, (iii) 1 to 70, preferably 1 to 50, more preferably 1 to 30, even more preferably 1 to the amino acid sequence in the protein of (a). an amino acid sequence in which ~20, even more preferably 1 to several (10, 9, 8, 7, 6, 5, 4, 3 or 2) amino acids have been inserted or added; (iv) the protein of (a); 1 to 70, preferably 1 to 50, more preferably 1 to 30, even more preferably 1 to 20, even more preferably 1 to several (10, 9, 8, 7, 6 , 5, 4, 3, or 2) amino acids are replaced with other amino acids, or (v) a combination thereof. However, (a) substitution of a mutated amino acid residue with a wild-type amino acid residue is excluded. For example, if the protein (a) is one in which lysine at position 239 of SEQ ID NO: 2 is substituted with another amino acid residue, the amino acid residue corresponding to the amino acid residue at position 239 is substituted with lysine. is excluded. The same applies to other mutation locations.
Furthermore, from the viewpoint of binding ability to Axin and/or GSK3β, mutations that result in substitution of the PPPSP motif, deletion of the Death domain, and/or deletion of the PS/T domain are not introduced into the mutant IRAK1 of the present invention. It is preferable.

Alternatively, variants of the protein (a) can also be defined by amino acid sequence identity, for example, 90% or more (e.g. 90%, 91%, 92%, 93%) with the amino acid sequence of (a). , 94%, 95%, 96%, 97%, 98%, 99% or more) identity (provided that the mutated amino acid residue in (a) is replaced by the wild-type amino acid residue) , and excluding the substitution of serine corresponding to position 173 of SEQ ID NO: 2 with another amino acid residue). Amino acid sequence identity was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) (https ://blast.ncbi.nlm.nih.gov/Blast.cgi).

Furthermore, substitutions between amino acids with similar properties (for example, glycine and alanine, valine, leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) If so, there may be a larger number of replacements, etc. When an amino acid is deleted, substituted, inserted, or added as described above, the position of the deletion, substitution, insertion, or addition is particularly determined as long as the activation-enhancing effect of the Wnt/β-catenin pathway is maintained. Not limited. The mutation can be introduced by a method known per se, such as site-directed mutagenesis, alanine scanning, or PCR-based mutagenesis.

The mutant IRAK1 of the present invention can be obtained from the above-mentioned 1. It can be produced in the same manner as the method for producing IRAK1, Axin, or GSK3β described in . However, it is more convenient to clone normal IRAK1 cDNA from human cells that have a normal IRAK1 gene without mutations using the same method as above, and then mutate it at the desired position using a well-known site-directed mutagenesis method. It can be obtained by introducing .

The mutant IRAK1 gene of the present invention is not particularly limited as long as it is a nucleic acid containing a nucleotide sequence encoding the mutant IRAK1 having the effect of enhancing activation of the Wnt/β-catenin pathway. The nucleic acid may be double-stranded DNA, single-stranded DNA, single-stranded RNA, double-stranded RNA, or DNA/RNA hybrid, but is preferably double-stranded DNA or single-stranded RNA. , more preferably double-stranded DNA. The mutant IRAK1 gene of the present invention can be used by being linked to a vector (cloning vector or expression vector). Furthermore, depending on the purpose (eg, protein expression), a vector suitable for the host cell into which the vector is introduced can be selected as appropriate, and the vector can be transformed into the host cell. The vector can further contain an appropriate promoter, a terminator region, a selection marker gene for selecting transformants (a drug resistance gene, a gene that complements an auxotrophic mutation, etc.), and the like.

In addition, the vector may contain a sequence encoding a tag sequence useful for protein separation and purification (for example, His tag, GST tag, FLAG (trademark) tag, etc.), and the vector may contain It may be integrated into the genome of

Types of vectors include, but are not limited to, pBTrp2, pBTac1, pBTac2, pBluescript II SK(+), pBluescript II SK(-), pSTV28, pUC118, pUC19, pKK233-2, pSE280, pSupex, pUB110, pTP5, pC194. , pET vector, pAGE vector, pcDNA vector, pGEX vector, pMC1neo, λgt11, pkk223-3, pBlue BacIII, pVL1392, pVL1393, and the like.

Host cells include any cell capable of expressing the vector, such as bacterial cells, fungal cells, insect cells, animal cells, and the like.
Examples of bacterial cells include, but are not limited to, Escherichia coli, Bacillus, Brevibacillus, Corynebacterium, Streptomyces, and the like. Examples of fungal cells include, but are not limited to, Saccharomyces cerevisiae, Aspergillus fungi, and the like. Examples of insect cells include, but are not limited to, Bombyx mori, Trichoplusia ni, and the like. Examples of animal cells include, but are not limited to, cell lines such as CHO cells, HeLa cells, COS-1 cells, COS-7 cells, HEK293 cells, BALL-1 cells, HCT-15 cells, and PC12 cells, and dorsal root ganglia. (DRG) Primary culture of neurons, ES cells, iPS cells, etc.

Vectors can be introduced into host cells using known transformation methods, such as competent cell method, protoplast method, calcium phosphate coprecipitation method, electroporation method, microinjection method, lipofection method, etc., depending on each host cell. It can be done using

When using transformed cells for the purpose of purifying mutant IRAK1, it is preferable to use cells that do not express the endogenous IRAK1 gene as host cells to facilitate purification, but even when using animal cells, human and Since there are some differences in the amino acid sequence of IRAK1 between mice, rats, etc., for example, when recombinantly producing a human-derived mutant IRAK1 protein, it is necessary to use mammalian cells other than primates such as mice or rats. You can also use Hereinafter, the thus obtained transformed cells may be referred to as model cells of the present invention.

The above-mentioned transformed cells can be cultured by methods known per se. The medium used for culturing can be appropriately selected for each desired cell. For example, for E. coli culture, LB medium, etc., for yeast culture, SD medium, etc., and for mammal culture, DMEM medium, EMEM medium, etc. can be used. Furthermore, the culture medium (semi-solid medium, liquid medium, powder medium) may contain various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, etc.), as necessary. Amino acids (including natural amino acids or synthetic amino acids), nucleobases (purines, pyrimidines), inorganic salts (MgSO4, MnSO4, FeSO4, NaCl, etc.), antibiotics (ampicillin, kanamycin, hygromycin), etc. can be added.

Culture time, culture temperature, etc. can be appropriately determined by those skilled in the art depending on the type of cells. For example, the culture temperature can be set to 10 to 45°C, preferably 20 to 40°C, and the culture time can be set to 1 hour to 14 days, preferably 10 hours to 7 days. The pH can also be adjusted as appropriate.

The mutant IRAK1 obtained by the above method can be purified by a method known per se. For example, after microbial cells collected by centrifugation are ground with ultrasound or glass beads, solids such as cell pieces are removed by centrifugation to prepare a crude enzyme solution, and then ammonium sulfate, sulfuric acid, etc. Salting out methods using sodium or the like, chromatographic methods such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography, gel electrophoresis methods, and the like can be used.

3. Model animals for tissue or organ fibrosis or tumor The mutant IRAK1 encoded by the mutant IRAK1 gene of the present invention promotes Wnt/β-catenin pathway activation and tissue or organ fibrosis or tumorigenesis in mammals. Since it has an enhancing effect, an animal model for tissue or organ fibrosis or tumor can be provided by introducing a nucleic acid encoding the mutant IRAK1 into non-human mammalian cells and producing a transgenic animal. Therefore, the present invention also provides transgenic (hereinafter also referred to as Tg) non-human mammals (hereinafter also referred to as "Tg") that exogenously express the mutant IRAK1 of the present invention and exhibit tissue or organ fibrotic or tumor phenotypes. (also referred to as "animal model for invention").

The Tg animals of the present invention include fertilized eggs, unfertilized eggs, sperm and their precursor cells (primordial germ cells, oogonia, oocytes, egg cells, spermatogonia, spermatocytes, spermatids) of non-human mammals. etc.), preferably at an early stage of embryonic development of a fertilized egg (more preferably before the 8-cell stage), calcium phosphate coprecipitation, electroporation, lipofection, agglutination, microinjection (microinjection), etc. It is produced by introducing the mutant IRAK1 gene of the present invention by a gene introduction method such as injection) method, gene gun (particle tumor) method, or DEAE-dextran method.
One embodiment of using a virus as an expression vector is a method of infecting early embryos or ES cells of non-human mammals with a virus containing DNA encoding mutant IRAK1 (for example, Proc. Natl. Acad. Sci. USA, 99(4): 2140-2145, 2002). For example, when using retroviruses or lentiviruses, cells are seeded in a suitable incubator such as a dish, a viral vector is added to the culture medium, and after culturing for 1 to 2 days, if the embryo is an early embryo, the recipient is pseudopregnant. The embryo is transplanted into the oviduct or uterus of a female non-human mammal, and if it is an ES cell, a selection agent such as G418 or hygromycin is added and culture is continued to select cells into which the vector has been integrated.
Furthermore, spermatogonia harvested from male non-human mammals were incubated with viral vectors during co-culture with STO feeder cells, as described in Proc. Natl. Acad. Sci. USA, 98: 13090-13095 (2001). To efficiently obtain heterozygous Tg (+/-) offspring with an exogenously mutated IRAK1 gene by injecting it into the seminiferous tubules of a male infertile non-human mammal and mating it with a female non-human mammal. I can do it.

Alternatively, Tg animals can also be produced using genome editing (eg, TALEN, CRISPR/Cas system, etc.). Specifically, for example, a nucleic acid encoding CRISPR-Cas9, a guide RNA targeting any region of the genome or the IRAK1 gene, and donor DNA into which a mutation has been introduced in advance are introduced, and the donor DNA and genomic DNA are introduced. Mutations can be introduced into genes by homologous recombination between them. By transplanting the fertilized egg thus obtained into the uterus of a pseudopregnant female animal, it is possible to obtain a mutant IRAK1 gene heterozygous Tg (+/-) offspring.

Furthermore, the Tg animal may be a knock-in (KI) animal in which the endogenous IRAK1 gene is replaced with a sequence encoding a mutant IRAK1. Therefore, the Tg animal of the present invention can be obtained by introducing the target DNA into ES or iPS cells using an appropriate targeting vector, and replacing the endogenous IRAK1 gene with a sequence encoding a mutant IRAK1 through homologous recombination. can also be made. Alternatively, mutant IRAK1 can be knocked into the genome of a fertilized egg by genome editing.

In the above nucleic acid to be incorporated into the genome, for example, a sequence encoding another protein such as a marker protein is incorporated upstream of the sequence encoding IRAK1, a STOP codon is incorporated at the end of the sequence, and a sequence encoding the other protein is added. By including sequences recognized by site-specific recombinase (e.g., Cre, FLP, R, Gin) (e.g., loxP sequence, FRT sequence, RS sequence, gix sequence, respectively) before and after the IRAK1 can also be expressed specifically in cells or tissues in which site-specific recombinant enzymes are expressed. By operably linking a sequence encoding the enzyme to a promoter specific to a particular cell or tissue, the enzyme can be expressed specifically in the cell or tissue. In addition, sequences encoding drug-responsive receptors, such as a modified version of the estrogen receptor (ERT2), are linked before and after the sequence encoding the enzyme, so that the site-specific recombinant enzyme translocates into the nucleus at a specific timing. You may let them.

Examples of prostate-specific promoters include prostate-specific antigen (PSA) promoter and its mutant ΔPSA, ARR2PB and probasin (PB) promoters, gp91-phox gene promoter, prostate-specific kallikrein (hKLK2) promoter, etc. can be mentioned.
Examples of liver-specific promoters include liver albumin promoter, α-fetoprotein promoter, α1-antitrypsin promoter, transferrin-transthyretin promoter, and the like.
Examples of colon-specific promoters include carbonic anhydrase I promoter, carcinoembryonic antigen promoter, and villin promoter.
Examples of ovary- or placenta-specific promoters include estrogen-responsive promoters, aromatase cytochrome P450 promoters, cholesterol side chain cleavage P450 promoters, and 17α-hydroxylase P450 promoters.
Breast-specific promoters include, for example, G. I. Examples include erb-B2 promoter, erb-B3 promoter, β-casein, β-lacto-globulin, WAB (whey acidic protein) promoter, and the like.
Examples of lung-specific promoters include surfactant protein C (SFTPC) promoter.
Examples of skin-specific promoters include K-14-keratin promoter, human keratin 1 or 6 promoter, and roicrin promoter.
Examples of brain-specific promoters include glial fibrillary acidic protein promoter, mature astrocyte-specific protein promoter, myelin promoter, tyrosine hydroxylase promoter, and GFAP promoter.
Examples of pancreatic-specific promoters include villin promoter, glucagon promoter, and insulin islet amyloid polypeptide (amylin) promoter.
Examples of thyroid-specific promoters include thyroglobulin promoter and calcitonin promoter.
Examples of kidney-specific promoters include renin promoter, liver/bone/kidney alkaline phosphatase promoter, erythropoietin (epo) promoter, and the like.

The present invention also provides a living body part of a model animal obtained by the above-described method and uses thereof. For example, blood may be collected from the model animal and blood or a preparation thereof may be used, or a portion of various tissues of the model may be collected and preparations such as tissue pieces or cultured cells may be used. These preparations can be carried out by methods known per se. Cells to be prepared include fibroblasts, myofibroblasts, dendritic cells, keratinocytes, heart cells, esophageal cells, muscle cells, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, and macrophages. , monocytes, osteocytes, bone marrow cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, etc., but are not limited to these, and may be prepared as appropriate depending on the purpose. can. Tissues to be prepared include skin, esophagus, lung, stomach, pancreas, liver, gallbladder, bile duct, small intestine, large intestine, kidney, bladder, prostate, uterus, ovary, blood vessel, bone marrow, brain, tongue, pharynx, etc. However, it is not limited to these, and can be appropriately prepared depending on the purpose. For example, in cells collected from the model animal of the present invention, activation of the Wnt/β-catenin pathway may be enhanced, and collagen deposition may be enhanced as in vivo.

As shown in the Examples below, when human IRAK1 was introduced into mouse cells and early Xenopus embryos, it was shown to have the ability to enhance Wnt/β-catenin activation in the cells and early embryos. Ta. Therefore, human IRAK1 may have the ability to enhance Wnt/β-catenin activation in a wide variety of animal species, including Xenopus, mice, and humans. The animals that can be targeted in the present invention are preferably non-human mammals, and the non-human mammal (recipient animal) is not particularly limited as long as it is a non-human mammal for which a transgenic system has been established. Examples include mice, rats, cows, monkeys, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, and hamsters. Preferably, from the viewpoint of producing disease model animals, rodent animals are more preferable as they have a relatively short ontogeny and biological cycle and are easy to breed. Preferred strains include FVB strain, DBA2 strain, etc., crossbred strains such as B6C3F1 strain, BDF1 strain, B6D2F1 strain, ICR strain, etc.), and rats (eg, Wistar, SD, etc.).

To date, the above-mentioned disease model animals are necessary to develop useful strategies for prevention and treatment of tissue fibrosis, tumor formation, and malignant transformation of tumors induced by chronic inflammation, but there are very few reports on such model animals. few. Since the model animal of the present invention can serve as a model animal for the above-mentioned pathological conditions, it can be used for prevention of the above-mentioned diseases, verification of treatment, elucidation of the molecular mechanisms of fibrosis and carcinogenesis, and the like.

4. Screening method using a model animal or a part of its living body Furthermore, the present invention also includes a screening method using a model animal or a part of its living body. The method is not particularly limited, but includes, for example, contacting a tissue or transgenic non-human mammal expressing the mutant IRAK1 with a test substance and not contacting the tissue or animal with the test substance. Examples include a method of measuring and comparing phenotypic changes in tissues and organs, and selecting test substances that improve the phenotype of tissue or organ fibrosis or tumors as candidates for preventive or therapeutic agents for tissue or organ fibrosis or tumors. It will be done.

In one embodiment, the above screening method includes (1) contacting a test substance with the model cells of the present invention, or the model animal of the present invention or a part of its living body, and (2) fibrosis or fibrosis of tissues or organs. It may include the step of selecting test substances that have improved the tumor phenotype.

Contact with the test substance can be carried out by adding the test substance to a culture medium or buffer solution in the case of cells, tissues, or organs, or by administering the test substance orally or parenterally in the case of transgenic non-human mammals. This can be done by administering. The amount added to the culture medium and the amount administered to animals can be appropriately selected depending on the test substance. Specific examples of test substances include 1. above. The same items as those described above are listed.

In the case of a fibrotic phenotype, the improvement of the above-mentioned phenotype can include, for example, fibrosis, thickening, collagen deposition, collagen I, and β-catenin expression in cells, tissues, or organs. In animals, in addition to the above, for example, alleviation of symptoms of tissue or organ fibrosis can be evaluated as an indicator. In addition, tumor phenotypes include, for example, abnormal cell proliferation, abnormal cell adhesion, abnormal movement, abnormal cell death, changes in angiogenesis induction, etc. In animals, in addition to the above, For example, alleviation of tumor symptoms can be evaluated as an index. Symptoms of fibrosis or tumor of the tissue or organ include, for example, in the case of pulmonary fibrosis, shortness of breath felt during exertion, cough without sputum, etc. In the case of lung tumor, in addition to the above, , difficulty breathing, weight loss, phlegm, and bloody sputum.

The prophylactic or therapeutic drug for tissue or organ fibrosis or tumor obtained by the above method is formulated by using the substance as an active ingredient and adding excipients, binders, disintegrants, lubricants, etc. It can also be used as a preventive or therapeutic agent for diseases or tumors.

5. A method for diagnosing fibrosis or tumor in a tissue or an organ, or a method for assisting the diagnosis In yet another embodiment, the present invention provides a method for diagnosing fibrosis or tumor in a tissue or organ, in which the p.G289V mutation, p.V412M mutation, and/or Q421H mutation ( If the subject is a non-human mammal, a mutation in which the amino acid residue corresponding to glycine at position 289 of human IRAK1 is substituted with another amino acid, preferably valine, or a mutation in which the amino acid residue corresponding to glycine at position 289 of human IRAK1 is substituted with valine at position 412 of human IRAK1 Mutations in which the corresponding amino acid residue is substituted with another amino acid, preferably methionine, or mutations in which the amino acid residue corresponding to glutamine at position 421 of human IRAK1 is substituted with another amino acid, preferably histidine. diagnosis of fibrosis or tumor in a tissue or organ in the subject, or an auxiliary method for the diagnosis (hereinafter, these may be collectively referred to as the "diagnostic method of the present invention, etc.") ) is also provided. As used herein, "assisting the diagnosis of tissue or organ fibrosis or tumor" refers to whether the subject is suffering from or is likely to suffer from tissue or organ fibrosis or tumor in the future. It refers to the provision of information that serves as an indicator for judgment, and refers to the process itself of determining whether or not a person is suffering from tissue or organ fibrosis or tumor, or is likely to suffer from it in the future, which is a medical procedure. means not included.

The types of tissue or organ fibrosis or tumor targeted by the diagnostic method of the present invention include the above-mentioned 1. The same items as those listed above are listed. Subjects to be diagnosed by the present invention include mammals (eg, humans, monkeys, cows, pigs, dogs, cats, mice, rats, etc.), but humans are preferred. Preferably, such a mammal exhibits symptoms of tissue or organ fibrosis or tumor, or is likely to be suffering from the disease.

The biological sample used in the diagnostic method of the present invention is not particularly limited as long as it is a sample collected from a subject, and examples include blood, plasma, serum, saliva, urine, skin, nails, hair, bone marrow fluid, oral mucosa, Examples include organizations. Preferably blood, plasma, serum or saliva. These samples can be obtained by methods known per se. For example, serum and plasma can be prepared by collecting blood from a subject and separating the liquid components according to a conventional method. DNA in a sample can be obtained by extracting genomic DNA from the sample; for example, genomic DNA can be isolated from blood collected from a subject through normal blood collection using a phenol extraction method or the like. At that time, commercially available genomic DNA extraction kits and devices such as QIAmp Circulating Nucleic Acid Kit may be used.

Detection of mutations in the IRAK1 gene can be carried out, for example, by amplifying the region of the IRAK1 gene by PCR using a primer set capable of amplifying the region containing the codon encoding p.G289, p.V412 or p.Q421, Mutations can be detected by determining the base sequence of the obtained amplification product by a method known per se. Examples of methods for determining such base sequences include the Sanger method and a method using a next generation sequencer (NGS). For methods using next-generation sequencing, for example, reference can be made to Experimental Medicine Special Edition "Next Generation Sequence Analysis Standards", 2014 (Yodosha), etc. NGS used for this purpose include Illumina instruments (e.g. MiSeq, HiSeq2500), Life Technologies instruments (e.g. Ion Proton, Ion PGM), and Roche Diagnostics. Examples include, but are not limited to, devices manufactured by Roche Diagnostic (eg, GS FLX+, GS Junior). For example, in the case of humans, the primer set can be a primer that has 15 or more consecutive nucleic acids having the base sequence shown in SEQ ID NO: 1 and can amplify the region.

6. Methods for diagnosing and treating fibrosis or tumors in tissues or organs The present invention also provides methods for diagnosing and treating fibrosis or tumors in tissues or organs. As mentioned above, based on the information obtained by the diagnostic method of the present invention, anti-inflammatory drugs are administered to subjects who are judged to be suffering from tissue or organ fibrosis or tumor, or who are likely to suffer from tissue or organ fibrosis or tumor in the future. Both fibrotic or anti-tumor therapy can be performed. Such therapies include surgery, radiotherapy, administration of anti-fibrosis drugs or anti-tumor drugs (chemotherapeutic drugs, other anti-tumor drugs), gene therapy, or combination therapy thereof.

Such antifibrotic agents include, for example, pantethine (D-bis-(N-pantothenyl-β-aminoethyl)-disulfide) (US Pat. No. 4,937,266), benzoylhydrazine (US Pat. No. 5,374,660 and US Pat. No. 5,571,846). ), combinations of angiotensin inhibitors and nitric oxide stimulants (US Pat. Nos. 5,645,839 and 6,139,847), A1 adenosine receptor antagonists and/or P2x purinergic receptor antagonists (US Pat. No. 6,117,445), somatostatin Examples include agonists, hepatocyte growth factor (HGF), chymase inhibitors, and IL-13 antagonists (US Pat. No. 6,268,342, US Pat. No. 6,303,126, US Pat. No. 6,500,835, and US Pat. No. 6,664,227).

Examples of antitumor drugs include acivicin, aclarubicin, acodazole, acronin, adzelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, trioxide Arsenic, asparaginase, asperline, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisanthrene, bisnafide dimesylate, vizerecin, bleomycin, brequinal, bropyrimine, busulfan, cactinomycin, carsterone, capecitabine, carasemide, carbetimer, carboplatin, Carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, sirolemycin, cisplatin, cladribine, cristnatole mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate , diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate, eflomitin, elsamitrucin, enloplatin, enpromate, and the like.

The above-mentioned anti-fibrotic drugs or anti-tumor drugs can be administered orally as a pharmaceutical composition in an appropriate dosage form by using the active ingredients alone or by mixing them with pharmaceutically acceptable carriers, excipients, diluents, etc. It may also be administered parenterally. Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Examples include formulations, emulsions, suspensions, and the like. On the other hand, as compositions for parenteral administration, for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may also be included. Furthermore, the dosage of the therapeutic agent can be appropriately determined depending on various conditions such as the type of compound, the symptoms of the subject to be administered, age, body weight, and drug acceptability.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[Example 1] Introduction of human IRAK1 mRNA and Wnt8 DNA into early Xenopus embryos Human IRAK1 mRNA or Wnt8 DNA was introduced into the expected head region of an early Xenopus embryo (4-cell stage) using a microinjection method. was injected to express IRAK1 protein or Wnt8 protein in the embryo.

As a result, all embryos that expressed human IRAK1 protein or Wnt8 protein exhibited a posterior axial axial shift phenotype, which is an indicator of activation of the Wnt/β-catenin pathway. That is, it was shown that human IRAK1 enhances the activation of the Wnt/β-catenin pathway (FIG. 2).

[Example 2] Construction of an assay for evaluating the activation of the Wnt/β-catenin pathway Based on the results of Reference Example 1 above, whether human IRAK1 can enhance the activation of the Wnt/β-catenin pathway even in human cells. I looked into it. First, lentiviral reporter genes were constructed by incorporating the Super TOPFLASH firefly luciferase reporter gene and the standardization-like Renilla reporter gene into lentiviral vectors, which can measure the activation of the Wnt/β-catenin pathway. By introducing these two reporter genes using the lentivirus method and selecting drugs, it has become possible to examine the presence or absence of these two reporter genes in a variety of human cell lines.

Various human cell lines (TOPFLASH/Renilla reporter-introduced cells: T/R cells) into which a reporter gene has been introduced, as well as human IRAK1 wild type (WT), human IRAK1 kinase activity-deficient mutant (KD, kinase dead), and mouse Wnt1 Two days later, a dual luciferase assay was performed using the Promega Dual-Glo Luciferase assay system to measure firefly luciferase activity, which was normalized to Renilla luciferase activity, which is a measure of the number of viable cells. (Figure 3). As a result, lung cancer cell lines (H157T/R , H 522T/R, H1299T/R), colorectal cancer cell lines RKOT/R, glioma cell lines ( U251T/R , U-87 MGT/R) , and ovarian cancer cells were found. IRAK1 activates the reporter gene in strain Ovca420T/R, prostate cancer cell line Du145T/R, breast cancer cell line MDA-MB231T/R, and mammary epithelial cell line MCF10AT/R, compared with negative control GFP as 1. In particular, the KD mutant showed stronger activity than wild-type IRAK1 (Figure 4).

[Example 3] Verification of activation of Wnt/β-catenin pathway in IRAK family
IRAK proteins belong to the IRAK1,2,3,4 family and are known to be involved in Toll-like receptor/IL-1 receptor-NFκB signaling. Its structure includes a death domain and a kinase domain that are highly conserved among families, and the death domain is said to be necessary for multimer formation between MyD88 and IRAK family members and for signal transmission to NFκB. However, regarding the Kinase domain, other than that IRAK4 phosphorylates IRAK1 and transmits the signal downstream, and that IRAK1 phosphorylates itself and regulates its activity and stability, there are no significant differences, including the presence or absence of kinase activity. There are many unknowns and many debates. In addition to these domains, there is a Proline-Serine/Threonine-rich region that undergoes phosphorylation and ubiquitination, and a C-terminal domain that has a TRAF6 binding site, which is a ubiquitin ligase, but there is no conservation among the families. low (Figure 5).

Based on the results shown in Figure 4, we investigated whether other IRAK proteins, along with IRAK1, also have the ability to activate the Wnt/β-catenin pathway by reporter assay, Western blotting, and quantitative RT-PCR. Using the same method as in Figure 3, wild-type IRAK protein (IRAK WT) and its kinase activity-deficient mutant (IRAK KD) were added to the human lung cancer cell line H1299T/R, and mouse Wnt1 (mWnt1) and Xenopus stable β were added as positive controls. -Catenin (Xβ-cateninSA) was expressed in the same amount, and Western blotting, reporter assay, and quantitative RT-PCR were performed using the same samples to examine the activation of the Wnt/β-catenin pathway. Note that proteins other than mWnt1, which is a secretory factor, have a flag label added to their N-terminus. As a result, among the IRAK family proteins, only IRAK1 1) activates TOPFLASH, 2) increases the mRNA expression level of the endogenous target gene Axin2, 3) increases the protein amount of Axin2, and 4) activates (inactive) It was revealed that phosphorylation caused an increase in β-catenin (Fig. 6).

Since activation of the Wnt/β-catenin pathway by IRAK1 was observed in multiple human lung cancer cell lines, we conducted a Kaplan study using a clinical database to investigate the relationship between IRAK family mRNA expression and prognosis in human lung adenocarcinoma. -Meier plotter (http://kmplot.com/analysis/index.php?p=background) revealed that only IRAK1 expression was a prognostic factor (Figure 7). As it has been previously reported that activation of the Wnt/β-catenin pathway is a factor that worsens the prognosis of lung cancer, this analysis result suggests that only IRAK1 can activate the Wnt/β-catenin pathway. This is considered to indicate that there is a response.

The main protein modifications and mutation analyzes of IRAK1 known so far include the following (Figure 9 ).
-W71 and R76 in the death domain are essential for multimer formation and downstream NFκB signaling through the death domain.
-S131, S144, and T173 phosphorylation sites within the -Proline-Serine/Threonine-rich region are phosphorylated by their own kinase activity, and are suggested to be involved in downstream NFκB signal transduction.
-The T209 site within the kinase domain is a phosphorylation site by IRAK4 located upstream and is essential for NFκB signaling.
-The K239 site within the kinase domain is the active center of the kinase and uses the above phosphorylation site within the Proline-Serine/Threonine-rich region as a substrate, but it is controversial whether the kinase activity is necessary for NFκB signal transduction. Divided (there are also recent reports that it is suppressive).
-Somatic mutations within the kinase domain, such as G289V (salivary gland cancer), V412M (glioma), and Q421H (breast cancer), have been reported in human cancer tissues, but their contribution to activity is unknown. be.

[Example 4] Verification of activation of the Wnt/β-catenin pathway by human IRAK1 using other mammalian cells Next, using mouse cells, we demonstrated that human IRAK1 activates the Wnt/β-catenin pathway. We investigated whether it enhances Using the human lung cancer cell line H1299 cells and mouse NIH3T cells, it was verified in the same manner as in Example 3 whether human IRAK1 or mouse IRAK1 has a Wnt/β-catenin activation-enhancing effect.

The results are shown in FIG. Human IRAK1 (hIRAK1) enhanced activation of the Wnt/β-catenin pathway in the mouse NIH3T3 cell line, but mouse IRAK1 (mIRAK1) did not enhance activation of the Wnt/β-catenin pathway.

[Example 5] Expression of point mutants of human IRAK1 and verification of Wnt/β-catenin pathway activity
In order to investigate the mechanism of action of IRAK1 on the Wnt/β-catenin pathway, we first analyzed the above-mentioned IRAK1 protein modification sites, point mutant function analysis, and somatic mutations that have been reported so far. Similar point mutants for lentivirus expression were created, and their physiological activities and expression patterns for the Wnt/β-catenin pathway were analyzed (FIG. 10).

The conclusions obtained from FIG. 10 are shown below (FIG. 11).
-Wild-type IRAK1 has relatively low protein expression compared to other mutants, has a slower electrophoretic mobility than expected from the protein's primary structure ( L(LOW), approximately 75 KDa), and an apparent molecular weight of Since it is approximately 120KDa (H(HIGH)), it is thought that it is highly post-translationally modified.
-The function of the death domain, which is essential for multimer formation, is required for Wnt/β-catenin pathway activity and advanced post-translational modification.
Many mutations within the kinase domain, such as -T209A, K239A, V412M, and Q421H, increase protein expression and Wnt/β-catenin pathway activity.
Somatic mutations discovered in glioma and breast cancer, such as -V412M and Q421H, enhance Wnt/β-catenin pathway activity.

Compared to wild-type IRAK1, the V412M and KD mutations, which show a marked increase in Wnt/β-catenin pathway activity, also have increased protein expression, indicating that they express the same amount of protein as the wild type. The activity was compared. As a result, both mutations had stronger Wnt/β-catenin pathway activity than wild-type IRAK1, suggesting that the ability to activate the Wnt/β-catenin pathway was increased not only quantitatively but also qualitatively. (Figure 12).

[Example 6] Verification of binding site with β-catenin inhibitory complex constituent factors
It was found that the Wnt/β-catenin activation ability of the KD mutant was dependent on the S173 site (Figures 10 and 11), so when we focused on the amino acid sequence near the S173 site, we found that the PPPSP sequence was found. It was done. Multiple sequences called PPPS/TP motifs exist at the C-terminus of LRP5/6, which is a coupled receptor for Wnt ligands, and when Wnt ligands bind to LRP5/6, they are phosphorylated and inhibit β-catenins such as Axin and GSK3β. It was known that β-catenin is stabilized by binding to complex constituent factors (FIG. 13). Interestingly, this motif is not conserved in other IRAK family members, nor is it present in mouse or Xenopus IRAK1, and was found to be unique to human IRAK1.

Therefore, based on the characteristics of this PPPSP, we investigated the binding of the Flag-labeled KD mutant, which shows the most ability to activate the Wnt/β-catenin pathway, with Axin and GSK3β by immunoprecipitation using anti-FLAG M2 antibody affinity beads and anti-Axin1 antibody ( CST) and Western blot analysis using anti-GSK3β antibody (CST). Since the function of the Death domain and the S173 site within the P-S/T domain are important for the ability to activate the Wnt/β-catenin pathway, we have added the KD mutant to the KD mutant, which has Death domain deficiency, and the P-S/T domain deletion. An IRAK1 mutant with deletion and S173A point mutation was added. As a result, it was shown that the KD mutant binds to endogenous Axin1 and GSK3β proteins, and in other mutants, the binding was attenuated or abolished (FIG. 14). This suggests that the function of the Death domain and the S173 site within the P-S/T domain are important for the binding of human IRAK1 to Axin and GSK3β, the ability to activate the Wnt/β-catenin pathway, and post-translational modification. .

[Example 7] Verification of upstream factors of IRAK1 by TOPFLASH assay From the above, it became clear that protein expression of IRAK1 is sufficient for activation of the Wnt/β-catenin pathway.Next, the necessity was investigated. Using the experimental system shown in Figure 3, we have shown that in the lung cancer cell line H1299T/R, Wnt3A, a ligand for the Wnt/β-catenin pathway, activates the Wnt/β-catenin pathway, and in the cell line U- 87 In MG/TR , the inflammatory cytokine IL1β, which is an upstream ligand of IRAK1, was found to activate the Wnt/β-catenin pathway, so we investigated whether IRAK1 is required in the activation mechanism by these cytokines. investigated. Two types of human IRAK1siRNA were introduced into the cells of H1299T/R and H358hpT/R using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and cultured for 2 days, and recombinant human Wnt3A and IL1β were added to each cell at 5 ng/ml. After culturing for an additional 1 day at a concentration of 100 ml, the cells were analyzed by TOPFLASH assay and Western blotting (FIG. 15). As a result, in H1299T/R cells treated with Wnt3a, although endogenous IRAK1 protein was efficiently reduced by siRNA, activation of TOPFALSH by Wnt3a was not suppressed, but in H1299T/R cells treated with IL1β. In H358hpT/R, TOPFALSH activation was suppressed by siRNA. As previously reported, it was confirmed that the IRAK1 protein activated by IL1β was rapidly degraded even in the control siRNA (Csi) treated group, which is one indicator of the action of IL1β. The above results suggest that IL1β ligand, rather than Wnt ligand, exists upstream of IRAK1 in the activation of β-catenin, similar to NFκB signals, and that IRAK1 is essential for this signal transduction (Fig. 15). All siRNAs used in this example were purchased from Thermo Fisher scientific, and their catalog numbers are as follows; IRAK1 siRNA1: siRNAID#: 322, IRAK1 siRNA2: siRNAID#: 323, Silencer Control siRNA (Csi ):catalog#:AM4611

Based on the above experimental results, without wishing to be bound by any theory, IRAK1 is a key factor that causes crosstalk between the IL-1R signal pathway and the Wnt/β-catenin pathway, which are activated in chronic inflammation. Assuming that there is, the following hypothesis is inferred as the mechanism of fibrosis/carcinogenesis induced by chronic inflammation (Figure 16).
(1) Inflammatory cells infiltrate into injured/inflamed tissues, resulting in normal production and stimulation of IL-1.
(2) When there are high IRAK1 expression sites or mutant cells in inflamed tissues, constant activation of β-catenin and expression of target genes that enhance epithelial-mesenchymal transition and proliferation occur.
(3) Constantly elevated expression of β-catenin target genes causes epithelial-mesenchymal transition and fibrosis, and enhanced cell proliferation greatly increases the risk of accumulation of genetic mutations and canceration.

[Example 8] Creation and analysis of mice that specifically express active IRAK1 and establishment of chronic inflammatory disease model mice Using genome editing technology, active IRAK1 (large intestine, lung, glial, and liver cell-specific) was generated. We will create transgenic mice in which the K239A and V412M mutations have been introduced, and attempt to analyze their phenotypes with a focus on fibrosis and carcinogenesis (Figure 17). As shown in FIG. 17, a mouse derived from a fertilized egg into which a targeting vector encoding active IRAK1 was introduced was produced using the CRISPR/Cas9 system. The target sequence of the guide RNA was ACTCCAGTCTTTCTAGAAGA -TGG ( TGG is the PAM sequence) (SEQ ID NO: 3) (Chu et. al., BMC biotechnology 164 2016). Once gene transfer into the germ cells of the mouse is confirmed, a mouse that expresses active IRAK1 in a tissue-specific manner can be obtained by crossing the mouse with a tissue-specific Cre mouse. By crossing the resulting mice with TOP-GAL mice or β-catenin ^flox mice that can quantify Wnt/β-catenin pathway activity, mice can be obtained to evaluate dependence on the Wnt/β-catenin pathway. . In addition, we will induce inflammation in these mice by administering drugs, such as pneumonia due to bleomycin administration and colitis due to AOM/DSS administration, and evaluate the effect of Wnt/β-catenin pathway activation on carcinogenesis due to IRAK1 enhancement under inflammation. .

The disease model animal provided by the present invention can be used for prevention of fibrosis or tumor in tissues or organs, verification of treatment, elucidation of molecular mechanisms of fibrosis and carcinogenesis, and the like. Further, according to the present invention, the candidate substance obtained by screening is useful as a prophylactic or therapeutic agent for fibrosis or tumor in tissues or organs.

Claims (6)

The following protein (a) or (b).
(a) A protein having a mutation of valine at position 412 of SEQ ID NO: 2, where the mutation is a substitution with methionine (b) In the protein of (a), one or more amino acids are deleted, substituted, or inserted and/or has an added amino acid sequence (excluding the substitution of the mutated amino acid residue in (a) with a wild-type amino acid residue), and has an activation-enhancing effect on the Wnt/β-catenin pathway. protein with A nucleic acid encoding the protein according to claim 1. A cell comprising the nucleic acid according to claim 2. A non-human mammal or a part of its living body that expresses the protein according to claim 1 and exhibits a tissue or organ fibrosis or tumor phenotype. The animal or part of its living body according to claim 4, wherein the mammal is a rodent. (1) A step of bringing a test substance into contact with the cells according to claim 3, or the animal or part of its living body according to claim 4 or 5, and (2) Phenotype of fibrosis or tumor of tissue or organ. A screening method for a preventive or therapeutic drug for tissue or organ fibrosis or tumor, the method comprising the step of selecting a test substance that has improved fibrosis or tumor.