JP2007314469A - Ubiquitin ligase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide and a ubiquitin ligase inhibitor having an action of ubiquitination inhibition of insulin receptor substrate-1 by ubiquitin ligase to prevent disuse muscle and bone atrophy and effective for the prevention/treatment of disuse muscle and bone atrophy. <P>SOLUTION: Ubiquitin ligase Cbl-b has a domain recognizing phosphorylated tyrosine. Based on the finding, the phosphorylated tyrosine-site of the substrate IRS-1 is searched by a computer, and a peptide composed of an amino acid sequence expressed by Asp-Gly-Tyr-Met-Pro or Leu-Asn-Tyr-Ile-Asp found from a potential peptapeptide capable of binding with Cbl-b and IRS-1 using ubiquitin ligase inhibiting activity as an index is used as the ubiquitin ligase Cbl-b inhibitor. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a peptide effective for the prevention / treatment of muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden based on the inhibitory action of ubiquitin ligase, or ubiquitin ligase The present invention relates to an inhibitor, a therapeutic agent and / or preventive agent for muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden, and a food for prevention and / or symptom improvement.

  The skeletal muscles of astronauts who have stayed in space for a long period of time will shrink so that they cannot stand by themselves after the period. The so-called “disused muscle / bone atrophy” is also seen in elderly patients who are bedridden due to fractures, and when the muscle-free condition persists, Is caused by a decrease in muscle strength. Preventing muscle and bone atrophy is an important issue that must be solved both in the future for humankind in space development and in our aging society.

  Based on the knowledge that polyphenols have an inhibitory effect on the above-mentioned disuse muscles and bone atrophy, it was purified from juices and extracts of fruits belonging to the family Rosaceae, in particular, immature fruits of apples, pears and peaches. Known are muscle atrophy-suppressing compositions characterized by containing fruit polyphenols comprising a polyphenol fraction as an active ingredient, and compositions for inhibiting bone weight and bone mineral content during disuse muscle atrophy (for example, patents). Reference 1).

  In recent years, it has been revealed that the ubiquitin / proteasome proteolytic pathway plays an important role in such disuse muscle / bone atrophy and neuromuscular degenerative diseases (for example, Non-Patent Document 1, Non-Patent Document 1, (See Patent Document 2). As a mechanism for the development of disuse muscular atrophy, muscles continue to be in a state where muscles are not used, and as a result, proteins that make up muscles such as myosin undergo degradation in the ubiquitin / proteasome degradation pathway, resulting in a decrease in muscle size. It is considered a thing.

  The ubiquitin is a protein consisting of 76 amino acids that is universally present in eukaryotes, and has a role of labeling a protein (target protein) to be degraded in a ubiquitin / proteasome proteolysis system. In the ubiquitin / proteasome proteolytic system, polyubiquitin chains play a role in labeling target proteins. A polyubiquitin chain is a chain of ubiquitins linked in a branched manner, and binds to a specific site of a target protein. When the polyubiquitin chain is bound to the target protein, the 26S proteasome recognizes the target protein and degrades the target protein.

  The proteasome is a high molecular weight protease (proteolytic enzyme) localized in the nucleus and cytoplasm, and is a protein having a molecular weight of 700 to 800,000 consisting of 28 subunits. The “26S proteasome” in which PA70 (activator) is bound to both ends of the proteasome has a function of degrading proteins in an ATP-dependent manner, and plays a central role in the ubiquitin / proteasome proteolytic system. The 26S proteasome selectively degrades a polyubiquitinated protein (modified by a polyubiquitin chain) (see, for example, Non-Patent Document 3).

  As described above, control of the ubiquitin / proteasome proteolytic pathway is thought to lead to the treatment of these disuse muscle / bone atrophy and neuromuscular degenerative diseases. Therefore, the proteasome and ZNF216 (or AWP1) A therapeutic agent for disuse muscular atrophy, and a screening method for a therapeutic agent for disuse muscular atrophy using a novel protein-protein interaction between polyubiquitin chain and ZNF216 (or AWP1), disease Diagnostic markers, onset risk evaluation methods (see, for example, Patent Document 2), novel ubiquitin specific protease (USP) having deubiquitination activity, and polypeptide or peptide derived therefrom, Polynucleotide, antibody against the polypeptide or peptide, physiological activity of novel USP Inhibitors, antagonists, activators, pharmaceutical compositions using these, methods for producing the polypeptides or peptides by genetic engineering techniques, methods for identifying the inhibitors, antagonists, activators, and novel USPs Methods and kits for diagnosis of diseases such as Alzheimer's disease and Parkinson's disease neurodegenerative disease (for example, see Patent Document 3) have been proposed.

  However, no inhibitor of ubiquitin ligase has been reported so far, and there is only a means to suppress the activity of the deubiquitination promoter after ubiquitination as described above and the proteasome activity which is the final stage of degradation. Since proteasome inhibitors are very toxic, they have been put to practical use only as therapeutic agents for the terminal stage of cancer. On the other hand, inhibition of ubiquitin ligase by the peptide of this patent has high substrate selectivity, small side effects, and is expected as a useful food ingredient. In addition, the amino acid sequence of the ubiquitin ligase inhibitory peptide is a very effective finding in constructing the three-dimensional structure of the inhibitor.

JP 2001-89387 A JP 2005-170796 Japanese Patent Application No. 2002-287039 Gomes et al “Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy”, Proc. Natl. Acad. Sci. USA, 2000, Vol. 98, No. 25, pp14440-14445 Bodine et al “Identification of ubiquitin ligases required for skeletal muscle atrophy”, Science, 2001, Vol.294, pp.1704-1708 Experimental Medicine, Yodosha, February 2003, Vol. 21, No. 3, p. 330-332

  An object of the present invention is to prevent disuse muscle / bone atrophy, and to have a peptide effective for prevention / treatment of disuse muscle / bone atrophy having an inhibitory action on ubiquitination of IRS-1 by ubiquitin ligase. It is in providing a ubiquitin ligase inhibitor. Insulin Receptor Substrate-1 (IRS-1; Insulin Receptor Substrate-1) is an important factor in insulin signaling, and inhibiting its ubiquitination and subsequent degradation increases sensitivity to insulin, It is expected to improve resistance to insulin.

  The present inventor discovered in a space shuttle experiment launched in 1998 that only a specific proteolytic pathway (ubiquitin-proteasome pathway) is activated in murine skeletal muscle atrophic by weightlessness. This proteolytic pathway is characterized in that the protein to be degraded is labeled with a peptide called ubiquitin (ubiquitination), and it has been revealed that many proteins are ubiquitinated and degraded in the skeletal muscle of this space flight rat. (FASEB J Express Article doi: 10.1096 / fj.03-0419fje Published online January 8, 2004). In addition, as a result of analyzing the space sample by DNA microarray and differential display method, IRS-1, which is an important intracellular signal transduction molecule of insulin and insulin-like growth factor, which is an activation factor of muscle cells and osteoblasts, was identified. Of ubiquitin ligase called Cbl-b (Casitus B lineage lymphoma-b), which is ubiquitinically activated, is found to increase about 10-fold by space flight, and is an intracellular transmitter molecule of growth factor by Cbl-b. It has been clarified that the increase in crystallization and degradation is an important cause of muscle and bone atrophy due to weightlessness.

  Therefore, as a result of synthesizing and screening a large number of oligopeptides, the present inventors obtained a pentamer peptide (an oligopeptide having 5 amino acids linked) that competitively inhibits the ubiquitination of IRS-1 by Cbl-b. As a result, the present invention has been completed.

  That is, the present invention relates to (1) a ubiquitin ligase inhibitor that inhibits binding between ubiquitin ligase and its substrate protein, and (2) ubiquitin ligase is Cbl-b, and the substrate protein is insulin receptor substrate-1 (IRS). -1) the ubiquitin ligase inhibitor described in (1) above, (3) (1) Asp-Gly-Tyr-Met-Pro (SEQ ID NO: 1) and / or (2) Leu-Asn-Tyr-Ile A peptide represented by the sequence of -Asp (SEQ ID NO: 2) and phosphorylated on a tyrosine (Tyr) residue, a substitution of at least one amino acid of the peptide, a salt thereof, and the peptide as an active domain A ubiquitin ligase inhibitor according to (1) or (2) above, comprising (4) (1) Asp-Gly-Tyr-Met-, comprising at least one substance selected from the group consisting of a compound having Pro (SEQ ID NO: And / or (2) Leu-Asn-Tyr-Ile-Asp (SEQ ID NO: 2), at least one substance selected from the group consisting of compounds spatially equivalent to some or all of the peptides or salts thereof The ubiquitin ligase inhibitor as described in (1) or (2) above.

  The present invention also includes (5) muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, comprising the ubiquitin ligase inhibitor according to any one of (1) to (4) as an active ingredient, An agent for treating and / or preventing insulin resistance syndrome or bedridden, or (6) further comprising an alkaline phosphatase inhibitor, said muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, A therapeutic and / or preventive agent for diabetes, insulin resistance syndrome or bedridden, or (7) muscular atrophy / bone atrophy, osteoporosis containing the ubiquitin ligase inhibitor according to any one of (1) to (4) above as an active ingredient, Food for preventing and / or improving symptoms of muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden, and (8) Muscle atrophy, bone atrophy (7) further comprising a Fataze inhibitors, osteoporosis, muscle wasting, muscle reduction, motor dysfunction, diabetes mellitus, a food for insulin resistance syndrome or bedridden the prevention and / or symptom improvement.

Furthermore, the present invention is represented by the sequence of (9) (1) Asp-Gly-Tyr-Met-Pro (SEQ ID NO: 1) or (2) Leu-Asn-Tyr-Ile-Asp (SEQ ID NO: 2), and A peptide or a salt thereof in which a tyrosine (Tyr) residue is phosphorylated, or (10) muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction using the peptide or salt thereof described in (9) above as an active ingredient, A therapeutic and / or preventive agent for lowering of motor function, diabetes, insulin resistance syndrome or bedridden, and (11) muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle, further comprising an alkaline phosphatase inhibitor Treatment, prevention and / or prevention agent for reduction, lowering of motor function, diabetes, insulin resistance syndrome or bedridden, (12) muscle atrophy / bone atrophy, osteoporosis, muscle containing peptide or salt thereof according to (9) above as an active ingredient Fatigue, muscle reduction, Food for prevention of dynamic function, diabetes, insulin resistance syndrome or bedridden and / or symptom improvement, and (13) Further comprising an alkaline phosphatase inhibitor, muscle atrophy / bone atrophy, osteoporosis, muscle fatigue The present invention relates to a food for preventing and / or improving symptoms of muscle reduction, lowering of motor function, diabetes, insulin resistance syndrome or bedridden.

  The present invention relates to a ubiquitin ligase inhibitor comprising a peptide or the like that suppresses ubiquitination of IRS-1, which is an information transmission substance. In particular, peptides are said to have almost no side effects and can be applied as meals. Therefore, drugs and meals (space foods) can be developed, and it will be an important means to prevent muscle atrophy due to weightlessness. Show. These results are also expected to be applied to prevent and / or prevent muscular atrophy due to bedridden, which is a social problem in Japan, and to treat and / or prevent disuse muscle and bone atrophy and diabetes.

  The novel peptide of the present invention comprises an amino acid sequence represented by SEQ ID NO: 1 (Asp-Gly-Tyr-Met-Pro) or SEQ ID NO: 2 (Leu-Asn-Tyr-Ile-Asp) in the sequence listing, and The peptide is not particularly limited as long as it is a peptide in which a tyrosine (Tyr) residue is phosphorylated or a salt thereof, and the novel peptide of the present invention has ubiquitin ligase Cbl-b inhibitory activity. In the present invention, the peptide salt is not particularly limited as long as it is a pharmacologically acceptable salt, but is preferably one that does not impair the ubiquitin ligase inhibitory activity. Specifically, hydrochloride, sulfate, Salts such as phosphate and citrate can be exemplified.

  Further, the ubiquitin ligase inhibitor of the present invention is not particularly limited as long as it inhibits the binding between the ubiquitin ligase and its substrate protein. The ubiquitin ligase is ubiquitin ligase Cbl-b, and the substrate protein is Insulin receptor substrate-1 (IRS-1) can be preferably exemplified. More specific examples of the ubiquitin ligase inhibitor of the present invention include the following compounds such as peptides.

  A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (Asp-Gly-Tyr-Met-Pro) in the Sequence Listing and having a tyrosine (Tyr) residue phosphorylated or at least one of the peptides ( However, a tyrosine residue is excluded) amino acid substitution, preferably 1-2 amino acid substitution, more preferably 1 amino acid substitution, or a salt thereof; a protein having the peptide as an active domain, etc. Or a salt thereof; a compound spatially equivalent to a part or all of the amino acid sequence shown in SEQ ID NO: 1 (a compound having the same three-dimensional configuration) or a salt thereof; SEQ ID NO: 2 (Leu-Asn in the sequence listing) -Tyr-Ile-Asp) and a tyrosine (Tyr) residue phosphorylated peptide or salt thereof; at least one of the peptides (excluding the tyrosine residue) No acid substitute, preferably 1 to 2 amino acid substitutes, more preferably 1 amino acid substitute, or a salt thereof; a compound such as a protein having the peptide as an active domain or a salt thereof; SEQ ID NO: 2, at least one substance selected from the group consisting of a compound spatially equivalent to a part or all of the amino acid sequence shown in FIG. 2 (a compound having the same three-dimensional configuration) and a salt thereof, for example, SEQ ID NO: 1 And a mixture of a peptide having a tyrosine residue phosphorylated and a peptide having the amino acid sequence represented by SEQ ID NO: 2 and having a tyrosine residue phosphorylated .

  The peptide of the present invention can be produced by a general chemical synthesis method based on amino acid sequence information. The method includes peptide synthesis methods by a normal liquid phase method and a solid phase method. More specifically, the peptide synthesis method is based on the amino acid sequence information, and a stepwise erosion method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. Then, a fragment condensation method in which each fragment is subjected to a coupling reaction is included. The peptide of the present invention can be synthesized by any of them. For the production of peptides by the solid phase method, for example, a peptide synthesizer model 430A apparatus manufactured by Applied Biosystems is used, and L-form amino acids are sequentially peptide-bonded from the carboxyl terminal side of the peptide to be synthesized to a polymeric solid support. After synthesizing the desired peptide chain by extending it, cleave it from the solid support using trifluoromethanesulfonic acid, etc., then remove the amino acid side chain protecting group and purify it by a method such as reversed-phase high-performance chromatography. Can be carried out by obtaining the desired peptide.

  In the peptide synthesis reaction, amino acids that are not involved in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, and tertiary butyl ester, such as benzyl ester, p-methoxybenzyl. It can be protected as an ester, p-nitrobenzyl ester aralkyl ester or the like. An amino acid having a functional group in the side chain, for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc., but such protection is not necessarily required. . Further, for example, a guanidino group of Arg is a suitable protecting group such as a nitro group, a tosyl group, a 2-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group, etc. Can be protected. The deprotection reaction of these protecting groups in the amino acids having the above-mentioned protecting groups, peptides and finally obtained peptides of the present invention is also carried out by conventional methods such as catalytic reduction, liquid ammonia / sodium, hydrogen fluoride, odor. It can be carried out according to a method using hydrogen fluoride, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

  Substitutes of at least one amino acid (excluding tyrosine residues) of the peptide, that is, one or several, preferably 1 to 2, more preferably 1 in the amino acid sequence shown in SEQ ID NO: 1 or 2 A peptide consisting of an amino acid sequence in which a single amino acid is deleted, substituted or added has an activity of inhibiting the binding between ubiquitin ligase and its substrate protein, and the amino acid sequence information and the base sequence encoding the amino acid Those skilled in the art can appropriately prepare or obtain the information based on the above information.

  If necessary, the peptide of the present invention can be combined with a marker protein and / or a peptide tag to form a fusion peptide. The marker protein is not particularly limited as long as it is a conventionally known marker protein. Specific examples include an enzyme such as alkaline phosphatase and HRP, an Fc region of an antibody, and a fluorescent substance such as GFP. As peptide tags, known peptide tags such as epitope tags such as HA, FLAG, and Myc, and affinity tags such as GST, maltose-binding protein, biotinylated peptide, oligohistidine, etc. It can be illustrated. Such a fusion peptide can be prepared by a conventional method. Purification of the peptide of the present invention utilizing the affinity between Ni-NTA and His tag, detection of the peptide of the present invention, and quantification of an antibody against the peptide of the present invention It is also useful as a research reagent in this field.

  The peptide of the present invention or a salt thereof, or the ubiquitin ligase inhibitor of the present invention, preferably a Cbl-b inhibitor is a disuse muscle / bone atrophy, a muscle atrophy other than disuse, a disease caused by insulin resistance, For example, it can be used as a prophylactic and therapeutic agent for diabetes, reducing muscle fatigue and strengthening muscles in healthy individuals, improving bone atrophy (improving osteoporosis), improving motor function (both bone and muscle simultaneously), and bedridden prevention Can be used. In addition, it can be used as a food for disuse muscle / bone atrophy, muscle atrophy other than disuse property, diseases caused by insulin resistance, such as diabetes prevention and / or symptom improvement, and reduce muscle fatigue in healthy subjects It can also be used as a functional food useful for improving muscle strength, improving bone atrophy (osteoporosis improvement), improving the function of exercising organs (both bone and muscle simultaneously), and bedridden prevention. When used as a prophylactic / therapeutic agent for disuse muscle / bone atrophy or diabetes, or as a food for prevention and / or symptom improvement of disuse muscle / bone atrophy or diabetes, etc. It is preferable to use an alkaline phosphatase inhibitor in combination with the ubiquitin ligase inhibitor of the invention, preferably a Cbl-b inhibitor. As such alkaline phosphatase inhibitors, known ones or commercially available products can be used, such as Auren, Abou (Nat. Med., 48, 165 (1994)), Bowie, Kyonin, Shochi, Yokuinin and Kansho (Tohoku Pharmaceutical). University Research Annual Report, 41, 149 (1994)) and bromoteramizole (BTO).

  As the preventive / therapeutic agent for muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden of the present invention, SEQ ID NO: 1 (Asp-Gly-Tyr) -Met-Pro), or a peptide comprising the amino acid sequence shown in SEQ ID NO: 2 (Leu-Asn-Tyr-Ile-Asp) and having a tyrosine (Tyr) residue phosphorylated, or a salt thereof, or the present invention The ubiquitin ligase inhibitor, preferably a Cbl-b inhibitor, is not particularly limited as long as it contains as an active ingredient. For example, these peptides may be used as a mixture of two kinds. When these prophylactic / therapeutic agents are used as pharmaceutical therapeutic / prophylactic agents, pharmaceutically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegrating agents, Various preparation compounding ingredients such as a solubilizer, a solubilizer, and an isotonic agent can be added. These therapeutic agents can be administered orally or parenterally, for example, can be administered orally in a dosage form such as powder, granules, tablets, capsules, syrups, suspensions, etc., or For example, a dosage form such as an emulsion or suspension can be administered parenterally as an injection.

  In addition, the food for preventing and / or improving symptoms of muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden according to the present invention is shown in SEQ ID NO: 1 in the above sequence listing. (Asp-Gly-Tyr-Met-Pro), or a peptide comprising the amino acid sequence shown in SEQ ID NO: 2 (Leu-Asn-Tyr-Ile-Asp) and having a tyrosine (Tyr) residue phosphorylated The peptide is not particularly limited as long as it contains the salt or the ubiquitin ligase inhibitor of the present invention, preferably a Cbl-b inhibitor, as an active ingredient, and these peptides may be used as a mixture of two kinds. Examples of the food or food material include yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, tea, sencha, oolong tea, sports drinks, pudding, cookies, bread, cakes, jelly, rice crackers, etc. Japanese confectionery such as baked confectionery, sheep candy, frozen confectionery, bread and confectionery such as chewing gum, noodles such as udon and soba, fish paste products such as kamaboko, ham, fish sausage, miso, soy sauce, dressing, mayonnaise, sweeteners, etc. It can be used as a food by blending it with dairy products such as cheese, butter and other dairy products, tofu, konnyaku, other boiled rice, dumplings, croquettes and salads. In addition, muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden prevention / treatment are used in packaging containers and instructions for muscle atrophy / bone atrophy, osteoporosis. , Muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or when bedridden is indicated to be effective for treatment or treatment.

  Hereinafter, the present invention will be described in detail using examples, but the technical scope of the present invention is not limited to these examples.

[1. Oligopeptide design]
Since the ubiquitin ligase Cbl-b has a domain that recognizes phosphorylated tyrosine, a computer search was performed for the phosphotyrosine site of the substrate IRS-1 (FIG. 1). As a result, six powerful pentapeptides (6 types in total: SEQ ID NOs: 1 to 6) capable of binding Cbl-b and IRS-1 were chemically synthesized using a peptide synthesizer “Symphony” manufactured by Rainin (see FIG. 2).

[2. Construction of Cell-free Ubiquitination system]
The ubiquitination of IRS-1 by Cbl-b was reconstituted within the study. The Cbl-b expression vector was obtained by using plasmid vector pCEFL-Cbl-b-HA (Oncogene. 1995 Jun 15; 10 (12): 2367-77.) Provided by Dr. S. Lipkowitz with restriction enzymes HindIII / KpnI. The fragment obtained by treatment was further subcloned into an expression vector (Biochem Biophys Res Commun 237 (2): 318-24, 1997) containing the cytomegalovirus immediate early enhancer-chicken b-globin hybrid (CAG) promoter. . The expression vector of IRS-1 was the one described in Biochem Biophys Res Commun. 1995 Nov 2; 216 (1): 321-8.
Heck293 cells were cultured in two dishes having a diameter of 10 cm. Plasmids containing Cbl-b and IRS-1 genes were strongly expressed in each of them, purified by immunoprecipitation with the respective antibodies, and reacted with the oligopeptides at 37 ° C. for 4 hours in the reaction system shown in Table 1. The reaction was stopped by adding 25 ml of reaction stop solution (200 mM Tris-HCl, pH 7.5, containing 8% SDS, 20% 2-mercaptoethanol, 20% glycerol and 0.02% BPB) to 50 ml of the reaction solution. . The entire amount was subjected to SDS-5% -PAGE, and Western blotting was performed with an anti-IRS-1 antibody (Calbiochem).

[3. result]
Using the Cell-free Ubiquitination system, it was examined whether Cbl-b promotes ubiquitination of IRS-1. As a result, it was revealed that Cbl-b significantly promotes ubiquitination of IRS-1 (FIG. 3). Moreover, when the inhibitory effect of the synthesized oligopeptide on IRS-1 ubiquitination was examined by the same method, no. 42 (SEQ ID NO: 1 Asp-Gly-Tyr-Met-Pro) and No. 42 Only 45 (SEQ ID NO: 2 Leu-Asn-Tyr-Ile-Asp) significantly inhibited IRS-1 ubiquitination (FIG. 4).

  Further, when the influence of an alkaline phosphatase inhibitor (Sigma: Posphatase inhibitor cocktail-II) on its ubiquitination inhibitory activity was examined, the activity disappeared when no alkaline phosphatase inhibitor was added (FIG. 5). . This suggests that phosphorylation of tyrosine residues in the synthetic oligopeptide is essential for the synthetic oligopeptide to exhibit ubiquitination inhibitory activity.

[4. Summary]
From the above, a synthetic oligopeptide having a structure very similar to that of the substrate inhibits the binding itself between the ubiquitin ligase Cbl-b and the substrate IRS-1, or binds to the substrate as a decoy. It was clarified that IRS-1 exhibits the ability to inhibit ubiquitination by competitively inhibiting (FIG. 6).

It is the figure which showed the result of having carried out the computer search for the phosphotyrosine site | part among the amino acid sequences of IRS-1. It is the figure which showed the peptide group which examined the inhibitory effect with respect to Cbl-b ubiquitin ligase. It is the figure which showed the result of having examined whether Cbl-b accelerates | stimulates the ubiquitination of IRS-1 using Cell-free Ubiquitination system. It is the figure which showed the result of having investigated the IRS-1 ubiquitination inhibitory effect of the synthesized oligopeptide using Cell-free Ubiquitination system. It is the figure which showed the result of having investigated the IRS-1 ubiquitination inhibitory effect of the synthesized oligopeptide in absence of ALP inhibitor using Cell-free Ubiquitination system. It is the model figure which showed the mechanism in which the oligopeptide of this invention develops the ubiquitination inhibitory effect of IRS-1.

Claims (13)

A ubiquitin ligase inhibitor that inhibits the binding of ubiquitin ligase and its substrate protein. The ubiquitin ligase inhibitor according to claim 1, wherein the ubiquitin ligase is Cbl-b and the substrate protein is insulin receptor substrate-1 (IRS-1). A peptide represented by the following sequence (1) and / or (2), wherein a tyrosine (Tyr) residue is phosphorylated, a substitution of at least one amino acid of the peptide, a salt thereof, the peptide The ubiquitin ligase inhibitor according to claim 1 or 2, comprising at least one substance selected from the group consisting of a compound having an active domain as an active domain and a salt of the compound.
(1) Asp-Gly-Tyr-Met-Pro (SEQ ID NO: 1)
(2) Leu-Asn-Tyr-Ile-Asp (SEQ ID NO: 2) The ubiquitin according to claim 1 or 2, comprising at least one substance selected from the group consisting of compounds or salts thereof that are spatially equivalent to some or all of the peptides having the sequence (1) and / or (2) below: Ligase inhibitor.
(1) Asp-Gly-Tyr-Met-Pro (SEQ ID NO: 1)
(2) Leu-Asn-Tyr-Ile-Asp (SEQ ID NO: 2) Treatment of muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden and / or comprising the ubiquitin ligase inhibitor according to any one of claims 1 to 4 as an active ingredient Preventive agent. Furthermore, the therapeutic and / or preventive agent for muscular atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden according to claim 5, comprising an alkaline phosphatase inhibitor. Prevention of muscular atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, motor function decline, diabetes, insulin resistance syndrome or bedridden and / or comprising the ubiquitin ligase inhibitor according to any one of claims 1 to 4 as an active ingredient Symptom improvement food. The food for preventing and / or improving symptoms according to claim 7, further comprising an alkaline phosphatase inhibitor, comprising muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden. A peptide represented by the following sequence (1) or (2) and having a tyrosine (Tyr) residue phosphorylated or a salt thereof.
(1) Asp-Gly-Tyr-Met-Pro (SEQ ID NO: 1)
(2) Leu-Asn-Tyr-Ile-Asp (SEQ ID NO: 2) A therapeutic and / or prophylactic agent for muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden, comprising the peptide according to claim 9 or a salt thereof as an active ingredient. The agent for treating and / or preventing muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden according to claim 10 further comprising an alkaline phosphatase inhibitor. A food for preventing and / or improving symptoms of muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden, comprising the peptide according to claim 9 as an active ingredient. The food for preventing and / or improving symptoms according to claim 12, further comprising an alkaline phosphatase inhibitor, comprising muscle atrophy / bone atrophy, osteoporosis, muscle fatigue, muscle reduction, decreased motor function, diabetes, insulin resistance syndrome or bedridden.