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WO2018135738A1 - COMPOSITION POUR INHIBER L'ACTIVITÉ DE LA MYOSTATINE, CONTENANT UNE PROTÉINE gla MATRICIELLE EN TANT QUE SUBSTANCE ACTIVE - Google Patents

COMPOSITION POUR INHIBER L'ACTIVITÉ DE LA MYOSTATINE, CONTENANT UNE PROTÉINE gla MATRICIELLE EN TANT QUE SUBSTANCE ACTIVE Download PDF

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WO2018135738A1
WO2018135738A1 PCT/KR2017/013190 KR2017013190W WO2018135738A1 WO 2018135738 A1 WO2018135738 A1 WO 2018135738A1 KR 2017013190 W KR2017013190 W KR 2017013190W WO 2018135738 A1 WO2018135738 A1 WO 2018135738A1
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myostatin
amino acid
gla protein
matrix gla
amino acids
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Korean (ko)
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최인호
이은주
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Research Cooperation Foundation of Yeungnam University
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Research Cooperation Foundation of Yeungnam University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/316—Foods, ingredients or supplements having a functional effect on health having an effect on regeneration or building of ligaments or muscles

Definitions

  • the present invention relates to a composition for inhibiting the activity of myostatin to induce muscle loss.
  • muscle weakness diseases that cause muscle weakness include muscular dystrophy and cardiac atrophy that progress with aging, muscular atrophy caused by imbalances in protein metabolism or decreased muscle use, starvation, and wasting diseases.
  • Muscular dystrophy refers to a decrease in muscle strength due to a decrease in muscle mass during aging, and not only a decrease in muscle mass, but also a change in the type of muscle fibers. This myotropia is induced by a decrease or growth of growth hormone, a change in physiological activity, a change in metabolism, an increase in the amount of sex hormone or fat or catabolic cytokines, and a balance change in protein synthesis and differentiation.
  • Satellite cells are small mononuclear cells located between the basement membrane and the muscles of myofibrils, which are activated by stimulation such as injury or movement, and proliferate into myoblasts. When differentiation progresses, they fuse with other cells to form multinucleated myofiber.
  • the decrease in the activity of the satellite cells the ability to regenerate damaged muscle or the response to the differentiation signal is reduced, resulting in a decrease in muscle formation.
  • Muscular dystrophy is caused by malnutrition or long-term muscle inactivity, resulting in a breakdown in the balance of normal protein synthesis and degradation.
  • Exercise may increase skeletal muscle protein synthesis in the short term, but is inadequate for long-term treatments, and testosterone or anabolic steroids may be used for medications, but this induces masculinity in women and prostate symptoms in men. Appears.
  • the present invention is to provide a composition containing the matrix gla protein as an active ingredient in order to inhibit the activity of myostatin to induce muscle loss to be used as a therapeutic agent for muscle diseases related to myostatin.
  • the present invention provides a composition for inhibiting myostatin activity comprising a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
  • MGP matrix gla protein
  • the present invention provides a pharmaceutical composition for preventing or treating muscle diseases, comprising as an active ingredient a matrix gla protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • the present invention comprises the steps of treating a candidate with a cell; And Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, in the myostatin amino acid sequence represented by SEQ ID NO: 2 in cells treated with the candidates Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to provide a myostatin inhibitor screening method comprising the step of identifying the binding level of any one or more amino acids and candidates selected from the group consisting of. Can be.
  • the present invention comprises the steps of treating the candidate material to the cell; And Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1, and Phe43 in the matrix gla protein amino acid sequence represented by SEQ ID NO: 1 in cells treated with the candidates.
  • Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 may provide a matrix gla protein and myostatin binding promoter screening method comprising the step of confirming the increase in the binding level of any one or more amino acids selected from the group consisting of have.
  • the present invention comprises a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, the matrix gla protein is Arg49, Pro46, Gln48, in the amino acid sequence represented by SEQ ID NO: 1, Leu20, Val22, Phe27, Trp29, Trp31 in the amino acid sequence of myostatin, wherein any one or more amino acids selected from the group consisting of Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 are represented by SEQ ID NO: 2 At least one amino acid selected from the group consisting of Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101. It can provide a reagent composition for inhibiting myostatin activity by
  • a composition containing the matrix gla protein as an active ingredient increases the expression of genes related to muscle differentiation, thereby promoting the differentiation of muscle satellite cells and inhibiting the binding of myostatin and ACVRIIB to induce muscle reduction.
  • the composition containing the matrix gla protein as an active ingredient can be effectively used as a therapeutic agent for muscle diseases such as myostatin activity modulators and muscle loss induced by myostatin.
  • FIG. 1 shows MGP expression in C2C12 cells grown in differentiation medium over time.
  • FIG. 1 (A) shows RT-PCR results confirming MGP mRNA levels
  • FIG. 1 (B) shows Western MGP protein levels.
  • Figure 1 (C) is the result of RT-PCR confirming the MGP mRNA level
  • Figure 1 (D ) Is a Western blot result
  • Figure 1 (E) is a result of confirming myotube cell formation in C2C12 cells with reduced MGP expression
  • Figure 1 (F) is a result of confirming the fusion index in C2C12 cells with reduced MGP expression
  • 1 (G) is an immunocytochemical result confirming MGP protein expression.
  • Figure 2 shows the results of confirming the expression of MGP protein in the cytoplasm of myotubes.
  • Figure 3 is a result of confirming the extracellular matrix (ECM) and myogenic marker gene expression in C2C12 cells (MGPkd) reduced MGP expression in order to confirm the effect of MGP expression on the root canal cell formation
  • Figure 3 (A) Is the result of confirming the RNA and protein expression levels of the myogenic marker gene MYOG
  • Figure 3 (B) is the result of confirming the RNA and protein expression levels of the myogenic marker gene MYOD
  • Figures 3 (C) and 3 (D) This is the result of confirming the expression of the extracellular matrix (ECM) genes COL1 ⁇ 1 and FMOD in cells with reduced MGP expression during the differentiation period (day 2).
  • Figure 4 is a result of confirming the MGP expression level in the cells suppressed FMOD, COL1 ⁇ 1, or MSTN gene
  • Figure 4 (A) is a result of confirming the MGP expression level in the FMOD reduced expression cells (FMODkd)
  • Figure 4 (B) is a result of confirming the MGP expression level in the COL1 ⁇ 1 expression reduced cells (COL1 ⁇ 1kd)
  • Figure 4 (C) is a result of confirming the MGP expression level in the MSTN expression reduced cells (MSTNkd)
  • E are immunochemical analyzes confirming the expression level of MSTN, a muscle development negative regulator, in cells with reduced MGP expression and in normal cells.
  • FIG. 5 is a result of confirming the correlation between MGP and MSTN and MGP and FMOD in in vitro conditions by complex immunoprecipitation and Western blot analysis
  • Figure 5 (A) is confirmed in normal cells
  • Figure 5 (B) 5 is a result of confirming the structure of the produced MGP
  • Figure 5 (D) is a result of confirming the correlation between MGP and MSTN by performing In silico experiments
  • 5 (E) shows the results of confirming the effect on the receptor ACVRIIB that binds to MSTN.
  • the present invention can provide a composition for inhibiting myostatin activity containing a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
  • MGP matrix gla protein
  • the matrix gla protein may inhibit myostatin activity by inhibiting the binding of myostatin and its receptor, activin receptor type IIB.
  • the matrix gla protein is any one selected from the group consisting of Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 in the amino acid sequence represented by SEQ ID NO: 1
  • the above amino acids are Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78 in the amino acid sequence of myostatin represented by SEQ ID NO: 2.
  • Myostatin activity can be regulated by binding to any one or more amino acids selected from the group consisting of Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 and inhibiting the binding of myostatin and its receptor, ACVRIIB.
  • the matrix gla protein amino acid Arg49 binds to the amino acid Leu20 of myostatin
  • the matrix gla protein amino acid Arg38 binds to any one of the amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin
  • the matrix gla Protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin
  • the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin
  • the matrix gla protein amino acid Gln24 is either amino acid Arg67 or Gly68 of myostatin
  • the matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin
  • the matrix gla protein amino acid Met26 binds to any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combine
  • the matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, As
  • the composition may be selected from the group consisting of a pharmaceutical composition and health food.
  • Myostatin (MSTN) of the present invention is one of transforming growth factor- ⁇ (TGF- ⁇ ) superfamily as an inhibitor of autocrine / paracrine which is very important for muscle growth It's working. Reportedly, the absence of myostatin expression in mice significantly increased the mass of skeletal muscle, resulting in muscular dystrophy and hyperplasia.
  • TGF- ⁇ transforming growth factor- ⁇
  • the activin receptor type 2B is a protein encoded by the ACVR2B gene and is involved in the activin signaling mechanism. Signal transduction by activin is known to be involved in the production, secretion of follicle stimulating hormone (FSH), regulation of the menstrual cycle, and in response to proliferation, differentiation and apoptosis of cells.
  • FSH follicle stimulating hormone
  • the matrix gla protein (MGP) of the present invention is another protein of the extracellular matrix (ECM), which is vitamin K2-dependent and includes a gla ( ⁇ -carboxyglutamate) domain. It has high affinity binding, serves as an inhibitor of blood vessel mineralization and plays an important role in bone tissue.
  • ECM extracellular matrix
  • the matrix gla protein was found to inhibit MSTN and ACVRIIB binding.
  • a protein-protein correlation study showed that the overall energy score of MSTN and ACVRIIB binding was -56.99.
  • the binding efficiency was reduced to -25.08 in the presence of MGP, and the amino acid residues included in MSTN and ACVRIIB binding were significantly reduced in the presence of MGP as shown in FIGS. 5E and 2.
  • MGP binds to MSTN and thereby inhibits MSTN and ACVRIIB binding to regulate MSTN activity.
  • MGP reduced cells in order to confirm the effect of MSTN expression regulation by MGP, as a result of confirming the mRNA and protein levels of MSTN in MGP reduced cells (MGPkd), MGP reduced cells as shown in Figure 4d The decrease in MSTN expression was confirmed, and as a result of immunostaining using the MSTN antibody, MSTN expression was reduced in cells with reduced MGP as shown in FIG. 4E.
  • the present invention can provide a pharmaceutical composition for preventing or treating muscle diseases containing the matrix gla protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
  • the pharmaceutical composition for preventing or treating muscle diseases the matrix gla protein is Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 in the amino acid sequence represented by SEQ ID NO: 1
  • at least one amino acid selected from the group consisting of Phe43 is Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, in the amino acid sequence of myostatin represented by SEQ ID NO: 2.
  • Myostatin is inhibited by binding to one or more amino acids selected from the group consisting of Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to inhibit the binding of myostatin and its receptor ACVRIIB. It can suppress induced muscle loss.
  • the matrix gla protein amino acid Arg49 binds to the amino acid Leu20 of myostatin
  • the matrix gla protein amino acid Arg38 binds to any one of the amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin
  • the matrix gla Protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin
  • the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin
  • the matrix gla protein amino acid Gln24 is either amino acid Arg67 or Gly68 of myostatin
  • the matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin
  • the matrix gla protein amino acid Met26 binds to any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combine
  • the matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, As
  • the matrix gla protein may promote the differentiation of muscle satellite cells by increasing the expression of the muscle differentiation genes MYOD, MYOG, Col1 ⁇ 1 and FMOD.
  • the muscular diseases include muscle dystrophy, rigid spine syndrome, muscle-eye-brain disease, amyotrophic lateral sclerosis, and Charco-Marie- It may be selected from the group consisting of Charcot-Marie-Tooth disease, chronic inflammatory neuropathy and distal myopathy.
  • the present invention comprises the steps of treating a candidate with a cell; And Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, in the myostatin amino acid sequence represented by SEQ ID NO: 2 in cells treated with the candidates Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to provide a myostatin inhibitor screening method comprising the step of identifying the binding level of any one or more amino acids and candidates selected from the group consisting of. Can be.
  • the screening method may further comprise the step of identifying the reduced level of binding of myostatin and its receptor activin receptor type IIB in combination with the myostatin candidate.
  • the binding level between the matrix gla protein and the myostatin receptor is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoprecipitation, immunocytochemistry , Western blotting and flow cytometry (FACS) can be identified by any one selected from the group consisting of.
  • RT-PCR reverse transcription-polymerase chain reaction
  • ELISA enzyme immunoassay
  • FACS flow cytometry
  • the myostatin activity inhibitor may be selected from the group consisting of a therapeutic agent for muscle disease, a meat enhancer and an anabolic agent for livestock.
  • the present invention can provide a pharmaceutical composition for preventing or treating muscle diseases, which contains a myostatin activity inhibitor selected by the screening method as an active ingredient.
  • the present invention comprises the steps of treating the candidate material to cells isolated from mammals; And Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1, and Phe43 in the matrix gla protein amino acid sequence represented by SEQ ID NO: 1 in cells treated with the candidates.
  • Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 may provide a matrix gla protein and myostatin binding promoter screening method comprising the step of confirming the increase in the binding level of any one or more amino acids selected from the group consisting of have.
  • the present invention contains a matrix gla protein (MGP) consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, the matrix gla protein is Arg49, Pro46, in the amino acid sequence represented by SEQ ID NO: 1; Any one or more amino acids selected from the group consisting of Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 are represented in SEQ ID NO: 2 by Leu20, Val22, Phe27, Trp29 At least one selected from the group consisting of, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 It is possible to provide a reagent composition for inhibiting myostatin activity by binding to amino acids to inhibit myostat
  • the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to conventional methods
  • suitable carriers, excipients, disintegrants, sweeteners, coatings, swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatics, diluents, conventionally used in the manufacture of the pharmaceutical compositions It may further comprise one or more additives selected from the group consisting of dispersants, surfactants, binders and lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules.
  • solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like
  • lubricants such as magnesium styrate and talc may also be used.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.
  • the preferred dosage of the matrix gla protein may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the health food is used with other food or food additives in addition to the matrix gla protein (MGP), and may be appropriately used according to conventional methods.
  • the mixed amount of the active ingredient can be suitably determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.
  • the effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for health and hygiene purposes or health control purposes It is evident that the component can be used in an amount above the range because there is no problem in terms of safety.
  • C2C12 cells from mouse myoblasts were purchased from Korean Cell Line Bank (KCLB), 10% fetal bovine serum (FBS; HyClone Laboratories) and 1% penicillin / streptomycin (P / S) (Invitrogen, Carlsbad, Incubated in DMEM (Dulbecco's modified Eagle's medium; HyClone Laboratories, Logan, UT) medium containing 37%, 5% CO 2 conditions.
  • KCLB Korean Cell Line Bank
  • FBS fetal bovine serum
  • P / S penicillin / streptomycin
  • C2C12 cells were grown to 70% confluence, replaced with DMEM differentiation medium containing 2% fetal bovine serum and 1% penicillin / streptomycin and grown for 1 to 5 days, and the differentiation medium was replaced every other day. .
  • Trizol TM reagent (Invitrogen) according to the manufacturer's instructions using a, and stored in C2C12 cells Total RNA was extracted diethyl fatigue from -80 °C condition until use carbonate (diethylpyrocarbonate) in treated H 2 O a.
  • Oligo (dT) 20 primer (Bioneer, Daejeon, Korea) was prepared from 20 ⁇ l of the reaction mixture containing total RNA (1 ⁇ g), and reverse transcription was performed at 42 ° C. for 50 minutes and 72 ° C. for 15 minutes. PCR was performed using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the cDNA (2 ⁇ l) and each gene specific primer (10 pmoles).
  • C2C12 cells were incubated for 2 or 4 days in differentiation medium in cover glass-bottom dishes and stained with MGP protein.
  • the cells were blocked with PBS containing 5% goat serum for 1 hour and then incubated with MGP antibody (rabbit polyclonal IgG MGP, 1:50; Proteintech) overnight at 4 ° C. wet conditions.
  • MGP antibody rabbit polyclonal IgG MGP, 1:50; Proteintech
  • Samples were washed with PBS and counterstained with nuclei using 4'6'-diamino-2-phenylindole (DAPI; Sigma-Aldrich) and photographed with a fluorescence microscope (Nikon) equipped with a digital camera.
  • DAPI 4'6'-diamino-2-phenylindole
  • the cells were washed with ice-cold PBS and lysed with RIPA buffer containing protease inhibitor cocktail (Thermo Scientific, NH, USA), followed by centrifugation at 4 ° C and 12,000 rpm for 10 minutes to separate whole protein and supernatant. Were collected and analyzed for protein by Bradford method.
  • mixed vectors or shRNAs for specific genes were transfected into C2C12 cells.
  • C2C12 cells grown to 30% confluence were transfected overnight with transfection reagent (Santa Cruz Biotechnology, Calif., USA) treated with 1 ng of gene specific shRNA and mixed vector (control), respectively, and the cells were 80% confluent. Transfected cells were treated with 2 ⁇ g / ml puromycin (Sigma Life Sciences).
  • siRNA 100 mM control or Col1 ⁇ 1 siRNA were transfected into C2C12 cells for 5 hours and then cultured in differentiation medium for 2 days.
  • RT-PCR and Western blots were performed to confirm mRNA and protein expression levels.
  • samples were obtained from normal C2C12 cells differentiated with 2% fetal bovine serum (FBS), C2C12 cells transfected with MGP shRNA, and C2C12 cells transfected with a complex vector.
  • FBS fetal bovine serum
  • the pellet was mixed with FMOD or MSTN antibody (Santa Cruz), incubated overnight, and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. to remove the supernatant.
  • the crystal structure of MSTN (pdb id: 3HH2) was retrieved from Protein Data Bank (PDB) and modeled with Modeller 9v14 using the crystal structure of human activin receptor type II kinase domain (pdb id: 2QLU) as a template. Five ACVRIIB models were made and the best model was selected based on the dope score.
  • MGP mRNA expression was confirmed to be 8-fold higher on the second day of muscle differentiation induction, and similar results were observed in the Western blot pattern of FIG. 1B.
  • MGP protein expression was confirmed in the cytoplasm of the myotubes.
  • MGP shRNAs were transfected into C2C12 cells and RT-PCR and Western blot analysis was performed.
  • MGP expression was reduced by 60% in the transfected cells as shown in Figs. 1c and 1d, and it was confirmed that myotube formation was reduced as shown in Figs. 1e and 1f by decreasing MGP expression.
  • FIG. 1G As a result of confirming the effect of reducing cell formation, as shown in FIG. 1G, more myotubes were found in normal cells (MGPwt) than in cells with reduced MGP (MGPkd).
  • MGP plays a role in regulating myotube cell formation during differentiation period.
  • MYOD and MYOG are known to regulate the progression of muscle stem cell differentiation in the course of muscle tissue development.
  • MGPkd MGP reduced cells
  • MGP was found to regulate the expression of genes that induce muscle differentiation.
  • C2C12 cells were transfected with shRNA / siRNA (shFMOD, siCol1 ⁇ , or shMSTN).
  • MGP expression was increased in FMODkd, Col1 ⁇ 1kd and MSTNkd cells as compared to the wild type as shown in FIGS. 4A to 4C.
  • MSTN expression was confirmed in cells with reduced MGP.
  • MGPkd MGP reduced cells
  • MGP is a gene regulating MSTN expression.
  • MSTN expression was decreased in cells with reduced MGP as shown in FIG. 4E. I could confirm that.
  • the protein-protein correlation study confirmed that the overall energy score of MSTN and ACVRIIB binding was -56.99. However, in the presence of MGP as shown in FIG. 5D, the binding efficiency was reduced to -25.08.
  • MGP MSTN MGP MSTN NIL R49, P46, Q48, L4, S45, E24, M26, S25, R38, T42, R37, M1F43 L20, V22, P27, W29, W31, A40, N41, Y42, Y55, R67, G68, S69, A70, G71, C73, P76, T77, K78, M79, Y86, N88, G89, P100, A100, M101

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Abstract

La présente invention concerne une composition pour inhiber l'activité de la myostatine, contenant une protéine gla matricielle en tant que substance active. Plus spécifiquement, il est confirmé qu'une composition contenant une protéine gla matricielle en tant que substance active stimule la différenciation de cellules satellites musculaires par augmentation de l'expression de gènes associés à la différenciation du muscle et inhibe l'activité de la myostatine par inhibition de la liaison de myostatine, qui induit une atrophie musculaire, avec ACVRIIB, et par conséquent, la composition contenant une protéine gla matricielle en tant que substance active peut être efficacement utilisable en tant qu'agent pour moduler l'activité de la myostatine et un agent pour traiter des maladies musculaires telles que l'atrophie musculaire induite par la myostatine.
PCT/KR2017/013190 2017-01-19 2017-11-20 COMPOSITION POUR INHIBER L'ACTIVITÉ DE LA MYOSTATINE, CONTENANT UNE PROTÉINE gla MATRICIELLE EN TANT QUE SUBSTANCE ACTIVE Ceased WO2018135738A1 (fr)

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US20150086636A1 (en) * 2006-11-29 2015-03-26 Nationwide Children's Hospital Myostatin inhibition for enhancing muscle and/or improving muscle function

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US20150086636A1 (en) * 2006-11-29 2015-03-26 Nationwide Children's Hospital Myostatin inhibition for enhancing muscle and/or improving muscle function

Non-Patent Citations (4)

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Title
AHMAD, S. ET AL.: "Matrix Gla Protein: An Extracellular Matrix Protein Regulates Myostatin Expression in the Muscle Developmental Program", LIFE SCIENCES, ELECTRONIC, vol. 172, 21 December 2016 (2016-12-21), pages 55 - 63, XP055505259 *
DATABASE Protein [O] 10 January 2013 (2013-01-10), "Chain A, Growth/differentiation Factor 8", XP055505442, Database accession no. 3HH2_A *
DATABASE Protein [O] 26 February 2014 (2014-02-26), "Matrix Gla Protein Precursor [Mus musculus]", XP055505263, Database accession no. NP_032623.1 *
VELLEMAN, S. G. ET AL.: "Versican, Matrix Gla Protein, and Death-associated Protein Expression Affect Muscle Satellite Cell Proliferation and Differentiation", POULTRY SCIENCE, vol. 91, no. 8, August 2012 (2012-08-01), pages 1964 - 1973, XP055518989 *

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