WO2022030907A1 - Composition destinée à améliorer ou traiter l'obésité comprenant le fragment polypeptidique du socs6 - Google Patents

Composition destinée à améliorer ou traiter l'obésité comprenant le fragment polypeptidique du socs6 Download PDF

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WO2022030907A1
WO2022030907A1 PCT/KR2021/010037 KR2021010037W WO2022030907A1 WO 2022030907 A1 WO2022030907 A1 WO 2022030907A1 KR 2021010037 W KR2021010037 W KR 2021010037W WO 2022030907 A1 WO2022030907 A1 WO 2022030907A1
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present
polypeptide
polypeptide fragment
sequence
composition
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PCT/KR2021/010037
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Korean (ko)
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박순형
한은수
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주식회사 에이엠메딕스
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A23V2200/332—Promoters of weight control and weight loss

Definitions

  • the present invention relates to a composition for treating obesity comprising a polypeptide fragment of SOCS6.
  • Drugs acting on the central nervous system include drugs such as fenfluramine and dexfenfluramine that inhibit the serotonin (5-HT) nervous system according to each mechanism, drugs such as ephedrine and caffeine via the noradrenergic nervous system, and recently serotonin and noradrenergic nervous system Drugs such as sibutramine, which act simultaneously to inhibit obesity, are on the market.
  • orlistat which is a drug that inhibits obesity by acting on the stomach/intestine, inhibits intestinal lipase to reduce fat absorption, and the like, is used as a representative drug.
  • drugs such as fenfluramine were recently banned because they caused primary pulmonary hypertension or heart valve lesions due to side effects. of patients, there is a problem that it cannot be used.
  • An object of the present invention is to provide a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a pharmaceutical composition and a food composition for preventing, improving, or treating obesity comprising the same as an active ingredient.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.
  • Another object of the present invention is to provide a host cell containing the recombinant vector.
  • the present inventors have tried to develop a polypeptide that inhibits the accumulation of fat and has an excellent decomposition effect of the accumulated fat.
  • a polypeptide having an amino acid sequence of “TRSSREH” was derived from the suppressor of cytokine signaling 6 (SOCS6) protein, and the present invention was completed after confirming that the polypeptide had an excellent fat accumulation inhibitory effect and lipolytic effect.
  • Information on the SOCS6 protein and the mRNA sequence encoding the same can be found in GenBank Access No. It can be confirmed in NM_004232.4.
  • the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention suppresses the accumulation of fat, AMPK1alpha, PPAR-gamma, and HSL (hormone-sensitive lipase) which are genes related to lipolysis ) to promote the expression of
  • the AMPK is an enzyme that acts as a sensor in maintaining energy homeostasis in cells.
  • ATP for example, fatty acid synthesis and cholesterol synthesis
  • promotes ATP production eg fatty acid oxidation and glycolysis
  • AMPK AMPK: an emerging drug target for diabetes and the metabolic syndrome.
  • target organs liver, muscle, fat, pancreas
  • AMPK an emerging drug target for diabetes and the metabolic syndrome.
  • Cell Metab 9:407-416, 2009 When AMPK is activated in the liver, it inhibits the synthesis of fatty acids and cholesterol and promotes the oxidation of fatty acids.
  • AMPK When AMPK is activated in skeletal muscle, it promotes the oxidation of fatty acids and absorption of sugar, and inhibits lipolysis and lipogenesis in adipocytes.
  • polypeptide refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonds.
  • Polypeptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques; Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al. , Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid phase synthesis technology (US Patent No. 5,516,891).
  • the polypeptide of the present invention may select a portion of the amino acid sequence and induce modifications at the N-terminus or C-terminus to increase its activity. Through this modification, the polypeptide of the present invention can have a high half-life with increased half-life upon in vivo administration.
  • the C-terminus of the polypeptide of the present invention may be modified with a hydroxyl group (-OH), an amino group (-NH 2 ), an azide group (-NHNH 2 ), etc., of the polypeptide
  • a protecting group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) may be bonded to the N-terminus.
  • Stability refers to storage stability (eg, room temperature storage stability) as well as in vivo stability.
  • the above-mentioned protecting group functions to protect the polypeptide of the present invention from attack by proteolytic enzymes in vivo.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention contains a pharmaceutically effective amount of the polypeptide.
  • the term “obesity” means excessive accumulation of body fat in the body and/or excessive increase in body weight than the standard.
  • the term “pharmaceutically effective amount” refers to an amount sufficient to achieve therapeutic or preventive efficacy of the above-described polypeptide for obesity.
  • prevention refers to any action that inhibits the onset of a disease or pathological condition by administration of the composition of the present invention.
  • treatment refers to inhibiting the development of a disease or pathological condition; alleviation of disease; and elimination of the disease.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stevia, glycerin, glycerin, glycerin,
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal administration, skin administration, intramuscular administration, intranasal administration, intramucosal administration, It may be administered by intrathecal administration, intraperitoneal administration, intraocular administration, etc., and specifically, oral administration.
  • a suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg/kg.
  • the daily dose of the pharmaceutical composition of the present invention is, for example, 0.1-100 mg/kg, 0.1-90 mg/kg, 0.1-80 mg/kg, 0.1-70 mg/kg, 0.1-60 mg/kg, 0.1 -50 mg/kg, 0.1-400 mg/kg, 0.1-30 mg/kg, 0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-50 mg/kg, 0.1-30 mg/kg, 0.1- 20 mg/kg, 0.1-10 mg/kg, 0.1-7 mg/kg, 0.1-5 mg/kg, 1-100 mg/kg, 1-90 mg/kg, 1-80 mg/kg, 1-70 mg/kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1 -7 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 1-2 mg/kg, more specifically 1, 2, 3, 4, 5, 6, 7, 8, It may be 9, or 10 mg/kg, but is not limited thereto.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
  • the pharmaceutical composition of the present invention may be administered in combination with a known compound or pharmaceutical composition having an effect of preventing and treating obesity.
  • the present invention provides a food composition for preventing or improving obesity comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the food composition may be used as a health functional food or added to various foods.
  • the term “improvement” refers to delaying progression to a disease or pathological condition by administration of the composition of the present invention; and any action that alleviates a disease or pathological condition.
  • the present invention also provides a health functional food comprising the food composition.
  • the health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, ice cream, alcoholic beverages, vitamin complexes, or health supplements.
  • the content of the polypeptide of the present invention contained in the food composition may be appropriately adjusted according to the type of food, desired use, etc., and there is no particular limitation.
  • the content of the polypeptide may be 0.001 to 30% by weight or 0.01 to 20% by weight of the total food weight, and in the case of a health beverage composition, 0.001 to 15 g, 0.02 to 10 g, or 0.3 to 1 based on 100 ml g, but is not limited thereto.
  • composition comprising the polypeptide of the present invention may include not only the polypeptide as an active ingredient, but also ingredients commonly added during food production.
  • additional ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents.
  • examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, bean extract, jujube extract, licorice extract, etc. may be additionally included in addition to the polypeptide of the present invention.
  • the food composition for preventing or improving obesity of the present invention contains a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient in the same manner as the above-described "pharmaceutical composition for preventing or treating obesity", the amount In order to avoid undue redundancy in the present specification, descriptions of matters common among the inventions are omitted.
  • the present invention provides a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 described above.
  • nucleic acid molecule has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).
  • nucleotide sequence encoding the polypeptide of the present invention is a nucleotide sequence encoding an amino acid sequence constituting the polypeptide, and is not limited to any specific nucleotide sequence. This is because, even if a nucleotide sequence mutation occurs, the protein sequence does not change when the mutated nucleotide sequence is expressed as a protein in some cases. This is called codon degeneracy.
  • the nucleotide sequence is a functionally equivalent codon or codon encoding the same amino acid (eg, due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid It contains a nucleotide sequence comprising a.
  • the nucleic acid molecule of the present invention encoding the polypeptide is construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence described above.
  • the substantial identity is at least 80% when the above-described nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. , more preferably at least 90% homology, and most preferably at least 95%, 97%, 98%, or 99% homology.
  • the nucleic acid molecule encoding the polypeptide of the present invention includes a sequence exhibiting substantial identity to the sequence shown in the sequence listing.
  • the substantial identity is at least 61% when the sequence of the present invention and any other sequence are aligned to the maximum correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the art.
  • Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol.
  • BLAST can be accessed through the BLAST page of the ncbi website. A method for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.
  • the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the above-described polypeptide.
  • the expression vector is a vector into which a nucleic acid molecule encoding the polypeptide is inserted, is operatively linked to a nucleotide sequence of the nucleic acid molecule, and is an RNA molecule in a host cell. It is a recombinant vector for host cell expression comprising a promoter that forms it and a poly A signal sequence that acts in the host cell to cause polyadenylation of the 3'-end of the RNA molecule.
  • operatively linked refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, and By this, the regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.
  • a nucleic acid expression control sequence eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites
  • the vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). , this document is incorporated herein by reference.
  • the vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • the vector of the present invention is an expression vector and a prokaryotic cell is used as a host
  • a strong promoter capable of propagating transcription eg, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.
  • a ribosome binding site for initiation of translation e.g, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.
  • E. coli When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage ⁇ (pL)
  • the ⁇ promoter Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)
  • Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980) can be used as a regulatory region.
  • vectors that can be used in the present invention include plasmids (eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (eg, ⁇ gt ⁇ 4B, ⁇ -Charon, ⁇ z1 and M13, etc.) or viruses (eg, SV40, etc.) can be manufactured.
  • plasmids eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.
  • phage eg, ⁇ gt ⁇ 4B, ⁇ -Charon, ⁇ z1 and M13, etc.
  • viruses eg, SV40, etc.
  • the vector of the present invention may be fused with other sequences to facilitate purification of the polypeptide expressed therefrom.
  • the sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequences for purification, the protein expressed in the host is rapidly and easily purified via affinity chromatography.
  • the vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • the vector of the present invention is an expression vector and a eukaryotic cell is a host
  • a promoter derived from the genome of a mammalian cell eg, a metallotionine promoter
  • a promoter derived from a mammalian virus eg, adeno viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV
  • the vector may additionally carry genes encoding reporter molecules (eg, luciferase and -glucuronidase).
  • the present invention provides a host cell transformed with the recombinant vector.
  • any host cell known in the art may be used, for example, E. coli Origami2, E. coli JM109, E. coli BL21 (DE3 ), E. coli strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus genus strains such as Bacillus subtilis, Bacillus thuringiensis, and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcesens, and various Pseudomonas species.
  • yeast Sacharomyce cerevisiae
  • insect cells and animal cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
  • yeast Sacharomyce cerevisiae
  • insect cells and animal cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
  • the method of delivering the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)) ), Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580). (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)).
  • the microinjection method (Capecchi, MR, Cell, 22:479 (1980)), the calcium phosphate precipitation method (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) ), and the like, to inject the vector into the host cell.
  • the microinjection method Capecchi, MR, Cell, 22:479 (1980)
  • the calcium phosphate precipitation method Graham, FL et al., Virology, 52:456 (1973)
  • the recombinant vector injected into the host cell can express the above-recombined polypeptide in the host cell, and in this case, a large amount of the polypeptide is obtained.
  • the expression vector includes the lac promoter
  • the host cell may be treated with IPTG to induce gene expression.
  • the transformed host cell can be cultured by a known host cell culture method or a modified method thereof.
  • a natural medium or synthetic medium can be used as the medium for culturing the transformed host cell if it contains a carbon source, nitrogen source, inorganic salt, etc. that can be efficiently used by E. coli. have.
  • Carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose; starch, a hydrolyzate of starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol, propanol, glycerol, and the like.
  • the nitrogen source is ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean extract, soybean hydrolyzate; various fermented cells and their lysates; and the like.
  • Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate, and the like.
  • the culture is usually carried out under aerobic conditions, such as by shaking culture or rotation by a rotary machine.
  • the culture temperature is preferably in the range of 10 to 40° C., and the culture time is generally 5 hours to 7 days.
  • the pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture.
  • the pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.
  • antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin and tetracycline may be added for maintenance and expression of the recombinant vector.
  • a suitable inducer may be added to the medium.
  • a suitable inducer may be added to the medium.
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • indoleacrylic acid may be added to the medium.
  • the present invention provides a composition for preventing, improving, or treating obesity comprising a SOCS6 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and the same.
  • the polypeptide fragment of SOCS6 of the present invention promotes the expression of genes related to lipolysis, suppresses the accumulation of lipids in adipocytes, and has excellent effects of decomposing triglycerides, so it can be usefully used as a preventive or therapeutic agent for obesity.
  • FIG. 1 is a diagram showing the results of a cytotoxicity test of a polypeptide fragment of the present invention.
  • FIG. 2 is a diagram showing the effect of a polypeptide fragment of the present invention on the expression of a lipolytic factor through RT-PCR results.
  • FIG. 3 is a diagram showing the fat accumulation inhibitory effect of the polypeptide fragment of the present invention through H&E staining results for adipose tissue.
  • % used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.
  • a polypeptide fragment for improving or treating obesity of the present invention consisting of the amino acid sequence of SEQ ID NO: 1 was prepared.
  • the amino acid sequence of the polypeptide fragment of the present invention consists of “TRSSREH”.
  • the polypeptide of the present invention was synthesized by Fmoc SPPS (solid phase peptide synthesis) method using ASP48S (Peptron, Inc., Korea), and Vydac Everest C18 column (250 mm ⁇ 22 mm, 10 ⁇ m, USA) was used. Separation was performed using high-performance reverse-phase HPLC (Shimadzu Prominence HPLC, Japan).
  • the 3T3-L1 cell line was placed in a 96-well plate with 1 ⁇ 10 4 cells in Dulbecco's modified Eagle's media (DMEM) containing 10% bovine calf serum (BCS) and 1% penicillin streptomycin at 37°C, 5% CO 2 condition. cultured in an incubator of After culturing for 12 hours, samples were treated at 1, 5, and 10 ug/ml, and after 24 hours of incubation, 10 ⁇ l of EZ-Cytox (DoGen, Korea, Cat. No. EZ-1000) was added to each well. . The reaction was carried out in an incubator for about 0.5 to 4 hours. Before measuring the absorbance of each sample, the absorbance was measured at 450 nm using a spectrophotometer after shaking gently for about 1 minute.
  • DMEM Dulbecco's modified Eagle's media
  • BCS bovine calf serum
  • penicillin streptomycin 37°C, 5% CO 2 condition.
  • factors related to lipolysis ATGL, PPAR-gamma, AMPK1a, and HSL
  • 50 uM/ml of TNF-alpha was used as a positive control.
  • 3 mg RNA, 2 mg of random hexamer, and DEPC-treated water were added and reacted at 65° C. for 5 minutes.
  • 5 x first strand buffer, 0.1M DTT, 10 mM dNTP, and reverse transcriptase were added to make a total of 20 ml and reacted at 42°C for 1 hour. After heating again at 95°C for 5 minutes, 20 ml of distilled water was added to make a final 40 ml of cDNA.
  • PCR was performed by mixing 3 ml cDNA, 10 pmole primers specific for each lipolysis-related gene (ATGL, PPAR-gamma, AMPK1a, and HSL), 10x Tag buffer, 10 mM dNTP, and i-Tag DNA synthetase. did PCR conditions were 30 seconds at 95°C, 30 seconds at 55-59°C, and 30 seconds at 72°C. Genes were analyzed under conditions in which PCR results can be amplified exponentially. 5-10 ul of the PCR product was obtained, electrophoresed on a 1% agarose gel, and stained with ethidium bromide to confirm. The results are shown in FIG. 2 .
  • the polypeptide fragment (AM-LIPOCUT-02) of the present invention increases the expression of HSL and AMPK1-alpha, which are lipolytic factors, in a concentration-dependent manner, thereby decomposing the accumulated fat, thereby exhibiting an excellent anti-obesity effect.
  • 3T3-L1 cells were seeded and cultured in a 24-well plate at 2x10 4 cells/well, and 10 ⁇ g/ml insulin, 0.1 ⁇ M dexamethasone and 0.5 ⁇ M IBMX were included. It was exchanged with the prepared differentiation medium and the polypeptide was treated at each concentration (1, 5, 10 ug/ml). After that, the medium containing 10 ⁇ g/ml insulin was exchanged every 2 days, and H&E staining was performed on the 9th day of differentiation induction.
  • 3T3-L1 cells treated with each experimental group were washed with PBS, fixed by treatment with 4% paraformaldehyde for 10 minutes, washed with distilled water, and incubated with 60% isopropanol for 5-10 minutes. Stained cells were observed under a light microscope. The results are shown in FIG. 3 .
  • polypeptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention is excellent in the effect of decomposing triglycerides.

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Abstract

La présente invention concerne une composition destinée au traitement de l'obésité comprenant le fragment polypeptidique du SOCS6. Le fragment polypeptidique du SOCS6 de la présente invention favorise l'expression de gènes associés à la lipolyse, supprime l'accumulation de lipides dans les adipocytes et présente d'excellents effets de décomposition des triglycérides et en conséquence peut être avantageusement utilisé en tant qu'agent de prévention ou de traitement de l'obésité.
PCT/KR2021/010037 2020-08-07 2021-08-02 Composition destinée à améliorer ou traiter l'obésité comprenant le fragment polypeptidique du socs6 WO2022030907A1 (fr)

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KR10-2020-0099319 2020-08-07
KR1020200099319A KR102427080B1 (ko) 2020-08-07 2020-08-07 Socs6의 폴리 펩타이드 단편을 포함하는 비만의 개선 또는 치료용 조성물

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Citations (3)

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WO2003031468A1 (fr) * 2001-10-05 2003-04-17 The Walter And Eliza Hall Institute Of Medical Research Molecules therapeutiques et diagnostiques pouvant interagir avec des proteines socs
WO2005086800A2 (fr) * 2004-03-04 2005-09-22 Vanderbilt University Polypeptides socs pour inhiber la signalisation induite par la cytokine
WO2011113048A2 (fr) * 2010-03-12 2011-09-15 Vanderbilt University Modulation du signalement des cytokines

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US9012723B2 (en) * 2009-01-16 2015-04-21 Monsanto Technology Llc Isolated novel acid and protein molecules from soy and methods of using those molecules to generate transgene plants with enhanced agronomic traits
WO2014089514A1 (fr) * 2012-12-07 2014-06-12 Solazyme, Inc. Souches microbiennes génétiquement modifiées comprenant des gènes de la voie des lipides de chlorella protothecoides
KR101887576B1 (ko) 2016-04-15 2018-08-13 (주)케어젠 항비만 및 항당뇨 효능을 갖는 펩타이드 및 이의 용도
KR101955511B1 (ko) * 2016-08-17 2019-03-11 (주)진셀팜 지방분해 촉진 효과를 가지는 펩타이드, 및 이의 용도
KR101920047B1 (ko) * 2018-01-03 2018-11-19 (주)케어젠 항비만 및 항당뇨 효능을 갖는 펩타이드 및 이의 용도

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WO2003031468A1 (fr) * 2001-10-05 2003-04-17 The Walter And Eliza Hall Institute Of Medical Research Molecules therapeutiques et diagnostiques pouvant interagir avec des proteines socs
WO2005086800A2 (fr) * 2004-03-04 2005-09-22 Vanderbilt University Polypeptides socs pour inhiber la signalisation induite par la cytokine
WO2011113048A2 (fr) * 2010-03-12 2011-09-15 Vanderbilt University Modulation du signalement des cytokines

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GALIC SANDRA, SACHITHANANDAN NIRUPA, KAY THOMASW, STEINBERG GREGORYR: "Suppressor of cytokine signalling (SOCS) proteins as guardians of inflammatory responses critical for regulating insulin sensitivity", BIOCHEMICAL JOURNAL, vol. 461, no. 2, 15 July 2017 (2017-07-15), pages 177 - 188, XP009533902, ISSN: 0264-6021, DOI: 10.1042/BJ20140143 *

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