WO2021145743A1 - Protéine ciblant les mitochondries et son utilisation - Google Patents

Protéine ciblant les mitochondries et son utilisation Download PDF

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WO2021145743A1
WO2021145743A1 PCT/KR2021/000651 KR2021000651W WO2021145743A1 WO 2021145743 A1 WO2021145743 A1 WO 2021145743A1 KR 2021000651 W KR2021000651 W KR 2021000651W WO 2021145743 A1 WO2021145743 A1 WO 2021145743A1
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mitochondrial
stat6
protein
disease
preventing
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PCT/KR2021/000651
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English (en)
Korean (ko)
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김현미
주일로
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아주대학교산학협력단
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Priority claimed from KR1020200005961A external-priority patent/KR102410556B1/ko
Priority claimed from KR1020210005717A external-priority patent/KR102527406B1/ko
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Publication of WO2021145743A1 publication Critical patent/WO2021145743A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • 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
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to STAT6 protein targeting mitochondria and uses thereof.
  • Mitochondria are important intracellular organelles in cells where the process of oxygen respiration in eukaryotes proceeds. It converts energy stored in various organic substances into the form of adenosine triphosphate (ATP) necessary for life activities through oxidative phosphorylation. Because it metabolizes energy, it acts as a power plant for cells. In addition, it regulates apoptosis by regulating the release of cytochrome c and other proapoptosis factors, and in addition, it is related to signal transduction, cell differentiation, calcium concentration control, reactive oxygen species (ROS) formation and utilization, and the like.
  • ROS reactive oxygen species
  • Neurodegenerative diseases such as Alzheimer's, Huntington's disease, Parkinson's disease, diabetes, which are recently known to be caused by dysfunction of mitochondria, for example, disruption of glycolysis, decrease of mitochondrial enzymes, increase of reactive oxygen species, change in number or size of mitochondria, etc.
  • dysfunction of mitochondria for example, disruption of glycolysis, decrease of mitochondrial enzymes, increase of reactive oxygen species, change in number or size of mitochondria, etc.
  • various diseases such as , cancer and aging-related diseases have been reported, interest in mitochondria and research on them are increasing.
  • Another object of the present invention is to provide a method for moving or distributing a target protein to the mitochondria, and a method for confirming the migration or distribution of the target protein to the mitochondria.
  • Another object of the present invention is to provide a carrier for mitochondrial drug delivery containing the peptide.
  • Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, including a complex to which the peptide and a pharmacologically active substance are bound.
  • Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, comprising the mitochondrial-targeting STAT6 protein as an active ingredient.
  • the present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.
  • the present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.
  • the present invention provides a carrier for mitochondrial drug delivery containing the mitochondrial targeting peptide as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which the mitochondrial targeting peptide and a pharmacologically active substance are bound.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, containing as an active ingredient a STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain.
  • STAT6 Signal transducer and activator of transcription 6
  • the present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases containing STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient.
  • STAT6 Synignal transducer and activator of transcription 6
  • the mitochondrial targeting peptide according to the present invention can effectively move or distribute a target protein or drug to the mitochondria by accurately targeting the mitochondria, and using it as a pharmaceutical composition for the prevention or treatment of diseases related to mitochondrial dysfunction can be utilized
  • mitochondrial targeting peptide Through the mitochondrial targeting peptide according to the present invention, while effectively delivering various substances targeting mitochondria, its movement or distribution can be easily and clearly observed or confirmed.
  • the mitochondrial-targeting peptide according to the present invention is also confirmed in mitochondria of other species than humans, and in this regard, it has the advantage of being able to conduct various studies through animal experiments, etc.
  • the STAT6 protein according to the present invention includes a mitochondrial internal target domain and a mitochondrial membrane permeation domain, and when the STAT6 protein is located in the mitochondrial outer membrane by these domains, it induces fragmentation of mitochondria and induces tumorigenesis.
  • the STAT6 protein can be used as a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, or a health functional food composition to more effectively prevent, treat or improve related diseases.
  • Example 1 shows the human STAT6 protein according to Example 1 of the present invention.
  • FIG. 2 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in a human U373-MG (malignant glioma) cell line.
  • FIG. 3 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in the mouse B16F10 cell line.
  • Figure 4 confirms the transmembrane sequence of the STAT6 protein to be located in the outer mitochondrial membrane according to Example 3 of the present invention.
  • FIG 5 shows the human STAT6 protein (MTS D) from which the human STAT6 protein and the mitochondrial target sequence have been removed according to Example 4 of the present invention.
  • FIG. 6 is a result of observing the effect on mitochondria when the STAT6 proteins of FIG. 5 were overexpressed in the 293T cell line.
  • Example 7 shows a stable cell line in which STAT6 expression is suppressed in the 293T cell line was prepared in order to confirm the effect of STAT6 on tumor formation according to Example 5 of the present invention, and this was performed as a Western blotting experiment. it has been confirmed
  • FIG. 9 is an observation by removing the tumor tissue after cell transplantation in FIG. 7 , the upper figure is a view of a mouse one month after cell transplantation, and the lower figure is the tumor tissue extracted from each numbered mouse in the upper figure. It is a photographed image.
  • FIG. 11 shows human STAT6 protein (STAT6 WT) and STAT6 protein (MTS D) from which the mitochondrial target sequence has been deleted, and each of these STAT6 proteins was overexpressed in the STAT6 KO stable cell line prepared according to FIG. 7 to reconstruct a stable cell line. Therefore, this was confirmed by Western blotting experiments.
  • FIG. 12 is a view of the STAT6 KO and STAT6 WT stable cell lines according to FIG. 11 were transplanted into mice, and the tumor tissue was excised and observed. Tumors are listed in order of size.
  • FIG. 13 is an observation of the tumor tissue excised after transplanting the STAT6 KO and MTS D stable cell lines according to FIG. 11 into mice.
  • the upper figure is a view of a mouse just before tumor extraction one month after transplantation, and the lower figure is the excised figure. Tumors are listed in order of size.
  • the present inventors have completed the present invention by confirming that the specific amino acid sequence of the STAT6 protein N-terminal sequence is a mitochondrial targeting sequence, and that the STAT6 protein is located in the mitochondria, particularly its outer membrane.
  • the present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the human STAT6 N-terminal 1-30 sequence among the human STAT6 internal sequences has a mitochondrial targeting (targeting) effect, which is indicated by SEQ ID NO: 4.
  • SEQ ID NO: 4 MSLWGLVSKMPPEKVQRLYVDFPQHLRHLL
  • the mitochondrial targeting peptide or functional equivalent thereof consisting of the amino acid sequence of SEQ ID NO: 4 according to the present invention may be derived from nature, chemically synthesized using a chemical synthesis method or genetic engineering method known in the art, or genetically recombination It may be obtained using technology, but is not limited thereto.
  • the genetic engineering method may produce a DNA sequence encoding the peptide or a functional equivalent thereof according to a conventional method.
  • the DNA sequence can be prepared by PCR amplification using appropriate primers.
  • the DNA sequence can be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (such as those sold by Biosearch or Applied Biosystems).
  • the synthesized DNA sequence is inserted into a vector containing one or more expression control sequences that are operably linked thereto to control the expression of the DNA sequence, and transform the host cell with the recombinant expression vector formed therefrom to transform create a transform
  • the "transformant” can be used interchangeably with “host cell”, etc., and is introduced into the cell by any means, for example, electroshock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc. It refers to a prokaryotic or eukaryotic cell containing heterologous DNA. Since the expression level and modification of the protein appear differently depending on the host cell, a person skilled in the art can select and use the most suitable host cell for the intended purpose.
  • a substantially pure peptide encoded by the DNA sequence can be recovered from the culture.
  • the recovery may be performed using a method known in the art (eg, chromatography).
  • substantially pure peptide means that the peptide according to the present invention does not substantially contain any other proteins derived from host cells.
  • the peptide according to the present invention may include a peptide having a native amino acid sequence as well as an amino acid sequence variant thereof.
  • amino acid sequence variant refers to a peptide having a different sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues in the amino acid sequence according to the present invention, substitution of an amino acid analog, or a combination thereof, By the above mutations, the activity of the molecule is not entirely changed.
  • the peptide according to the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.
  • the present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.
  • the gene encoding the amino acid sequence of SEQ ID NO: 4 is a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the recombinant vector is a means for expressing the gene, and may include a known expression vector such as a plasmid vector, a cosmid vector, or a bacteriophage vector, but is not limited thereto.
  • the vector can be easily prepared by those skilled in the art according to any known method using DNA recombination technology.
  • the recombinant vector according to the present invention may be one in which a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a gene encoding a target protein are operably linked.
  • the "operably linked” means that an expression control sequence is linked to control transcription and translation of a gene encoding the amino acid sequence of SEQ ID NO: 4, and the gene under the control of an expression control sequence (eg, promoter, enhancer, etc.) and maintaining the correct reading frame so that the peptide according to the invention is expressed and encoded thereby.
  • an expression control sequence eg, promoter, enhancer, etc.
  • the gene encoding the amino acid sequence of SEQ ID NO: 4 is effective for mitochondrial targeting, the gene encoding the amino acid sequence of SEQ ID NO: 4 of the present invention is operably linked to the gene encoding the target protein to be targeted to the mitochondria. In the case of inserting and expressing it, the target protein can be targeted to the mitochondria.
  • FP fluorescent protein
  • the fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.
  • GFP green fluorescent probe
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.
  • the introduction into the cell means transforming the cell using a recombinant vector, and the transformation method according to this may use a well-known method well known to those skilled in the art.
  • the present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.
  • the fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.
  • GFP green fluorescent probe
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the cell By using a fluorescence microscope to continuously image the fluorescence of a specific wavelength emitted from a protein expressed in a cell into which the recombinant vector is introduced, that is, a protein expressed in the transformed cell, the cell is in a living state, and thus the target protein in the cell can be visualized in detail step by step in the process of expression and mitochondrial migration.
  • the 31-50th sequence and the 1-20th sequence, respectively (GFP) was bound to prepare 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids, and by transfecting them into cells and observing them under a microscope, migration to mitochondria could be confirmed.
  • the present invention provides a mitochondrial drug delivery carrier containing a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient.
  • the peptide is effective at mitochondrial targeting, drugs related to various diseases targeting mitochondria can be delivered directly to the mitochondria, thereby improving drug absorption in the mitochondria.
  • the present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which a mitochondrial targeting peptide and a pharmacologically active substance are bound, which consists of the amino acid sequence of SEQ ID NO: 4.
  • the pharmacologically active substance is a substance capable of inducing a desired pharmacological effect by promoting or inhibiting a physiological function in an animal or human body, and refers to a chemical or biological substance or compound suitable for administration to an animal or human.
  • the pharmacologically active substance has a preventive effect of preventing unwanted biological effects such as preventing infection, alleviating pain or infection resulting from a disease, and can play a role of completely eliminating a disease, so that the pharmacologically active substance is a “drug ” or “therapeutic agent” may be used interchangeably.
  • the complex includes a peptide capable of directing a target to the mitochondria, so that a substance capable of acting on the mitochondria and exhibiting direct pharmacological activity can be effectively delivered to the mitochondria, moved and stayed there. Therefore, it can be usefully used for the prevention or treatment of diseases related to mitochondrial dysfunction.
  • the pharmacologically active substance may be a natural product, compound, protein, or gene (DNA, RNA or PNA) capable of acting on mitochondria to exhibit specific activity, but is not limited thereto.
  • the compound is doxorubicin (doxorubicin), epirubicin (epirubicin), idarubicin (idarubicin), docetaxel (docetaxel), paclitaxel (paclitaxel), cabazitaxel (cabazitaxel), vinblastine (vinblastine), vincristine (vincristine) And it may be one or more anticancer agents selected from the group consisting of cisplatin (cisplain), but is not limited thereto.
  • the compound is selected from the group consisting of flavonoids, polyphenols, resveratrol, carotenoids, alpha-lipoic acid, tocopherol and ascorbic acid. It may be one or more antioxidants, but is not limited thereto.
  • the compound may be an antibiotic, an anti-inflammatory agent, etc., but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases.
  • ALS amyotrophic lateral sclerosis
  • the present invention provides a STAT6 (Signal transducer and activator of transcription 6) protein, specifically, a mitochondrial dysfunction-related disease prevention or treatment pharmaceutical containing STAT6 protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient A composition is provided.
  • STAT6 Signal transducer and activator of transcription 6
  • STAT6 Signal transducer and activator of transcription 6
  • STAT6 Signal transducer and activator of transcription 6
  • the STAT6 protein may consist of an amino acid sequence represented by SEQ ID NO: 1.
  • the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.
  • the human STAT6 N-terminal 1-30 amino acid sequence among the human STAT6 internal sequences has a mitochondrial targeting effect.
  • the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11.
  • the mitochondrial membrane permeation domain may localize the STAT6 protein targeting the inside of the mitochondria to the outer mitochondrial membrane.
  • the 624-698 amino acid sequence among the human STAT6 internal sequence serves as a transmembrane domain that allows the protein to be located in the outer mitochondrial membrane, which is SEQ ID NO: 11 indicated as
  • the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the outer mitochondrial membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and tumor formation can be inhibited, the STAT6 protein can be utilized as a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.
  • composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be prepared according to a conventional method in the pharmaceutical field.
  • the drug delivery carrier or pharmaceutical composition may be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. It can be prepared by including more.
  • the "pharmaceutically acceptable” means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the mitochondrial targeting peptide or STAT6 protein according to the present invention. It may be selected differently depending on the dosage form and dosage form.
  • the carrier or pharmaceutical composition for drug delivery may be applied in any formulation, and more specifically, it may be formulated into oral formulations, topical formulations, suppositories, and parenteral formulations of sterile injection solutions according to conventional methods.
  • the solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may be included.
  • the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.
  • liquid formulations include suspensions, solutions, emulsions, syrups, etc.
  • various excipients for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. there is.
  • the parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • As the base of the suppository witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.
  • composition according to the present invention may further add calcium, vitamin D3, or the like to enhance therapeutic efficacy.
  • the pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.
  • the effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be administered to any animal capable of developing a disease related to mitochondrial dysfunction, and the animal may be, for example, not only humans and primates, but also cattle, pigs, horses, dogs, etc. of livestock and the like.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be administered by an appropriate administration route depending on the form of the formulation, and may be administered via oral or parenteral various routes as long as it can reach the target tissue.
  • the administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .
  • the pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of diseases related to mitochondrial dysfunction, or may be used in combination with surgery or other drug treatment.
  • the present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases, which contains STAT6 (Signal transducer and activator of transcription 6) protein including a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient.
  • STAT6 Signal transducer and activator of transcription 6
  • the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.
  • the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11, and the STAT6 protein targeting the inside of the mitochondria may be located in the outer mitochondrial membrane.
  • the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the mitochondrial outer membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and inhibits tumor formation. As a result, the STAT6 protein can be used as a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.
  • composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.
  • the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limitation in the form that the health functional food can take, It can include any food with meaning.
  • beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.
  • the health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. Whether or not it is suitable as a food additive can be judged according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
  • the items listed in the 'Food Additives Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.
  • chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid
  • natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum
  • Mixed preparations such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.
  • the other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. may further contain.
  • natural carbohydrate monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used.
  • natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.
  • the health functional food composition according to the present invention has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, unlike general drugs, and has excellent portability, preventing or improving mitochondrial dysfunction-related diseases It can be taken as a supplement for
  • Example 1 is a human STAT6 protein according to Example 1 of the present invention, in detail, the approximate domain constituting the human STAT6 protein (STAT6 wild type, STAT6 WT), STAT6 in which the amino acid sequence at positions 624-847 has been deleted ( TAD-deleted STAT6, TAD D), STAT6 with the 699-847 amino acid sequence deleted (699-847 aa-deleted STAT6, 699-847 D), and human STAT6 N-terminal sequence (1-50 amino acid sequence) it has been shown
  • sequences predicted to act as mitochondrial target sequences are green at the C-terminus, respectively.
  • sequences 1-30, sequences 31-50, sequences 1-20 are green at the C-terminus, respectively.
  • fluorescent protein Green fluorescent protein; GFP
  • 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids were prepared.
  • the specific preparation method of each plasmid is as follows.
  • the STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 2 and 3 in Table 1 to obtain human STAT6 N-terminal 1-30 insertion sequences (SEQ ID NO: 4).
  • SEQ ID NO: 2 AGATCTGCCGCCGCGATCGCCATGTCTCTGTGGGGTCTGGTCT (Forward)
  • SEQ ID NO: 3 GCGGCCGCGTACGCGTCAGAAGATGCCGCAGGTGT (Reverse)
  • pCMV6-AC-GFP Origene was also digested with restriction enzymes Sgf1 and Mlu1, and then the inserted sequences 1-30 were subcloned between the cut sequences.
  • the completed 1-30-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 5 and 6 in Table 2 to obtain human STAT6 N-terminal 31-50 insertion sequences (SEQ ID NO: 7).
  • SEQ ID NO: 5 AGATCTGCCGCCGCGATCGCCGGTGACTGGCTGGAGAGC (Forward)
  • SEQ ID NO: 6 GCGGCCGCGTACGCGTGCAGCAGAAGGCGTC (Reverse)
  • pCMV6-AC-GFP (Origene) was also digested with restriction enzymes Sgf1 and Mlu1, and the 31-50th insertion sequence was subcloned between the cut sequences.
  • the completed 31-50-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • the completed 1-20-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • the three types of plasmids and one type of control GFP plasmid (Origene) prepared by the method 1 above were transfected into human U373-MG malignant glioma cell line and mouse B16F10 melanoma cell line. Each GFP-attached amino acid was overexpressed.
  • the transfection method is as follows.
  • the culture medium of human U373-malignant glioma cell line and mouse B16F10 melanoma cell line distributed from Korea Cell Line Bank was exchanged with OPTI-MEM, treated with the above mixture, and cultured for 48 hours.
  • STAT6 WT TAD D, 699-847 D
  • FIG. 1 stable cell lines expressing each of the STAT6 proteins were prepared.
  • STAT6 TAD-deleted STAT6
  • STAT6 TAD-deleted STAT6
  • mCherry fluorescence was first attached to the C-terminus of human STAT6 protein (STAT6 WT) and STAT6 (MTS D) in which the mitochondrial target sequence was deleted, as shown in FIG. Mitochondrial labeling was stained with TOM20 protein present in the outer mitochondrial membrane.
  • tumor tissues were extracted from mice and the size of the tumor was compared.
  • the size of the tumor was larger and the weight of the tumor was significantly increased than in the case of the 293T parental cells. That is, it can be confirmed that inhibition of STAT6 expression induces tumorigenesis promotion.
  • STAT6 KO stable cell line prepared above
  • human STAT6 protein (STAT6 WT) and STAT6 (MTS D) from which the mitochondrial target sequence was deleted were overexpressed, respectively, to construct a stable cell line as shown in FIG. 11 .
  • the prepared STAT6 KO stable cell line was transplanted into the left leg of a mouse, and the stable cell line overexpressing human STAT6 protein (STAT6 WT) was transplanted into the right leg, and tumor growth was observed for 1 month. As shown in Fig. 12, it was confirmed that the size of the tumor was significantly smaller in the case of transplanting the cells overexpressing STAT6 WT again than in the case of transplanting the STAT6 KO cells.
  • the STAT6 KO stable cell line prepared above was transplanted into the left leg of a mouse, and a stable cell line re-expressing STAT6 (MTS D) with the mitochondrial target sequence deleted was transplanted into the right leg, and the tumor was grown for 1 month.
  • MTS D stable cell line re-expressing STAT6
  • the size of the tumor was measured once every 3 days, and after 1 month, the tumor was excised from the mouse and weighed, and the graph was shown.

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Abstract

La présente invention concerne une protéine STAT6 ciblant les mitochondries et son utilisation. Plus particulièrement, la présente invention concerne un peptide ciblant les mitochondries, une composition pharmaceutique comprenant un complexe dans lequel le peptide ciblant les mitochondries et une substance pharmaceutiquement active sont couplés, en tant que principe actif pour la prévention ou le traitement d'une maladie associée à un dysfonctionnement mitochondrial, une composition pharmaceutique comprenant la protéine STAT6 en tant que principe actif pour la prévention ou le traitement d'une maladie associée à un dysfonctionnement mitochondrial, et une composition alimentaire fonctionnelle de santé. La protéine STAT6 cible avec précision les mitochondries et, en tant que tel, peut migrer ou disperser de manière efficace une protéine ou un médicament d'intérêt dans les mitochondries, permettant de prévenir ou de traiter une maladie associée à un dysfonctionnement mitochondrial.
PCT/KR2021/000651 2020-01-16 2021-01-18 Protéine ciblant les mitochondries et son utilisation WO2021145743A1 (fr)

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KR10-2020-0005961 2020-01-16
KR1020200005961A KR102410556B1 (ko) 2020-01-16 2020-01-16 미토콘드리아 타겟팅용 펩타이드
KR1020210005717A KR102527406B1 (ko) 2021-01-15 2021-01-15 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 조성물
KR10-2021-0005717 2021-01-15

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014010985A1 (fr) * 2012-07-13 2014-01-16 한양대학교 산학협력단 Peptide pour l'introduction et la conservation hautement efficaces d'une protéine intracellulaire
KR20140032896A (ko) * 2012-08-31 2014-03-17 경희대학교 산학협력단 미토콘드리아 타겟팅 펩타이드
KR101490580B1 (ko) * 2013-02-15 2015-02-05 경북대학교 산학협력단 미토콘드리아 타겟팅용 펩타이드

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Publication number Priority date Publication date Assignee Title
WO2014010985A1 (fr) * 2012-07-13 2014-01-16 한양대학교 산학협력단 Peptide pour l'introduction et la conservation hautement efficaces d'une protéine intracellulaire
KR20140032896A (ko) * 2012-08-31 2014-03-17 경희대학교 산학협력단 미토콘드리아 타겟팅 펩타이드
KR101490580B1 (ko) * 2013-02-15 2015-02-05 경북대학교 산학협력단 미토콘드리아 타겟팅용 펩타이드

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AVALLE LIDIA; CAMPOREALE ANNALISA; MORCIANO GIAMPAOLO; CAROCCIA NATASCIA; GHETTI ELENA; ORECCHIA VALERIA; VIAVATTENE DANIELE; GIOR: "STAT3 localizes to the ER, acting as a gatekeeper for ER-mitochondrion Ca2+fluxes and apoptotic responses", CELL DEATH & DIFFERENTIATION, NATURE PUBLISHING GROUP, GB, vol. 26, no. 5, 24 July 2018 (2018-07-24), GB, pages 932 - 942, XP036861305, ISSN: 1350-9047, DOI: 10.1038/s41418-018-0171-y *
KHAN RASEL, LEE JASON E., YANG YANG-MING, LIANG FENG-XIA, SEHGAL PRAVIN B.: "Live-Cell Imaging of the Association of STAT6-GFP with Mitochondria", PLOS ONE, vol. 8, no. 1, 30 January 2013 (2013-01-30), pages e55426, XP055829856, DOI: 10.1371/journal.pone.0055426 *

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