WO2021025527A1 - Polypeptide ciblant les mitochondries et son utilisation - Google Patents

Polypeptide ciblant les mitochondries et son utilisation Download PDF

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WO2021025527A1
WO2021025527A1 PCT/KR2020/010489 KR2020010489W WO2021025527A1 WO 2021025527 A1 WO2021025527 A1 WO 2021025527A1 KR 2020010489 W KR2020010489 W KR 2020010489W WO 2021025527 A1 WO2021025527 A1 WO 2021025527A1
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polypeptide
slc1a5
mitochondria
syndrome
mitochondrial
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PCT/KR2020/010489
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Korean (ko)
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한정민
유희찬
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention relates to a polypeptide for targeting mitochondria and its use.
  • This study is related to the identification of the role of ASCT2 variants in cancer metabolism conducted by Yonsei University with funding from the Ministry of Education in 2018-2019 with the support of the National Research Foundation of Korea, and the development of antagonists for them as anticancer drugs (No. 1345279574).
  • this study is related to Yonsei University's Comprehensive Pharmacy Research Institute (No. 1345295623) conducted by Yonsei University with support from the National Research Foundation of Korea with the funding of the Ministry of Education in 2019-2020.
  • Mitochondria play a key role in many critical intracellular processes, including energetic metabolism and metabolism of certain substances (eg, fatty acids) within cells.
  • mitochondria are directly involved in the formation and use of free radicals (hereinafter referred to as'FR') and reactive oxygen species (hereinafter referred to as'ROS'). Because of this, it has been reported that mitochondria play a key role in the process of programmed cell death in relation to the reactive moieties that can affect many processes in living cells.
  • Non-Patent Document 1 mitochondrial abnormalities may be the cause of the development of type 2 diabetes, a metabolic syndrome.
  • These diseases which are caused by dysfunction of mitochondria, are caused by abnormalities in the energy supply system of cells, and most of them are accompanied by muscle diseases and brain diseases.
  • an effective concentration of the substance can be obtained by repeatedly accumulating the desired substance in the target target, increasing the application efficiency and reducing the overall dosage, thereby reducing the likelihood and intensity of side effects. There is a strong point.
  • Patent Document 1 US Patent No. 7,109,189
  • An object of the present invention is to provide a polypeptide targeting mitochondria.
  • Another object of the present invention is to provide a composition and a method for detecting mitochondria comprising the polypeptide.
  • Another object of the present invention is to provide a composition for imaging mitochondria.
  • Another object of the present invention is to provide a mitochondrial-specific drug delivery composition comprising the polypeptide.
  • Another object of the present invention is to provide a polynucleotide encoding the polypeptide, a recombinant vector comprising the same, and a cell transformed with the recombinant vector.
  • An aspect of the present invention for achieving the above object is a polypeptide comprising the 27th to 46th amino acid in the amino acid sequence represented by SEQ ID NO: 1; Or a variant having a sequence homology of 90% or more with the polypeptide; which includes, provides a polypeptide.
  • polypeptide may be for mitochondrial targeting, but is not limited thereto.
  • a specific transcriptional variant derived from the SLC1A5 (solute carrier family 1 member 5) gene targets mitochondria after protein translation (targeting ) was first identified.
  • the "transcript variant of SLC1A5" is a mitochondrial targeting sequence and glutamine transporter essential for the activity of the glutamine transporter in mitochondria It may mean a protein having activity.
  • the polypeptide may include one or more selected from the amino acid sequence represented by SEQ ID NO: 3, 5 or SEQ ID NO: 8, and more specifically, may include the amino acid sequence of SEQ ID NO: 8, but is not limited thereto. .
  • peptide is used interchangeably with “protein” or “polypeptide”, and, for example, refers to a polymer of amino acid residues as commonly found in proteins in nature.
  • the polypeptide of the present invention may be derived from nature, and may be synthesized using a known peptide synthesis method (genetic engineering method, chemical synthesis). Construction by a genetic engineering method, for example, according to a conventional method, a nucleic acid encoding the peptide or a functional equivalent thereof (eg, a polynucleotide of SEQ ID NO: 4, 6 or SEQ ID NO: 9 is constructed. Nucleic acids can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences can be prepared by standard methods known in the art, for example, using an automatic DNA synthesizer (available from Biosearch or Applied Biosystems).
  • the constructed nucleic acid is operatively linked thereto to be inserted into a vector containing one or more expression control sequences (eg, promoters, enhancers, etc.) that control the expression of the nucleic acid.
  • expression control sequences eg, promoters, enhancers, etc.
  • the recovery can be carried out using a method known in the art (eg, chromatography)
  • "substally pure peptide” means that the peptide according to the present invention is derived from cells.
  • the genetic engineering method for synthesizing the peptides of the present invention may refer to the following documents: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymolo gy, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.
  • polypeptides of the present invention can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press). , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).
  • polypeptides of the present invention include the functional equivalents and salts thereof of the polypeptides of the present invention described above.
  • the "functional equivalent” refers to having sequence homology (ie, identity) of at least 80% or more, specifically 90%, and more specifically 95% or more with the aforementioned polypeptide of the present invention, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, 100% sequence homology, and refers to a peptide exhibiting substantially the same physiological activity as the polypeptide of the present invention.
  • substantially homogeneous physiological activity refers to an activity targeting mitochondria (ie, activity to move, distribute and/or bind to mitochondria).
  • the functional equivalent may be one generated as a result of addition, substitution or deletion of some of the amino acid sequences of the polypeptide of the present invention described above.
  • the amino acid substitution in the above is preferably a conservative substitution.
  • conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met).
  • the functional equivalent includes a variant in which a part of an amino acid is deleted from the amino acid sequence of the polypeptide of the present invention.
  • the deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity (mitochondrial targeting) of the polypeptide provided by the present invention.
  • variants in which several amino acids are added in the sequence or at both ends of the amino acid sequence of the polypeptide of the present invention are also included.
  • the scope of the "functional equivalent” of the present invention includes derivatives in which some of the chemical structures of the peptides are modified while maintaining targeting activity on the basic skeleton and mitochondria of the polypeptide of the present invention. This includes, for example, structural changes to alter the stability, storage, volatility or solubility of the peptide.
  • the peptide of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.
  • One letter (three letters) of amino acids used in the present invention means the following amino acids according to the standard abbreviation regulations in the field of biochemistry:
  • amino acid (one amino acid position) (one amino acid position)' as used in the specification of the present invention means that the amino acid previously indicated at the corresponding amino acid position of the natural (wild type) polypeptide is substituted with the amino acid indicated later.
  • R44A represents a point mutation in which arginine at the 44th amino acid sequence is substituted with alanine.
  • Another aspect of the present invention provides a composition for detecting mitochondria comprising the polypeptide.
  • the polypeptide of the present invention may be provided in a labeled state to facilitate the identification, detection, and quantification of the migration, distribution, or/and binding of the polypeptide into the mitochondria.
  • the detectable label is a chromogenic enzyme (eg peroxidase, alkaline phosphatase), a radioactive isotope (eg 18 F, 123 I, 124 I, 125 I, 32 P, 35 S, 67 Ga), and A chromophore, a luminescent material, or a fluorescent material (e.g., FITC, RITC, Fluorescent Protein (GFP); EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein)); DsRed (Discosoma sp.
  • a chromogenic enzyme eg peroxidase, alkaline phosphatase
  • a radioactive isotope eg 18 F, 123 I, 124 I, 125 I, 32 P, 35 S, 67 Ga
  • a chromophore e.g., FITC, RITC, Fluorescent Protein (GFP); EGFP (Enhanced Green Fluorescent Protein), RFP (Red
  • red fluorescent protein CFP (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), Cy3, Cy5, and Cy7.5
  • magnetic resonance imaging material e.g. Gadolinium (Gd, gadolinium), paramagnetic It may be particles (super paramagnetic particles) or super paramagnetic particles (ultrasuper paramagnetic particles).
  • the detection method according to the label is widely known in the art, but may be performed, for example, by the following method. If a fluorescent substance is used as a detectable label, immunofluorescence staining can be used. For example, after reacting the peptide of the present invention labeled with a fluorescent substance with a sample and removing unbound or non-specific binding products, fluorescence by the peptide can be observed under a fluorescence microscope. In addition, when an enzyme is used as a detectable label, the absorbance is measured by a color reaction of a substrate through an enzymatic reaction, and in the case of a radioactive substance, the radiation emission amount can be measured. In addition, the detected result may be imaged according to a known imaging method according to the detection mark.
  • Another aspect of the present invention (a) mixing the polypeptide with a sample; (b) removing the unbound or non-specifically bound polypeptide; And (c) it provides a method for detecting mitochondria comprising the step of confirming the binding of the polypeptide and the position.
  • step (c) the movement, distribution, or/and binding of the polypeptide of the present invention into the mitochondria and the location of the polypeptide of the present invention are confirmed by a method known in the art with reference to the description in the composition for detecting mitochondria. It can be done by detecting the polypeptide.
  • sample may mean a biological sample, and for example, may be any one selected from the group consisting of a cell sample, a biopsy sample, a solid tissue sample such as tissue culture, blood, and the like, but is not limited thereto.
  • the sample can be pretreated prior to use for detection. For example, it may include extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
  • composition for imaging mitochondria comprising the polypeptide.
  • intracellular mitochondria can be imaged in vitro or in vivo together with any labeling means (imaging means). Although not limited thereto, for example, it is possible to diagnose or monitor morphological abnormalities of mitochondria and related diseases according to the imaging.
  • the imaging and diagnosis of the mitochondrial-related disease is not limited thereto, but may include the purpose of the initial examination of the disease, the progress of the disease, the course of treatment for the treatment, and monitoring the response to the treatment.
  • the peptide of the present invention may be provided in a labeled state in order to facilitate identification, detection, quantification, etc. of migration, distribution or/and binding in the mitochondria.
  • Another aspect of the present invention provides a mitochondrial-specific drug delivery composition comprising the polypeptide.
  • the peptide may be in a state associated with a drug
  • the drug may be one or more selected from the group consisting of a pharmacologically active compound, a polypeptide, or a polynucleotide, but is not limited thereto.
  • the linkage of the drug (or drug preparation) and the polypeptide of the present invention may be performed through a method known in the art, such as covalent bonding, crosslinking, and the like.
  • the polypeptide of the present invention may be chemically modified within a range in which its activity is not lost.
  • the linkage is meant to include both direct bonds (eg, by covalent bonds) between the drug and the polypeptide of the present invention, as well as indirect bonds including a linker or the like.
  • the amount of the peptide of the present invention contained in the composition of the present invention may vary depending on the type and amount of the therapeutic agent to be bound.
  • the drug may be a drug used for the prevention or treatment of diseases related to mitochondrial dysfunction.
  • the disease associated with the mitochondrial dysfunction is a state in which the biological activity of mitochondria in normal cells is decreased or increased, but is not limited thereto, but is, for example, a disease caused by mutation, deletion, or rearrangement of mitochondrial DNA; Diseases caused by the nuclear-coding defective protein component of the mitochondrial respiratory chain; Age-related diseases; Diseases resulting from administration of cytotoxic cancer chemotherapeutic agents; Diseases caused by defects in activity of mitochondrial complexes I, II, III, IV or V; Congenital mitochondrial disease; Neurodegenerative diseases; Neuromuscular degenerative diseases; And cancer diseases.
  • the disease is a decrease in mitochondrial enzyme activity, decrease in electron transport chain activity, decrease in membrane potential, increase in production of reactive oxygen species, mitochondria fragmentaion, calcium dysregulation, And mitochondrial DNA (mtDNA) may be due to any one mitochondrial dysfunction selected from the group consisting of mutations, but is not limited thereto.
  • Such diseases include cancer, Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophy, muscular dystrophy, chronic fatigue syndrome, Friedrich's ataxia, epilepsy, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neuropathy.
  • Intestinal dysfunction sensorineural hearing loss, nerve-derived bladder dysfunction, migraine, ataxia, renal tubular acidosis, dilated cardiomyopathy, steatohepatitis, liver failure, lactic acidemia, mitochondrial encephalopathy with lactic acidemia and strokelike episodes; MELAS), Leber's hereditary optic neuropathy (LHON), MERRF syndrome (Myoclonic Epilepsy with Ragged-Red Fibers syndrome), MNGIE syndrome (Mitochondrial neurogastrointestinal encephalopathy syndrome), NARP syndrome ((neuropathy, ataxia) retinitis pigmantosa), Barth Syndrome, Leigh Syndrome, Kearns-Sayre syndrome, degenerative brain disease, Multiple Sclerosis-like Syndrome, Maternally hereditary cardiomyopathy Inherited CardioMyopathy), Progressive External Ophthalmoplegia, Pearson Marrow syndrome, Aminoglycoside-associated deafness, Diabetes with deafness, Lucas disease ), Alpers Disease, medium chain acyl-CoA
  • the drug provides a drug used for the prevention or treatment of diseases related to mitochondrial dysfunction together with the polypeptide of the present invention, so that the drug can act more efficiently. Therefore, the composition of the present invention can be usefully used in the prevention or treatment of diseases related to mitochondrial dysfunction.
  • the drug may be an antioxidant.
  • antioxidants are acetylcysteine (N-Acetylcysteine), glutathione (glutathione), SOD-like (SOD-mimicking) peptide, Zeto-Schiller-peptide (Szeto-Schiller-peptides), vitamin E (Vitamine E). It may be one or more selected from the group, but is not limited thereto.
  • the drug may be an anticancer agent.
  • the anticancer agent is gemcitabine, paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D (actinomycin- D), docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplain, 5-flo 5-fluouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard (nitrogen mustard), nitrosourea, streptokinase, urokinase,reteplase, angiotensin II inhibitor, aldosterone receptor inhibitor, eryopothrietin , NMDA (N-methyl-d-aspartate) receptor inhibitor, Lovastatin, Rapamycin, Celebrex, Ticlopin Marim
  • composition for drug delivery of the present invention may be formulated with a suitable carrier according to the route of administration.
  • compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the effective amount of the drug delivery composition may be administered through various routes.
  • "effective amount” refers to an amount of substance that enables diagnosis or tracking of therapeutic effects when administered to a patient.
  • the dosage of the composition according to the present invention may be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex, weight, individual differences, and disease states.
  • composition containing the polypeptide of the present invention can vary the content of the active ingredient depending on the severity of the disease, but is usually administered several times a day in an effective dose of 1 mg to 1000 mg per administration based on an adult. It can be administered repeatedly.
  • the route of administration of the composition according to the present invention is not limited thereto, but may be administered orally or parenterally.
  • the parenteral route of administration may include, for example, transdermal, nasal, abdominal, intramuscular, subcutaneous or intravenous routes.
  • composition of the present invention may be formulated in various ways according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier.
  • 'Pharmacologically acceptable refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans, and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions.
  • the carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
  • the composition of the present invention may be formulated according to a method known in the art in the form of an injection, a transdermal administration and a nasal inhalation agent together with a suitable parenteral carrier.
  • the injection must be sterilized and protected from contamination by microorganisms such as bacteria and fungi.
  • suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can.
  • the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
  • composition according to the present invention may include one or more buffers (e.g. saline or PBS), carbohythrate (e.g. glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.
  • buffers e.g. saline or PBS
  • carbohythrate e.g. glucose, mannose, sucrose or dextran
  • antioxidants e.g. glucose, mannose, sucrose or dextran
  • bacteriostatic agents e.g.
  • chelating agents e.g. For example, EDTA or glutathione
  • adjuvants e.g, aluminum hydroxide
  • suspending agents e.g., aluminum hydroxide
  • thickening agents and/or preservatives may further be included.
  • Another aspect of the present invention provides a polynucleotide encoding the polypeptide for targeting mitochondria of the present invention.
  • the polynucleotide is not particularly limited in base combination of the polynucleotide as long as it can encode the polypeptide of the present invention.
  • the polynucleotide may be provided as a single-stranded or double-stranded nucleic acid molecule including all DNA, cDNA and RNA sequences.
  • polynucleotide encoding the polypeptide represented by SEQ ID NO: 3 may include the nucleotide sequence of SEQ ID NO: 4, and the polynucleotide encoding the polypeptide represented by SEQ ID NO: 5 represents the nucleotide sequence of SEQ ID NO: 6
  • the polynucleotide encoding the polypeptide represented by SEQ ID NO: 8 may include the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.
  • Another aspect of the present invention provides a recombinant vector comprising the polynucleotide.
  • the vector of the present invention may include, but is not limited to, a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector.
  • the vector of the present invention may be a recombinant viral vector.
  • the recombinant viral vector of the present invention may be used without limitation as long as it is a viral vector commonly used to deliver genes in the field of gene therapy.
  • the recombinant viral vector may be selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, a retroviral vector, a herpes virus vector, a lentiviral vector, a vaccinia virus vector, and a poxvirus vector.
  • AAV adeno-associated virus
  • the recombinant viral vector in the present invention may preferably be an adenovirus vector.
  • Adenovirus is treated in the field of gene therapy due to its medium genome size, ease of genetic manipulation and manufacture, ease of production and separation due to high titer, and high infection efficiency with a wide range of target cells. It is widely used as a carrier for delivering the yong gene. For gene therapy, adenoviruses, which lack the ability to self-replicate and produce viruses, are widely used.
  • the vector of the present invention may be a conventional cloning vector or expression vector, and the expression vector is for membrane targeting or secretion in addition to expression control sequences such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer (promogene). It includes a signal sequence or a leader sequence and can be prepared in various ways according to the purpose.
  • the polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, and the operably linked gene sequence and expression control sequence are one expression including a selection marker and a replication origin. It can be contained within a vector. “Operably linked” can be a gene and expression control sequence linked in a manner that allows gene expression when an appropriate molecule is bound to an expression control sequence.
  • “Expression control sequence” means a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters to effect transcription, any operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation.
  • the vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable vector, it includes a source of replication.
  • the vector provided in the present invention includes a promoter, the polynucleotide of the present invention, and a polynucleotide encoding the protein of interest, and the promoter, the polynucleotide of the present invention and the gene encoding the protein of interest are operably linked It can be a recombinant vector.
  • the "target protein” refers to a polypeptide molecule intended for movement, delivery, distribution, or/and binding into the mitochondria in practicing the present invention by those skilled in the art, but is not limited thereto, but an example protein; And it may be selected from the group consisting of disease treatment proteins.
  • the recombinant vector may further include a labeling means (a gene encoding the labeling protein), which is also operably linked.
  • the recombinant vector is a promoter, a polynucleotide encoding the amino acid sequence of the polypeptide of the present invention (representative example, SEQ ID NO: 3, 5 or SEQ ID NO: 8), a gene encoding the protein of interest, and a marker protein (e.g. For example, a fluorescent protein) gene can be operably linked.
  • a marker protein e.g. For example, a fluorescent protein
  • Another aspect of the present invention provides a method of moving or/and distributing a protein of interest to mitochondria comprising the step of introducing the recombinant vector into cells.
  • This method may be performed, including the following steps, for example:
  • a promoter a polynucleotide encoding the amino acid sequence of the polypeptide of the present invention (representative example, SEQ ID NO: 3 or SEQ ID NO: 5), a gene encoding a protein of interest, and a gene encoding a label protein (these work
  • the cell type is not particularly limited as long as it has mitochondria, and may preferably be a eukaryotic cell, particularly a human or mammalian cell.
  • the cells may be isolated from an individual.
  • introduction of a recombinant vector (especially hybrid plasmid) into a cell can be understood as transforming a cell using a recombinant vector, and the transformation method is well known to those skilled in the art. Method can be used. For example, microprojectile bombardment, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion, microinjection ) And a liposome-mediated method, but are not limited thereto.
  • CaPO4 calcium phosphate
  • CaCl2 calcium chloride
  • PEG-mediated fusion calcium chloride
  • microinjection microinjection
  • a liposome-mediated method but are not limited thereto.
  • the fluorescence of a specific wavelength emitted from the protein expressed in the transformed cell can be continuously imaged using a fluorescence microscope. By doing so, it is possible to visualize the expression process of the target protein in the cell and the migration process to the mitochondria in detail step by step.
  • Another aspect of the present invention provides a cell transformed with the vector.
  • transformation with the vector can be performed by a transformation technique known to those skilled in the art, which is referred to as the foregoing.
  • the term'cell' may be used interchangeably with'transformants', etc., and introduced into cells by any means (e.g., the aforementioned electric shock method, calcium phosphatase precipitation method, microinjection method, viral infection, etc.) It means a cell containing heterologous DNA (in the present invention, preferably a eukaryotic cell).
  • the transformant is all kinds of single-celled organisms commonly used in the field of cloning, such as prokaryotic microorganisms such as various bacteria (eg, Clostridia genus, E. coli, etc.), lower eukaryotic microorganisms such as yeast, and insect cells.
  • prokaryotic microorganisms such as various bacteria (eg, Clostridia genus, E. coli, etc.)
  • lower eukaryotic microorganisms such as yeast
  • insect cells Cells derived from higher eukaryotes including, plant cells, mammals, and the like may be used as cells, but are not limited thereto. Since the expression level and modification of the protein differ depending on the cell, one of ordinary skill in the art can select and use the most suitable cell for the purpose.
  • microorganisms used as transformants in the present invention are Escherichia coli , Bacillus subtilis , Streptomyces spp., Pseudomonas spp., Proteus mirabilis, and Proteus mirabilis. It may be Proteus mirabilis , a microorganism of the genus Staphylococcus spp., Agrobacterium tumefaciens , and the like, but is not limited thereto.
  • polypeptide of the present invention exhibits a feature that is targeted to mitochondria, it can be utilized as an application for delivering a target substance to mitochondria by using it.
  • Figure 1 shows the two novel SLC1A5 transcript variants, SLC1A5 and SLC1A5_var, identified in the present invention (A: the constitution of the exon and intron of the human SLC1A5 gene, and the two transcript variants SLC1A5 (NM_005628. 2) and SLC1A5_var (NM_001145145.1)
  • A the constitution of the exon and intron of the human SLC1A5 gene, and the two transcript variants SLC1A5 (NM_005628. 2) and SLC1A5_var (NM_001145145.1)
  • B As the exon structure of the mRNA transcripts of SLC1A5 (blue) and SLC1A5_var (red), the binding site of siRNA and the RT-PCR amplification product are indicated).
  • FIG. 2 shows the results of analyzing the expression pattern of SLC1A5_var in various cancer cells (A, B: pancreatic cancer cell line; C, D: colon cancer cell line; E, F: lung cancer cell line).
  • FIG. 3 shows the results of confirming the distribution of SLC1A5_var in the mitochondria in the cells by immunofluorescence (A: results of confirming HeLA cells transformed with HA-taggged SLC1A5_var or HA-taggged SLC1A5 by immunofluorescence.
  • A results of confirming HeLA cells transformed with HA-taggged SLC1A5_var or HA-taggged SLC1A5 by immunofluorescence.
  • B SLC1A5_var and cells Quantitative analysis of the presence of organelle markers using Zen colocalization analysis).
  • Figure 4 shows the results of confirming the distribution of SLC1A5_var in the mitochondria in the cell through the cell organelle fractionation experiment (A: immunoblot result of the cell organelle fractionation experiment of SLC1A5_var obtained from MiaPaCa2 cells. B: Mitochondria of MiaPaCa2 against SLC1A5_var Organelle fractionation test results).
  • Figure 5 shows the experimental results confirming that the SLC1A5 transcript variant is a mitochondrial glutamine transporter targeting mitochondria isolated from MiaPaCa2 cells expressing the control vector, SLC1A5, SLC1A5_var, or SLC1A5_var D186A mutation
  • A time course Degree of glutamine uptake (Gln uptake) according to B: degree of amino acid uptake
  • C control vector, SLC1A5, SLC1A5_var, or SLC1A5_var The degree of glutamine uptake in the case of treatment with siRNA for the D186A mutation
  • D siRNA The degree of amino acid uptake after treatment
  • E Experimental results showing that inhibitors of the SLC1A5 transcript variant inhibit the glutamine transporter activity of the SLC1A5 transcript variant
  • F ⁇ -KG levels and mitochondria in each cell, in the whole cell side ⁇ -KG level at).
  • FIG. 6 shows the results of testing whether or not mitochondrial targeting of polypeptide fragments isolated from SLC1A5_var protein was tested (A: Preliminary results of predicting the mitochondrial target region of SLC1A5_var total protein using the rediSi program. B: Mitochondrial targeting of the present invention.
  • Figure 7 shows each experimental group and control polypeptide conjugated to the N-terminus of GFP (representatively, NT-WT fragment, NT_3A fragment, NT_2A fragment, NT_(1-26) fragment, NT_(27-46) fragment, and CT. Fragment) is shown in the confocal microscopy (confocal microscopy) observation image of the transformed living HeLa cells.
  • the human SLC1A5 gene consists of eight exons, and there are two transcript variants with different transcription start sites (NM_005628.2 and NM_001145145.1; Fig. 1A).
  • Long-length transcript variants (SLC1A5/ASCT2, NM_005628.2) lack exon 2 and consist of 541 amino acids
  • short transcript variants (SLC1A5_var, NM_001145145.1) lack exon 1, 339 It consists of dog amino acids (Fig. 1B).
  • the SLC1A5 transcript variant was named SLC1A5_var.
  • the mRNA level of each transcript variant was analyzed using RT-PCR.
  • RNA for the RT-PCR was isolated using an RNA extraction kit (MiniBEST Universal RNA Extraction Kit, Takara) and synthesized into cDNA using a cDNA synthesis kit (PrimeScriptTM 1st strand cDNA Synthesis Kit, Takara).
  • the synthesized cDNA uses a primer set consisting of primers having nucleotide sequences of SEQ ID NOs: 10 to 13 shown in Table 1 below, and reverse transcription polymer chain reaction (RT) at 95° C., 45° C. and 72° C. After amplification through PCR), the reaction result was confirmed through 1% agarose gel electrophoresis method. The results were quantitatively analyzed using ImageJ software and GAPDH was used as a quantification criterion.
  • SLC1A5_var in all pancreatic cancer cell lines was increased compared to that of normal pancreatic ductal epithelial cells (HPDE), and more particularly, more overexpressed in Panc-1, MiaPaCa-2, AsPC1, and Panc10.05 cell lines. It was confirmed (A, B in Fig. 2). In addition, even in various colon cancer cell lines, the expression level of SLC1A5_var was measured higher than that of human colon epithelial cells (FHC) (FIG. 2C and D).
  • HPDE pancreatic ductal epithelial cells
  • FHC human colon epithelial cells
  • SLC1A5_var unlike SLC1A5_var, SLC1A5 did not increase its expression in pancreatic or colon cancer cell lines.
  • the intracellular distribution pattern of the SLC1A5_var transcript variant protein which was confirmed to have a high expression level in cancer cells, was confirmed.
  • HeLa cells were transformed by conjugating HA-tag to the cDNA of the SLC1A5_var transcript mutant, and the colocalization pattern with organelle markers was analyzed.
  • the cells used in the experiment were HeLa cells and were labeled with anti-Cox4 antibody, anti-Na+-K+ ATPase antibody, anti-ERp72 antibody, anti-GM130 antibody, and anti-LAMP2 antibody as primary antibodies, respectively, after fixation with methanol. Thereafter, labeling was performed using an antibody labeled with Alexa-488 or Alexa-594 fluorescence as a secondary antibody. Cell nuclei were stained with DAPI and then observed with a confocal microscope. To determine the degree of image overlap, 10 or more images per sample were analyzed using Zen imaging software.
  • the SLC1A5_var protein containing the HA-tag was observed to coexist with the mitochondrial marker (COX4), but the cell membrane (Na, K-ATPase), endoplasmic reticulum (ERp72), Golgi apparatus (GM130) or Ly It was confirmed that the SLC1A5_var protein was present in the mitochondria because the expression pattern was different from that of markers such as smallsome (LAMP2) (Fig. 3A, B).
  • the SLC1A5_var protein is present in the mitochondria, and an organelle fractionation experiment was performed in order to examine the distribution pattern in more detail (FIG. 4).
  • the mitochondrial inner membrane was separated under low osmotic pressure conditions.
  • the mitochondrial fraction was put in a swelling buffer (10M KH 2 PO 2 , digitonin 2 mg/ml, pH 7.4) and then stored on ice for 1 hour. And the same volume of iso-osmotic solution (32% sucrose, 30% glycerol, and 10mM MgCl 2 ) was added. Thereafter, centrifugation was performed at 10000 g and 10 min, and the resulting supernatant was used as the mitochondrial outer membrane fraction, and the pellet was used as the mitochondrial inner membrane and matrix fraction.
  • the pellet was resuspended again using a swelling buffer without digitonin, stored on ice for 1 hour, and the same volume of iso-osmotic solution was added again, followed by centrifugation at 17000 g for 1 hour. After separation, the supernatant was used as a matrix fraction and the pellet was used as a mitochondrial inner membrane fraction.
  • Immunoblotting was performed on the mitochondrial organelle fraction.
  • cells were crushed using lysis buffer (40mM HEPES at pH 7.4, 0.5% Triton X-100, 10mM ⁇ -glycerol phosphate, 10mM pyrophosphate, 2.5mM MgCl 2 ) and ultrasonic waves.
  • PNGase F was treated for observation of SLC1A5_var.
  • the sample was not boiled, and at least about 30 ug of protein per sample was separated by SDS-PAGE.
  • the primary antibody corresponding to each protein is treated at 4°C for 8 hours, and the secondary antibody with HRP that recognizes each primary antibody is attached to each existing membrane. Protein was identified.
  • the SLC1A5_var protein was isolated together with Tim23, a marker of the mitochondrial inner membrane, but was fractionated independently from Tom20, a marker of the outer mitochondrial membrane, and MnSOD2, a marker of the mitochondrial matrix (FIG. 4B). Therefore, it was clearly confirmed that the SLC1A5_var protein was distributed in the mitochondria and the SLC1A5 protein was distributed in the cell membrane.
  • SLC1A5_var or SLC1A5_var D186A were stably expressed in MiaPaCa2 cells, which are pancreatic cancer cell lines, respectively, and glutamine uptake activity in mitochondria was measured.
  • Example 3 After obtaining a mitochondrial fraction by the method of Example 3, resuspended in a buffer containing 10 mM NaCl, 100 mM glutamine, 100 mM serine, 100 mM alanine or 100 mM glutamic acid in KPBS buffer, and then stored at 37°C. Started amino acid absorption. Thereafter, 20 mM HgCl 2 was added to terminate the reaction, and after completion, each sample was centrifuged for 10000 g and 5 min. Thereafter, the supernatant was taken and the remaining amino acids consumed compared to the first were measured using an amino acid assay kit (Glutamine assay kit, Serine assay kit, Alanine assay kit, Glutamate assay kit, Biovision). The measured value was corrected by measuring the amount of mitochondrial protein in each sample and using it as a reference value for quantification.
  • an amino acid assay kit Glutamine assay kit, Serine assay kit, Alanine assay kit, Glutamate assay kit, Biovision
  • each of SLC1A5 and SLC1A5_var was knocked down using siRNA.
  • the control siRNA was composed of SEQ ID NO: 14
  • the siRNA against SLC1A5 was composed of SEQ ID NO: 15
  • the siRNA against SLC1A5_var was composed of the nucleotide sequence represented by SEQ ID NO: 16.
  • the uptake of glutamine was inhibited in mitochondria isolated from cells that inhibited the overexpression of SLC1A5_var, but this phenomenon was not observed in mitochondria isolated from cells that suppressed the overexpression of SLC1A5 (Fig. 5C).
  • the same results were observed for alanine and serine (FIG. 5D).
  • the inhibitors are known HgCl 2 also mitochondrial glutamine absorption as that was shown to inhibit mitochondrial glutamine absorption in which the basal levels (basal level) and SLC1A5_var parameter, terminating the transport induced by SLC1A5 in proteosome liposomes (proteoliposome) Suppressed (Fig. 5E).
  • SLC1A5_var is a mitochondrial glutamine transporter.
  • SLC1A5_var protein is present in the mitochondrial inner membrane, whether SLC1A5_var contains a mitochondrial targeting sequence (MTS), and if so, where is its location and separates it. It was confirmed whether it can be used alone.
  • MTS mitochondrial targeting sequence
  • the present inventors used the PrediSi program (http://www.predisi.de) to identify the main motifs for mitochondrial targets in the N-terminal amino acid region from the 39th to the 51st amino acid region of the entire SLC1A5_var protein. It was checked for existence.
  • NT_(27-46) in Fig. 6B consisting of positively charged amino acids (arginine, R; lysine, K) immediately following a non-hydrophobic ⁇ -helical structure is targeted to mitochondria. It was confirmed that it is a key sequence for.
  • FIG. 6B shows the location of the fragment corresponding to the N-terminal 1 to 46aa (NT-WT fragment) from the total protein SLC1A5_var and the C-terminal 235 to 339aa fragment (CT fragment) used as a control, and the basicity of the NT-WT fragment.
  • the R9A/R15A/K17A mutation (referred to as NT_3A) and the R44A/K45A mutation (referred to as NT_2A) introducing a point mutation in the amino acid are shown.
  • NT-WT fragment was divided in half, and a fragment of NT_(1 ⁇ 26) which is the first half of the N-terminal and NT_(27 ⁇ 46) which is the second half of the N-terminus were respectively shown.
  • the sequence of each fragment described above is specifically described in Table 2 below.
  • EGFP Enhanced green fluorescent protein, GenBank: AFA52654.1
  • GenBank: AFA52654.1 Enhanced green fluorescent protein
  • cells were used for confocal microscopy only. While culturing in a cell culture dish, each cell was transfected with a control vector or a plasmid capable of expressing NT_WT, NT_3A, NT_2A, NT_(1 ⁇ 26), NT_(27 ⁇ 46) and CT and EGFP, which are a part of SLC1A5_var. (transfection). After 48 hours, mitochondria were labeled with MitoTracker Red, a reagent capable of staining mitochondria, and observed with a confocal microscope.
  • MitoTracker Red a reagent capable of staining mitochondria
  • EGFP bound to the NT-WT fragment and NT_3A (R9A/R15A/K17A mutation) fragment was targeted to mitochondria
  • EGFP bound to the NT_2A (R44A/K45A mutation) fragment was not targeted to mitochondria. It was not and was dispersed in the cytoplasm.
  • NT_(1 ⁇ 26) consisting of the 1st to 26th amino acid sequence consisting of the hydrophilic amino acid sequence of the first half of NT-WT was not targeted to the mitochondria, but NT_(27 ⁇ 46) consisting of the hydrophobic amino acid sequence and the positively charged amino acid of the latter half was targeted to mitochondria, and it was confirmed that the fragment corresponds to the smallest unit fragment targeted to mitochondria.
  • Figure 8 shows the results of quantitatively evaluating the colocalization of the mitochondrial marker Mitotracker and each fragment on a confocal microscope, and as shown in Figure 8, the NT-WT fragment, NT_3A (R9A/R15A/K17A mutation) ) Fragment and NT_(27 ⁇ 46) confirmed that the mitochondrial targeting effect was remarkably excellent.
  • EGFP conjugated to NT_WT fragment, NT_3A fragment or NT_(27 ⁇ 46) fragment was detected with COX4 in the fraction from which mitochondria are separated, whereas NT_2A fragment EGFP conjugated to or control EGFP and NT_(1-26) were detected in a fraction from which the cytoplasm or endomembrane was separated.
  • NT_(27 ⁇ 46) fragment having the amino acid sequence of SEQ ID NO: 8 is a key sequence for mitochondrial targeting.

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Abstract

La présente invention concerne un polypeptide ciblant les mitochondries et son utilisation.
PCT/KR2020/010489 2019-08-07 2020-08-07 Polypeptide ciblant les mitochondries et son utilisation WO2021025527A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007071962A1 (fr) * 2005-12-23 2007-06-28 Medical Research Council Ciblage de polypeptides sur les mitochondries
WO2012174452A1 (fr) * 2011-06-17 2012-12-20 Shire Human Genetic Therapies, Inc. Ciblage mitochondrial et utilisation thérapeutique associée
KR20130109966A (ko) * 2010-05-14 2013-10-08 애브비 인코포레이티드 Ｉｌ－１ 결합 단백질
KR20150131360A (ko) * 2013-03-15 2015-11-24 애브비 인코포레이티드 ＴＮＦα에 대해 지시된 이원 특이적 결합 단백질

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US6984636B2 (en) 2002-08-12 2006-01-10 Medical Research Council Mitochondrially targeted antioxidants
JP5812869B2 (ja) * 2010-01-15 2015-11-17 協和発酵キリン株式会社 抗システムａｓｃアミノ酸トランスポーター２（ａｓｃｔ２）抗体

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Publication number Priority date Publication date Assignee Title
WO2007071962A1 (fr) * 2005-12-23 2007-06-28 Medical Research Council Ciblage de polypeptides sur les mitochondries
KR20130109966A (ko) * 2010-05-14 2013-10-08 애브비 인코포레이티드 Ｉｌ－１ 결합 단백질
WO2012174452A1 (fr) * 2011-06-17 2012-12-20 Shire Human Genetic Therapies, Inc. Ciblage mitochondrial et utilisation thérapeutique associée
KR20150131360A (ko) * 2013-03-15 2015-11-24 애브비 인코포레이티드 ＴＮＦα에 대해 지시된 이원 특이적 결합 단백질

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DATABASE Protein 19 January 2018 (2018-01-19), ANONYMOUS: "SLC1A5 isoform 3 [Pan troglodytes]", XP055777885, retrieved from NCBI Database accession no. PNI28894.1 *

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