WO2023177261A1 - Nouveau peptide de pénétration cellulaire ayant une perméabilité cellulaire améliorée et son utilisation - Google Patents

Nouveau peptide de pénétration cellulaire ayant une perméabilité cellulaire améliorée et son utilisation Download PDF

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WO2023177261A1
WO2023177261A1 PCT/KR2023/003593 KR2023003593W WO2023177261A1 WO 2023177261 A1 WO2023177261 A1 WO 2023177261A1 KR 2023003593 W KR2023003593 W KR 2023003593W WO 2023177261 A1 WO2023177261 A1 WO 2023177261A1
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peptide
cell
target substance
endoplasmic reticulum
present
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PCT/KR2023/003593
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English (en)
Korean (ko)
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신재민
박정현
박혜진
김민석
장재근
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주식회사 에이조스바이오
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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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • 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

Definitions

  • the present invention relates to a new cell-penetrating peptide with improved cell permeability and its use.
  • the cell membrane functions as a barrier that prevents most foreign substances from entering the cell.
  • This function of the cell membrane is essential for protecting cells, but from a pharmaceutical perspective, it is a major factor impeding the delivery of drugs for treatment.
  • Methods used to deliver the drug into cells are divided into a method using an active transport system through ATP-binding transport molecules present in the cell membrane, and a method of forcibly penetrating the cell membrane.
  • the method using the active transport system is Since the drugs that can be used are extremely limited, most methods that forcefully penetrate the cell membrane are used.
  • Specific methods for forcibly permeating the cell membrane include methods of delivering drugs into cells using cationic lipids/liposomes, cell-penetrating peptides, micro-injection, viruses, etc., which methods can be used depending on the drug.
  • research is continuously being conducted to develop new methods to forcibly permeate cell membranes.
  • CPP cell penetrating peptides
  • Cell-penetrating peptides are cell membrane-penetrating peptides composed of about 10 to 30 short peptides, most of which are derived from protein-transduction domains or membranetranslocating sequences.
  • CPP is an oligopeptide that reacts with the cell membrane and undergoes endocytosis or directly penetrates the cell membrane, and has electrochemical and physicochemical properties that allow it to penetrate the cell membrane.
  • CPPs are well known in the pharmaceutical field as they have been studied in connection with their application to drug delivery systems, and have been widely studied and used as a tool to regulate gene expression by being applied in specific biological systems.
  • the present invention relates to a new cell-penetrating peptide with improved cell permeability and its use.
  • the present invention provides a novel cell-penetrating peptide.
  • the present invention provides a composition containing a cell-penetrating peptide and a target substance.
  • the present invention provides a composition for delivering a target substance into cells, including a cell-penetrating peptide and a target substance.
  • the present invention provides a medicinal use of a composition containing a cell-penetrating peptide and a target substance.
  • One aspect of the present invention to achieve the above object is to provide a peptide containing an amino acid sequence represented by the following general formula 1:
  • X 1 is any one selected from the group consisting of P, W, K, L, A, R and H,
  • X 2 is any one selected from the group consisting of G, K, L, A, H, W and R,
  • X 3 is any one selected from the group consisting of T, K, W, R, L, H and A.
  • the peptide according to the present invention can efficiently deliver the desired peptide into cells, and this effect is superior to that of known wild-type peptides. Accordingly, it can be used as a drug carrier and therapeutic agent, and has an excellent advantage in that it can be used for preventive and therapeutic purposes by controlling various physiological phenomena in the living body.
  • the numbering of the peptide sequence according to the present invention is based on the peptide sequence of SEQ ID NO: 1 below.
  • SEQ ID NO: 1 MLPGLALLLLAAWTARA (Amyloid-beta precursor protein derived signal sequence, Human (wild type))
  • amino acid is an organic compound containing an amino group and a carboxylic acid group.
  • Polypeptides contain two or more amino acids.
  • amino acids include the 20 naturally occurring amino acids, non-natural amino acids and amino acid analogs (i.e. amino acids in which the ⁇ -carbon carries a side chain).
  • naturally occurring amino acids refers to the 20 L-amino acids that occur in peptides.
  • peptide refers to a molecule formed by combining amino acid residues with one another through an amide bond (or peptide bond).
  • the peptide may be synthesized using a genetic recombination and protein expression system, and preferably may be synthesized in vitro using a peptide synthesizer.
  • the peptide of the present invention includes derivatives thereof. Amino acid fragments or parts are substituted or deleted, part of the amino acid sequence is modified into a structure that can increase stability in vivo, part of the amino acid sequence is modified to increase hydrophilicity, or part or all of the amino acids are L- or Fragments in which a D-amino acid is substituted or part of an amino acid is modified may be mentioned.
  • non-natural amino acids include, for example, amino acids other than the 20 naturally occurring amino acids or analogs of other amino acids, including, but not limited to, D-isostereomers of amino acids. Examples of non-natural amino acids are known to those skilled in the art and may be included in peptides according to the invention.
  • conservative amino acid substitutions may be further made within the above-mentioned peptides, provided that the resulting peptides exhibit cell-penetrating activity.
  • Conservative substitutions of suitable amino acids are known in the art and are generally made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, usually, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Other substitutions are also permitted and can be determined experimentally or based on known conservative substitutions.
  • Constant amino acid substitution is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain.
  • Classes of amino acid residues with similar side chains are defined and well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), and amino acids with uncharged polar side chains (e.g., glycine).
  • amino acids with non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains amino acids with aromatic side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • the peptide according to the present invention has substantially the same function and/or effect as the peptide according to the present invention, and has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or It is interpreted to include sequences with more amino acid sequence homology.
  • the peptide containing the amino acid sequence represented by General Formula 1 according to the present invention is represented by SEQ ID NO: 2.
  • the peptide according to the present invention excludes the wild-type sequence MLPGLALLLLLAAWTARA in General Formula 1 above. That is, it includes amino acid substitution in at least one selected from the group consisting of X 1 , X 2 and X 3 . More specifically, X 1 is not P, X 2 is not G, or X 3 is not T.
  • the numbering of the residues relative to the numbering in SEQ ID NO: 1 provides peptides containing one or more amino acid substitutions as described below in this specification.
  • Non-limiting examples of amino acid substitutions in peptides according to the invention are at any one or more of amino acid residue positions 3, 4, or 14 as numbered for the peptide set forth in SEQ ID NO:1.
  • Amyloid-beta precursor protein can be determined through sequence alignment with the peptide corresponding to SEQ ID NO: 1 above.
  • substitution of any one or more amino acid residues at positions 3, 4, or 14 by numbering for the peptide shown in SEQ ID NO: 1 can also be made based on other Amyloid-beta precursor proteins.
  • the numbering for the peptide shown in SEQ ID NO: 1 may include an amino acid substitution in at least one residue selected from the group consisting of P3, G4, and T14.
  • At least one or more selected from the group consisting of P3K, P3R, P3A, P3H, P3W, P3L, G4R, G4K, G4A, G4H, G4W, G4L, T14R, T14K, T14A, T14L, T14H and T14W. includes substitution.
  • any combination containing at least one, at least two, or at least three of the above substitutions may be mentioned.
  • This can greatly improve efficiency in terms of delivering target substances to cells, including therapeutic effects. Accordingly, it can be used for various purposes based on its increased solubility and cell delivery ability.
  • any of the sequences mentioned above are specifically shown in SEQ ID Nos: 3 to 40.
  • sequences on the above-mentioned SEQ ID NOs correspond to some of one or more mutant sequences, and the entire sequence of the present invention is not limited to these sequences.
  • the peptide according to the present invention has improved cell permeability and delivery ability to the endoplasmic reticulum (or endoplasmic reticulum targeting ability).
  • the peptide itself not only has cell permeability, but also has the property of penetrating the cell membrane, reaching the inside of the cell, and then reaching the endoplasmic reticulum.
  • cell permeability refers to the ability or property of a peptide to penetrate the cell membrane and enter the cell.
  • endoplasmic reticulum targeting refers to the delivery of peptides from within cells to the endoplasmic reticulum.
  • Sequences comprising, consisting essentially of, or consisting of the above-mentioned peptides may have the above-mentioned cell penetration ability and/or endoplasmic reticulum delivery ability.
  • the cell-penetrating peptide according to the invention preferably comprises, consists essentially of, or consists of the above-mentioned amino acid sequence.
  • the peptide sequence according to the present invention is a peptide sequence for which improved cell permeability was confirmed through cell permeability analysis.
  • the peptide sequence according to the present invention is a peptide sequence that has improved cell permeability and endoplasmic reticulum delivery ability.
  • the present invention also provides a composition comprising the above-mentioned peptide and a target substance.
  • a peptide comprising an amino acid substitution in at least one residue selected from the group consisting of P3, G4 and T14, numbered relative to the peptide set forth in SEQ ID NO: 1; and a composition containing the target substance.
  • the peptide sequences are designated as P3K, P3R, P3A, P3H, P3W, P3L, G4R, G4K, G4A, G4H, G4W, G4L, T14R, T14K, T14A, T14L, T14H and T14W.
  • peptide sequence set forth in SEQ ID NO: 1 P3A/G4K, P3A/G4H, P3A/T14K, P3A/T14R, P3K/G4K, P3K/G4H, P3K/T14K, P3K/T14R, G4K/T14K , G4K/T14R, G4H/T14K, G4H/T14R, P3A/G4K/T14K, P3A/G4H/T14K, P3K/G4K/T14K, P3K/G4H/T14K, P3A/G4K/T14R, P3A/G4H/T14R, P3A/G4H/T14R, P3K A peptide containing any one substitution selected from the group consisting of /G4K/T14R and P3K/G4H/T14R; and a composition containing the target substance.
  • the target substance according to the present invention includes any substance for endoplasmic reticulum delivery.
  • the target substances include, for example, compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs, lipid-based formulations, viruses, and quantum dots. ) and fluorochrome, and is preferably a protein.
  • FITC was linked to a peptide sequence and delivered to intracellular endoplasmic reticulum.
  • the “target substance” is a component that can form a composition together with the cell-permeable peptide, and is a general term for substances that have certain physiological functions in vivo, and have various physiological activities.
  • the target substance may be a drug.
  • the drug includes a chemical drug, a biodrug, a nucleic acid drug, a peptide drug, a protein drug, and a natural product drug. ), semi-synthetic drug, lipid drug, enzyme, regulatory factor, growth factor, hormone, contrast agent and antibody ( It may be one or more selected from the group consisting of antibodies, but is not limited thereto.
  • it may be an antibody, compound, protein or nucleic acid.
  • the drug delivery may be delivering the drug into cells.
  • the drug delivery may be delivering the drug to intracellular endoplasmic reticulum.
  • the peptide will be described in more detail as an example of one embodiment, but the scope of the target substance is not limited to this description.
  • any peptide that can exhibit pharmacological or physiological activity that modulates intracellular activity after being delivered into the cell can be included in the scope of the present invention.
  • protein (peptide) substances may include, but are not limited to, immunogenic peptides, antigens, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors. No.
  • bioactive peptides include, for example, GLP-1 receptor agonists, glucagon receptor agonists, GIP (Gastric inhibitory polypeptide) receptor agonists, FGF (Fibroblast growth factors) receptor agonists (FGF1, FGF19, FGF21, FGF23) etc.), Cholecystokinin receptor agonist, gastrin receptor agonist, melanocortin receptor agonist, human growth hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, interferons and interferon receptors ( Examples: interferon-alpha, -beta and -gamma, soluble type I interferon receptors, etc.), colony stimulating factors, interleukins (e.g.
  • beta-glucosidase ( betaglucosidase), alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, galactose- Galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinsase, galactocerebrosidase (galactocerebrosidsase), arylsulfatase A, B, beta-hexosaminidase A, B, heparin Nsulfatase, alpha-D-manosidase (alpha- D-mannosidase), beta-glucuronidase, N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N -Acetyl-glucosamini
  • macrophage activator macrophage peptide, B cell factor, T cell factor, protein A, allergy suppressor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin (alpha-lactalbumin), apolipoprotein-E, erythropoietin, hyperglycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activation peptide, thrombomodulin , blood factor VII, blood factor VIIa, blood factor VIII, blood factor IX, blood factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein , renin inhibitor, collagen
  • Protein calcitonin, insulin, atriopeptin, cartilage-inducing factor, elcatonin, connective tissue activator, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone , nerve growth factors (e.g.
  • glial-derived neurotrophic factor glial derived neurotrophic factor, netrin, neurophil inhibitor factor, neurotrophic factor, neuturin, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortex Hormones, glucagon, cholecystokinin, pancreatic peptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, ADNP (activity-dependent neuroprotective protein), BACE1 (beta-secretase1), APP (Amyloid Precursor Protein), NCAM (Neural cell adhesion molecule), Amyloid beta, Tau, RAGE (receptor for advanced glycation endproducts), alpha-synuclein ), or their agonists or antagonists, their receptors (
  • the peptide and the target substance according to the present invention which have cell permeability, may be used after simply mixing.
  • the cell-permeable peptide and the target substance may be linked, if necessary, by methods known in the art for binding the protein and the target substance, for example, covalent bonding, chemical bonding, They can be connected through peptide bonds, etc.
  • a peptide comprising an amino acid substitution in at least one residue selected from the group consisting of P3, G4 and T14, numbered for the peptide shown in SEQ ID NO: 1; and a biologically active peptide linked thereto.
  • a peptide comprising an amino acid substitution at any one or more residues selected from the group consisting of P3, G4 and T14, numbered relative to the peptide shown in SEQ ID NO: 1;
  • a composition for intracellular peptide delivery comprising a physiologically active peptide linked thereto is provided.
  • the biologically active protein may be linked to the cell-penetrating peptide directly or via a linker or spacer peptide.
  • linker refers to a short amino acid sequence used to separate two peptides with different functions when constructing a fusion protein.
  • the absence of a linker between two or more individual domains in a protein can result in reduced or inappropriate function of the protein domain due to steric hindrance, for example, reduced catalytic activity or binding affinity for the receptor/ligand.
  • Connecting protein domains in chimeric proteins using artificial linkers can increase the space between the domains.
  • the linker or spacer is not particularly limited as long as it has the effect of improving the activity of the conjugate of the cell-permeable peptide and the physiologically active peptide according to the present invention.
  • the constituent amino acids affect some properties of the molecule, such as folding, net charge, or hydrophobicity. can be selected to affect.
  • the linker may be a peptide linker or a non-peptide linker.
  • a “peptidic linker” includes a peptide bond or polymer of amino acids that connects two fusion partners.
  • a protease-cleavable sequence may be included as a peptide linker.
  • the protease cleavage site may be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site.
  • the peptide sequence is cleaved through a peptide sequence recognized by a signal peptidase enzyme.
  • a sequence that can be recognized by the signal peptidase enzyme can be added between the cell-penetrating peptide and the biologically active peptide sequence.
  • the peptide linker is an amino acid sequence in which the GGGGS motif (SEQ ID NO: 41), GS motif (SEQ ID NO: 42), GGGS motif (SEQ ID NO: 43), or GGSG motif (SEQ ID NO: 44) are repeated. Constituent peptides may also be considered.
  • the motif may be repeated 1 to 10 times, but is not particularly limited thereto.
  • signal peptidase cleavage peptide refers to a linker sequence that is recognized by signal peptidase and can provide cleavage between the biologically active peptide and the endoplasmic reticulum target cell-penetrating peptide sequence. “Signal peptidase cleavage peptide” can recognize the C-terminus of a cell-penetrating peptide sequence and the upper cleavage peptide sequence and provide peptide cleavage between them.
  • the cell-penetrating peptide sequence according to the present invention is derived from a signal sequence, “AX 4 A (SEQ ID NO: 45)” or “VX 4 A (SEQ ID NO: 46)” at the C-terminus (where X 4 is any amino acid sequence) Lim) can have.
  • the linker sequence may additionally include an “EA” sequence.
  • composition according to the invention specifically comprises a peptide exhibiting the above-mentioned cell permeability properties; a linker or spacer peptide connected thereto; and a physiologically active peptide linked thereto.
  • a peptide comprising an amino acid substitution in at least one residue selected from the group consisting of P3, G4, and T14, numbered relative to the peptide shown in SEQ ID NO: 1; A signal peptidase cleaved peptide of SEQ ID NO: 47 or 48 linked thereto; and a physiologically active peptide linked thereto.
  • the peptide sequences set forth in SEQ ID NO: 1 are as follows: A peptide containing at least one or more substitutions selected from the group consisting of; A signal peptidase cleaved peptide of SEQ ID NO: XX linked thereto; and a physiologically active peptide linked thereto.
  • Non-peptide linker refers to a biocompatible linker in which two or more repeating units are combined, and the repeating units are linked to each other through any covalent bond rather than a peptide bond.
  • Non-peptide polymers which are non-peptide linkers, include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, and PLA (polylactic acid).
  • polylactic acid and PLGA (polylactic-glycolic acid), biodegradable polymers, lipid polymers, chitins, hyaluronic acid, oligonucleotides, and combinations thereof.
  • biodegradable polymers an example of which may be polyethylene glycol, but is not limited thereto.
  • derivatives thereof already known in the art and derivatives that can be easily produced at the level of technology in the art are also included in the scope of the present invention.
  • composition can be utilized for various purposes through the use of the peptide according to the present invention that exhibits cell permeability.
  • the composition may include the previously mentioned cell-penetrating peptide; And it may be a composition for intracellular drug delivery containing a drug.
  • the composition may be a composition for delivering a target substance into cells.
  • it may be a composition for delivering a target substance to endoplasmic reticulum.
  • the term “to the endoplasmic reticulum” of the present invention means that a substance can be delivered to the endoplasmic reticulum, the endoplasmic reticulum lumen, and/or the interior of the endoplasmic reticulum existing within a cell.
  • composition for delivery according to the present invention can directly deliver a cell-penetrating peptide and a target substance linked directly or through a linker or spacer to the target cell and/or to the endoplasmic reticulum.
  • Cells for the purpose of transferring the above substances include epithelial cells, muscle cells, nerve cells, glandular cells, glial cells, germ cells, stem cells, mesenchymal cells, mesenchymal stem cells, osteoblasts, osteocytes, osteoblasts, osteoclasts, Various cells such as blood cells, hematopoietic cells, lung cells, hepatocytes, fibroblasts, immune cells, endothelial cells, adipocytes, chondrocytes, and cancer cells may be included, but are not limited to the examples above.
  • the epithelial cells include mucosal epithelial cells, hair follicle epithelial cells, digestive tract epithelial cells, respiratory epithelial cells, genital epithelial cells, and urinary epithelial cells.
  • the endothelial cells include vascular endothelial cells or lymphatic endothelial cells.
  • Vascular endothelial cells may include, but are not limited to, endothelial cells of various arteries, veins, and capillaries, including cerebral blood vessels that form the blood-brain barrier (BBB), lymphatic vessel endothelial cells, and endothelial cells of the inner wall of the heart.
  • BBB blood-brain barrier
  • the cancer cells may be various cancer cells, including cervical cancer, breast cancer, liver cancer, lung cancer, etc., but are not limited thereto.
  • the target substance is introduced into cells through contact between the cell membrane and the peptide in vitro or in vivo. And these substances may target the endoplasmic reticulum more specifically.
  • the purpose of using the peptide according to the present invention is to deliver the target substance into cells.
  • the target substance can be delivered to the endoplasmic reticulum more specifically using the above sequence.
  • a substance when delivering a substance to the endoplasmic reticulum, it can exert various effects through regulating protein expression in the endoplasmic reticulum.
  • membrane proteins secreted from cells or distributed in the inner cell membrane necessarily move to the lumen of the endoplasmic reticulum (ER) and undergo appropriate folding and processing.
  • chaperone proteins such as PDI (protein disulfide isomerase), BiP (heavy chain binding protein), calnexin (CNX), and calreticulin (CRT) exist to help ensure proper folding and processing of proteins biosynthesized in the endoplasmic reticulum.
  • PDI protein disulfide isomerase
  • BiP heavy chain binding protein
  • CNX calnexin
  • CRT calreticulin
  • the present invention has the advantage of being able to achieve various desired actions by appropriately delivering a specific target substance to the endoplasmic reticulum in terms of delivering the target substance to the endoplasmic reticulum as described above.
  • Peptides according to the invention can be prepared using available techniques known in the art. Peptides can be synthesized using any suitable procedure known to those skilled in the art, i.e., known peptide synthesis methods (e.g. genetic engineering methods, chemical synthesis).
  • the peptide according to the present invention can be produced by recombinant techniques using genetic engineering methods.
  • a nucleic acid polynucleotide
  • the nucleic acid can be prepared by amplification by PCR using appropriate primers.
  • the DNA sequence may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer.
  • the constructed nucleic acid is operably linked to the nucleic acid and inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that control the expression of the nucleic acid to produce a recombinant expression vector.
  • expression control sequences e.g., promoter, enhancer, etc.
  • the cell is cultured under appropriate media and conditions for expression of the desired peptide, and substantially pure peptide expressed from the nucleic acid is recovered from the culture.
  • the recovery can be performed using methods known in the art. It is not limited thereto, but is separated by methods known in the art, such as extraction, recrystallization, various chromatographies (gel filtration, ion exchange, precipitation, adsorption, reversed phase), electrophoresis, and countercurrent partitioning. and can be purified.
  • the term 'substantially pure polypeptide' means that the peptide according to the present invention substantially does not contain any other proteins derived from host cells.
  • the peptide according to the present invention can be prepared by chemical synthesis methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
  • the peptide of the present invention can be prepared by direct peptide synthesis using solid phase techniques.
  • the solid-phase peptide synthesis (SPPS) method can initiate synthesis by attaching functional units called linkers to small porous beads to connect the peptide chain.
  • the peptide is covalently bonded to the bead and is prevented from falling off during the filtration process until it is cleaved by a specific reactant such as TFA (trifluoroacetic acid).
  • the protection process where the N-terminal amine of the peptide attached to the solid phase binds to the N-protected amino acid unit, the deprotection process, the re-revealed amine group and the new Synthesis occurs by repeating the cycle of coupling process (deprotection-wash-coupling-wash) in which amino acids combine.
  • the SPPS method can be performed using microwave technology, which can shorten the time required for coupling and deprotection of each cycle by applying heat during the peptide synthesis process. The heat energy can prevent folding or aggregation of the extended peptide chain and promote chemical bonding.
  • the present invention provides a polynucleotide encoding the peptide.
  • the peptides include the above-mentioned peptides themselves, which have cell-permeable properties, or the above-mentioned peptides, which have cell-permeable properties and physiologically active peptides linked thereto.
  • the polynucleotide may be mutated by substitution, deletion, insertion, or combination of one or more bases.
  • a synthesis method well known in the art for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used. , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on a solid support.
  • the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.
  • expression vector refers to a recombinant vector capable of expressing a target peptide in a target host cell, and refers to a gene construct containing essential regulatory elements operably linked to express the gene insert.
  • the expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator.
  • the start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the peptide, and when the gene construct is administered, the It must be functional and be in frame with the coding sequence.
  • the promoter of the vector may be constitutive or inducible.
  • operably linked refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA are functionally linked to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence.
  • Operational linkage with an expression vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can be done using enzymes generally known in the art.
  • the expression vector may contain a signal sequence for peptide release to promote separation of the protein from the cell culture medium.
  • a specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences.
  • an exogenous translation control signal which may include an ATG start codon, must be provided. These exogenous translation control signals and initiation codons can be from a variety of natural and synthetic sources. Expression efficiency can be increased by introduction of appropriate transcription or translation enhancers.
  • the expression vector may further include a protein tag that can be optionally removed using endopeptidase to facilitate detection of the peptide according to the present invention.
  • tag refers to a molecule that exhibits quantifiable activity or characteristics, and includes a chemical fluorescent substance such as fluorescein, a peptide fluorescent substance such as a fluorescent protein (GFP), or a related protein. It may be a fluorescent molecule containing; It may be an epitope tag such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or straptavidin tag. In particular, when using an epitope tag, a peptide tag preferably composed of 6 or more amino acid residues, more preferably 8 to 50 amino acid residues, can be used.
  • the expression vector may include a nucleotide sequence encoding the peptide of the present invention described above.
  • the vector used in this case is not particularly limited as long as it can produce the peptide of the present invention, but is preferably It can be plasmid DNA, phage DNA, etc., and more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), or Bacillus subtilis-derived plasmids.
  • Plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), animal virus vectors (retrovirus, It can be an adenovirus, vaccinia virus, etc.) or an insect virus vector (baculovirus, etc.). Since the expression level and modification of the protein in the expression vector varies depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.
  • the transformant provided by the present invention can be produced by introducing the expression vector provided by the present invention into a host and transforming it, and can be used to produce the peptide of the present invention by expressing the polynucleotide contained in the expression vector. You can.
  • the transformation can be performed by various methods, but is not particularly limited as long as the peptide of the present invention can be produced, but by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl 2 precipitation method or CaCl 2 precipitation method.
  • DMSO dimethyl sulfoxide
  • the host used in the production of the transformant is not particularly limited as long as it can produce the cell-penetrating peptide of the present invention, but includes bacterial cells such as E.
  • coli coli , Streptomyces, and Salmonella Typhimurium
  • Yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe
  • Fungal cells such as Pichia pastoris
  • Insect cells such as Drosophila and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, NSO, 293, Bow's melanoma cells; Or it could be a plant cell.
  • composition according to the present invention can be used for the prevention and treatment of various diseases by delivering physiologically active substances into cells.
  • exemplary diseases include, for example, type 1 diabetes, type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis ( amyotrophic lateral sclerosis (ALS), hemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt -Kreutzfeldt-Jakob disease, familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidosis hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, Coron's disease, ulcerative colitis, breast cancer, and prostate cancer.
  • type 1 diabetes type 2 diabetes
  • Alzheimer's disease amyotrophic lateral sclerosis
  • ALS amyotrophic lateral s
  • composition of the present invention can be used in various medical fields by specifically and efficiently delivering the desired biologically active substance into cells.
  • the pharmaceutical composition according to the present invention can be used in mammals, preferably humans, for example intravein, intraperitoneal, intramuscular, subcutaneous, intradermal, intranasal (
  • the target substance i.e., bioactive substance
  • the target substance can be delivered into cells by injection through routes such as nasal, mucosal, inhalation, and oral routes.
  • treatment refers to the inhibition or alleviation of a disease or condition.
  • therapeutically effective amount refers to an amount sufficient to achieve the above pharmacological effect.
  • the pharmaceutical composition may be formulated and provided in an appropriate form.
  • the above preparations are administered in oral dosage forms such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, and aerosols, or in parenteral dosage forms in the form of transdermal preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. It can be formulated and used.
  • the preparation may additionally contain pharmaceutically suitable and physiologically acceptable auxiliaries such as carriers, excipients, and diluents.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium.
  • Silicates cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used.
  • the preparation may include a carrier for formulating the pharmaceutical composition (active ingredient) in addition to the pharmaceutical composition.
  • the carrier may include binders, lubricants, suspending agents, solubilizers, buffers, preservatives, lubricants, isotonic agents, excipients, stabilizers, dispersants, suspending agents, colorants, fragrances, etc.
  • the composition may be administered alone, but may be administered mixed with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice.
  • topical administration such as liquid, gel, cleansing composition, tablet for insertion, suppository form, cream, ointment, dressing solution, spray, and other coating agents.
  • topical administration such as liquid, gel, cleansing composition, tablet for insertion, suppository form, cream, ointment, dressing solution, spray, and other coating agents.
  • External skin preparations such as solutions, gels, cleansing compositions, and tablets for insertion may be included.
  • the formulation can be prepared by adding solubilizers, emulsifiers, buffers for pH adjustment, etc. to sterilized water.
  • the non-aqueous solvent or suspension may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable este
  • the preparation when provided for oral use, for example, in the form of tablets containing starch or lactose, in the form of capsules alone or containing excipients, or as elixirs containing chemicals for flavoring or coloring.
  • it may be administered orally, intraorally, or sublingually in the form of a suspension.
  • the administered dose of the above agent may vary depending on the patient's age, weight, gender, dosage form, health condition, and disease level, and may be administered in divided doses from once to several times a day at certain time intervals, depending on the judgment of the doctor or pharmacist. there is.
  • the daily dosage is 0.001 to 10000 mg/kg, 0.01 to 10000 mg/kg, 0.1 to 10000 mg/kg, 0.5 to 10000 mg/kg, 0.001 to 1000 mg/kg, 0.01 to 1000.
  • mg/kg 0.1 to 1000 mg/kg, 0.5 to 1000 mg/kg, 0.001 to 500 mg/kg, 0.01 to 500 mg/kg, 0.1 to 500 mg/kg, 0.5 to 500 mg/kg, 0.001 to 300 mg /kg, 0.01 to 300 mg/kg, 0.1 to 300 mg/kg, or 0.5 to 300 mg/kg.
  • the above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.
  • the daily dosage of the agent is less than the above dosage, no significant effect can be obtained, and if it exceeds the dosage, it is not only uneconomical but also outside the range of the usual dosage, so there may be a risk of undesirable side effects. It is better to use a range.
  • the subject of administration of the agent may be a mammal such as a human, a cell, tissue, body fluid isolated from a mammal, or a culture thereof.
  • the present invention provides a method of treating or preventing a disease or disorder comprising administering a therapeutically effective amount of the composition to a subject in need thereof.
  • the present invention provides a method for treating or preventing disease, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof.
  • the invention also provides uses of the compositions.
  • the present invention relates to a peptide according to the present invention; and providing a method of delivering a target substance to endoplasmic reticulum within a cell, including the step of processing the target substance into the cell.
  • the present invention relates to a peptide according to the present invention; and providing a method of delivering a target substance to an endoplasmic reticulum, including the step of processing the target substance into a target object.
  • the present invention relates to a peptide according to the present invention; and a method of selectively delivering a target substance to intracellular endoplasmic reticulum, including the step of processing the target substance into the cell.
  • the present invention relates to a peptide according to the present invention; and providing a method for selectively delivering a target substance, including the step of treating the target substance to an object.
  • the present invention provides a peptide according to the invention for use in the treatment or prevention of the diseases or disorders mentioned above; and a pharmaceutical composition containing the target substance.
  • the present invention provides a peptide according to the present invention for delivering a target substance to intracellular endoplasmic reticulum; and a composition containing the target substance.
  • the present invention relates to the peptide according to the invention for the preparation of a medicament for use in the treatment or prevention of the diseases or disorders mentioned above. and a use of a composition containing the target substance.
  • the present invention relates to the preparation of an agent for delivering a target substance to intracellular endoplasmic reticulum, including the peptide according to the present invention; and a use of a composition containing the target substance.
  • the novel cell-penetrating peptide with improved cell permeability of the present invention can efficiently deliver a target substance into cells. Accordingly, it can be used as a drug carrier and therapeutic agent, and has an excellent advantage in that it can be used for preventive and therapeutic purposes by controlling various physiological phenomena in the living body.
  • Figure 1 shows the results of providing amino acid mutations to the wild-type sequence based on the Amyloid-beta precursor protein sequence and scoring the cell penetration potential.
  • Figure 2 shows the results of predicting information about sequences with high cell permeability by performing a sequence logo plot on approximately 6,000 sequences with high cell permeability according to the scoring results.
  • Figure 3 shows the results of confirming cell permeability to MCF-7 cells using cell permeable peptide sequences prepared through actual synthesis (Peptide No. 1 to Peptide No. 9 in Table 3 (corresponding to Peptides 1 to 9) ).
  • Figure 4 shows the results of confirming cell permeability to SH-Sy5Y cells using cell permeable peptide sequences prepared through actual synthesis (Peptides No. 1 to No. 9 (corresponding to Peptides 1 to 9) in Table 3). .
  • Figure 5 shows the results of confirming cell permeability to hBMSCs using cell permeable peptide sequences prepared through actual synthesis (Peptides No. 1 to No. 9 (corresponding to Peptides 1 to 9) in Table 3).
  • Figure 6 shows the results of confirming the endoplasmic reticulum target of the mutant CPP sequence using a fluorescence microscope (Peptides No. 1 to No. 5 in Table 3 (corresponding to Peptides 1 to 5)).
  • Figure 7 shows the results of confirming the endoplasmic reticulum target of the mutant CPP sequence using a fluorescence microscope (Peptides No. 6 to No. 9 in Table 3 (corresponding to Peptides 6 to 9)).
  • Example 1 Prediction of difference in permeability according to amino acid mutation of wild-type cell permeable peptide using artificial intelligence
  • top-ranked sequences according to deep learning-based cell permeability analysis are as follows:
  • SEQ ID NO: 1 MLPGLALLLLAAWTARA (Amyloid-beta precursor protein derived signal sequence, Human (wild type))
  • WT CPP wild-type cell-penetrating peptide
  • the peptides with the highest probability of being CPP were collected to determine which positions had the most changes, and the positions with the most modifications in the high-ranking peptides were determined to be the positions of amino acids that needed to be modified to improve the performance of CPP.
  • a sequence logo plot was drawn using the above 6,000 modified CPPs, and the major modification locations were specifically identified.
  • amino acid preference at each position can be confirmed through the Sequence logo plot. The Since only a maximum of 3 amino acids were changed from the wild-type sequence, the wild-type peptide sequence appears to be the most abundant.
  • the third amino acid, P was replaced in the following order: W, K, L, A, R, and H.
  • the fourth amino acid, G was replaced in the following order: K, L, A, H, W, and R.
  • the 14th amino acid, T was replaced in the order of K, W, R, L, and A. Overall, it was confirmed that many of the amino acids were replaced with K.
  • peptides were synthesized by modifying the 3rd, 4th, and 14th amino acids to the above-mentioned mutations, reflecting the amino acid preference, and then the cell permeability of each mutant peptide was confirmed.
  • the peptide sequence used in the present invention was requested to be synthesized by Peptron Co., Ltd., and the following experiments were performed using it.
  • WT CPP wild-type cell-penetrating peptide
  • FITC Fluorescein
  • the cells were treated with the nine types of peptides at a concentration of 1 ⁇ M for 2 hours. Afterwards, the intensity of FITC was analyzed using FACS and the cell permeability of each peptide was confirmed, and the results are shown in Figures 3 to 5 for each cell.
  • WT CPP wild-type cell-penetrating peptide
  • FITC Fluorescein
  • the sequence of the mutant CPP targets the endoplasmic reticulum, allowing the target sequence to be delivered to the endoplasmic reticulum within the cell and/or intracellular organelles.
  • intracellular material transfer can be carried out efficiently.
  • Example 4 Analysis of the inhibitory effect of endoplasmic reticulum-targeted antioxidants on cell death induced by endoplasmic reticulum stress
  • Peptides were prepared by fusing antioxidants to nine types of peptides (Peptide No. 1 to No. 9) shown in Table 3 above. Specifically, glutathione ( ⁇ E-CG, GSH) was bound to the C terminus of the nine types of peptides, and the endoplasmic reticulum-targeted antioxidant efficacy was confirmed using this.
  • the synthesized peptide and the well-known antioxidants glutathione (GSH) or N-acetyl-cysteine (NAC) were applied to neurotumor cells. After pretreatment for 1 hour, the cells were treated with thapsigargin at a concentration of 20 ⁇ M for 24 hours, and changes in cell death were analyzed to confirm the improved effect of the peptides according to the present invention.
  • the endoplasmic reticulum targeting cell-permeable peptide sequence of the present invention is transmitted to the endoplasmic reticulum to target substances (e.g., compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs). , lipid-based formulations, viruses, quantum dots, and fluorochromes, etc.).
  • target substances e.g., compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs.
  • target substances e.g., compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs.
  • target substances e.g., compounds, proteins, glycoproteins, peptides, nucleotides

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Abstract

La présente invention concerne un nouveau peptide de pénétration cellulaire ayant une perméabilité cellulaire améliorée, et son utilisation. Le nouveau peptide de pénétration cellulaire ayant une perméabilité cellulaire améliorée selon la présente invention peut administrer de manière efficace des substances dans des cellules. En conséquence, le peptide présente d'excellents avantages en tant que système d'administration de médicaments et agent thérapeutique, et peut être utilisé à des fins prophylactiques et thérapeutiques en raison de sa capacité à contrôler divers phénomènes physiologiques in vivo.
PCT/KR2023/003593 2022-03-18 2023-03-17 Nouveau peptide de pénétration cellulaire ayant une perméabilité cellulaire améliorée et son utilisation WO2023177261A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20140335613A1 (en) * 2010-06-04 2014-11-13 Toagosei Co., Ltd. Cell growth-promoting peptide and use thereof
US20150273018A1 (en) * 2012-10-18 2015-10-01 Toagosei Co., Ltd. Synthetic peptide for inhibiting expression of type 2 tnf receptor and use thereof
US20160326488A1 (en) * 2013-12-25 2016-11-10 Toagosei Co., Ltd. Method for inducing differentiation of pluripotent stem cells into endodermal cells
JP2017063669A (ja) * 2015-09-29 2017-04-06 東亞合成株式会社 合成ペプチドを用いた神経幹細胞の生産方法
WO2019157131A1 (fr) * 2018-02-07 2019-08-15 Dana-Farber Cancer Institute, Inc. Modules peptidiques agrafés perméables aux cellules pour administration cellulaire

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Publication number Priority date Publication date Assignee Title
US20140335613A1 (en) * 2010-06-04 2014-11-13 Toagosei Co., Ltd. Cell growth-promoting peptide and use thereof
US20150273018A1 (en) * 2012-10-18 2015-10-01 Toagosei Co., Ltd. Synthetic peptide for inhibiting expression of type 2 tnf receptor and use thereof
US20160326488A1 (en) * 2013-12-25 2016-11-10 Toagosei Co., Ltd. Method for inducing differentiation of pluripotent stem cells into endodermal cells
JP2017063669A (ja) * 2015-09-29 2017-04-06 東亞合成株式会社 合成ペプチドを用いた神経幹細胞の生産方法
WO2019157131A1 (fr) * 2018-02-07 2019-08-15 Dana-Farber Cancer Institute, Inc. Modules peptidiques agrafés perméables aux cellules pour administration cellulaire

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PARK HYEJIN, PARK JUNG-HYUN, KIM MIN SEOK, CHO KWANGMIN, SHIN JAE-MIN: "In Silico Screening and Optimization of Cell-Penetrating Peptides Using Deep Learning Methods", RM D-724 HYUNDAI KNOWLEDGE INDUSTRY CENTER, AZOTHBIO. INC., 520 MISA-DAERO, HANAM-SI 12927, REPUBLIC OF KOREA, vol. 13, no. 3, 13 March 2023 (2023-03-13), pages 522, XP093092669, DOI: 10.3390/biom13030522 *

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