WO2022139070A1 - Nouveau peptide de pénétration cellulaire et son utilisation - Google Patents

Nouveau peptide de pénétration cellulaire et son utilisation Download PDF

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WO2022139070A1
WO2022139070A1 PCT/KR2021/003945 KR2021003945W WO2022139070A1 WO 2022139070 A1 WO2022139070 A1 WO 2022139070A1 KR 2021003945 W KR2021003945 W KR 2021003945W WO 2022139070 A1 WO2022139070 A1 WO 2022139070A1
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cell
cells
peptide
group
present
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Korean (ko)
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남지영
노영현
최호재
이복수
김용호
서민아
이재철
이은아
김한주
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(주)아임뉴런
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components

Definitions

  • the present invention relates to an intracellular delivery technology for delivering a biologically active substance into a cell, and more particularly, to a novel cell-penetrating peptide having excellent cell-penetrating ability and uses thereof.
  • the cell membrane prevents macromolecules such as peptides, proteins, and nucleic acids from entering the cell, and even if it enters the cell through a physiological mechanism called endocytosis by cell membrane receptors, it is fused with the lysosomal compartment of the cell. As it is eventually degraded, there are many restrictions in the treatment and prevention of diseases using the macromolecules. In addition, in the case of anticancer drugs, in order to deliver the drug into cells, obstacles such as multidrug resistance must be overcome.
  • liposomes and micelles are artificially made phospholipid carriers that can encapsulate both lipophilic and hydrophilic drugs, and since they are biocompatible materials, they are non-toxic and protect the drugs from the external environment. However, absorption is delayed, distribution is limited, metabolic rate is low, and it is captured by cells of the liver or spleen and is rapidly removed from the blood.
  • micelles have the ability to increase drug solubility and bioavailability, many studies are still needed on the effects of substances on the movement of substances into cells and their basic medical and clinical applicability. Because of these limitations, there is a need for new agents that can effectively deliver biomaterials into the body, have no cytotoxicity, and do not enter through endocytosis in particular.
  • a cell-penetrating peptide is a kind of signal peptide and is a peptide that is a combination of a specific amino acid sequence used for the purpose of delivering high molecular substances such as protein, DNA, RNA, etc. into cells.
  • a cell-penetrating peptide is a kind of signal peptide and is a peptide that is a combination of a specific amino acid sequence used for the purpose of delivering high molecular substances such as protein, DNA, RNA, etc. into cells.
  • Antennapedia Pierin
  • VP22 derived from HSV-1 virus
  • Simian virus 40 giant antigen T (Simian) Pep-1, derived from Virus 40 large antigen T)
  • Simian Simian virus 40 giant antigen T
  • peptides in which several cationic amino acids such as arginine and lysine are repeatedly linked, such as poly arginine and poly lysine have also been reported to have excellent cell permeability, and are being applied to various mass transfers.
  • most of these cell-penetrating peptides contain the possibility of immunogenicity and toxicity, and are considered to have poor efficacy when delivered to human cells. Therefore, it is necessary to develop a cell-penetrating peptide that does not cause toxicity, has safety in vivo, and can effectively deliver substances.
  • the present inventors designed and synthesized cell-permeable peptides composed of various amino acid sequences according to the present invention and confirmed their excellent cell-permeability, thereby The present invention was completed.
  • an object of the present invention is to provide a novel cell-penetrating peptide.
  • Another object of the present invention is to provide a complex comprising the cell-penetrating peptide and a biologically active substance.
  • Another object of the present invention is to provide a composition for mass transfer comprising the complex, and a mass transfer method comprising the step of treating the composition to cells.
  • Another object of the present invention is to provide a polynucleotide encoding the cell-penetrating peptide.
  • the present invention provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I] or an amino acid sequence having 70% or more homology thereto.
  • X 1 is GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE or GHHG;
  • X a is Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) or Gln(Q);
  • X b is Lys(K), Arg(R), His(H), Asn(N) or Gln(Q);
  • X 2 is SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS;
  • X 3 is TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG or NSG.
  • the cell-penetrating peptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 62.
  • the cells are brain blood barrier endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells.
  • cell; iPSC), neural stem cell (NSC), T cell, B cell, natural killer cell (NK cell), macrophage, microglia, neuron ), glial cells, astrocytes, and muscle cells may be selected from the group consisting of.
  • the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.
  • the biologically active material is a compound (chemical compound), protein, glycoprotein, peptide, antibody (antibody), enzyme (enzyme), nuclease (nuclease), hormone, cytokine (cytokine), transcription factors, toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots dots) and may be at least one selected from the group consisting of fluorochromes.
  • the nuclease is CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (Zinc-finger nucleases) and TALEN ( Transcription activator-like effector nuclease) may be selected from the group consisting of.
  • the nucleic acid is DNA, RNA, ASO (Antisense oligonucleotide), microRNA (microRNA; miRNA), small interfering RNA (siRNA), aptamer (aptamer), LNA (locked) nucleic acid), PNA (peptide nucleic acid), and morpholino (morpholino) may be selected from the group consisting of.
  • ASO Antisense oligonucleotide
  • microRNA microRNA
  • miRNA small interfering RNA
  • siRNA small interfering RNA
  • aptamer aptamer
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • morpholino morpholino
  • the present invention provides a composition for mass transfer comprising the complex.
  • the present invention provides a material delivery method comprising the step of treating the cells with the composition.
  • the present invention provides a polynucleotide encoding the peptide.
  • the cell-penetrating peptide according to the present invention is expected to be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, etc. because it can effectively deliver a biologically active substance into a living body such as cells and tissues.
  • 1A and 1B show the excellent cell permeability of the peptide according to the present invention through in vitro cell permeability analysis of a peptide candidate group in which amino acids are substituted in the region of positions 1 to 4 from the N-terminus in a peptide consisting of 16 amino acids. The confirmed results are shown.
  • Figure 1c shows the results for the control peptide group that did not show cell permeability through in vitro cell permeability analysis on the peptide candidate group in which amino acids were substituted in the region of positions 1 to 4 from the N-terminus in the peptide consisting of 16 amino acids. will be.
  • Figure 2a is the result of confirming the excellent cell permeability of the peptide according to the present invention through the in vitro cell permeability analysis result of the peptide candidate group in which amino acids are substituted in the region of positions 5 to 7 from the N-terminus in the peptide consisting of 16 amino acids; is shown.
  • Figure 2b shows the results of the control peptide group that did not show cell permeability through the in vitro cell permeability analysis result of the peptide candidate group in which amino acids were substituted in the region of positions 5 to 7 from the N-terminus in the peptide consisting of 16 amino acids. it has been shown
  • 3A and 3B show excellent cell permeability of the peptide according to the present invention through the results of in vitro cell permeability analysis on the peptide candidate group in which amino acids are substituted in the region of positions 8 to 12 from the N-terminus in the peptide consisting of 16 amino acids. shows the results of checking .
  • Figure 3c shows the results of the control peptide group that did not show cell permeability through the in vitro cell permeability analysis result of the peptide candidate group in which amino acids were substituted in the region of positions 8 to 12 from the N-terminus in the peptide consisting of 16 amino acids. it has been shown
  • Figure 4a is the result of confirming the excellent cell permeability of the peptide according to the present invention through the results of in vitro cell permeability analysis on the peptide candidate group in which amino acids are substituted in the region of positions 13 to 16 from the N-terminus in the peptide consisting of 16 amino acids; is shown.
  • Figures 4b and 4c show the control peptide group that did not show cell permeability through in vitro cell permeability analysis on the peptide candidate group in which amino acids were substituted in the region of positions 13 to 16 from the N-terminus in the peptide consisting of 16 amino acids. results are shown for
  • 5A and 5B show the results of confirming the excellent cell permeability of the peptide according to the present invention through the results of in vitro cell permeability analysis on the peptide candidate group in which one or more amino acids are simultaneously substituted in the entire region of the peptide consisting of 16 amino acids. .
  • FIG. 5c shows the results of the control peptide group in which cell permeability was not observed through in vitro cell permeability analysis results for the peptide candidate group in which one or more amino acids were simultaneously substituted in the entire region of the peptide consisting of 16 amino acids.
  • the present invention relates to a cell-permeable peptide that can be usefully used in the field of basic research, diagnosis and treatment of various diseases, and the like, and relates to a basic platform peptide structure that can be expanded with an unlimited number of designs.
  • the present invention provides a cell-permeable peptide consisting of an amino acid sequence represented by the following [General Formula I] or an amino acid sequence having at least 70% homology thereto.
  • X 1 is GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE or GHHG;
  • X a is Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) or Gln(Q);
  • X b is Lys(K), Arg(R), His(H), Asn(N) or Gln(Q);
  • X 2 is SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS;
  • X 3 is TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG or NSG.
  • amino acid sequence used in the present invention is abbreviated as follows according to the IUPAC-IUB nomenclature.
  • cell permeability refers to the ability or property of a peptide to penetrate a cell (membrane) and penetrate into the cell.
  • peptide is a polymer of amino acids, usually a form in which a small number of amino acids are linked is called a peptide, and when many amino acids are linked, it is called a protein.
  • linkage between amino acids consists of an amide bond or a peptide bond.
  • a peptide bond is a bond between a carboxyl group (-COOH) and an amino group (-NH 2 ) through which water (H 2 O) escapes and forms -CO-NH-.
  • the novel peptide of [General Formula I] may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 62, but is not limited thereto.
  • the cell-penetrating peptide is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NOs: 1 to 62, respectively. %, 94%, 95%, 96%, 97%, 98%, 99% or more of an amino acid sequence having sequence homology.
  • various amino acids capable of enhancing the effect as the cell-permeable peptide may be additionally added or deleted to the N-terminus and C-terminus of the cell-permeable peptide represented by the [General Formula I].
  • the types of cells permeable to the cell-penetrating peptide include, but are not limited to, brain endothelial cells, cancer cells, blood cells, lymphocytes, and immune cells.
  • Cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), T cells, B cells, natural killer cells (NK cells), macrophages (macrophage) ), microglia, neurons, glial cells, astrocytes, and muscle cells may be any one selected from the group consisting of.
  • the peptide of the present invention can be manufactured so that the purity of each peptide is 90% or more through a conventional peptide synthesis method or manufacturing method known to those skilled in the art.
  • the peptide can be used by producing both D-form or L-form, a peptide composed of only a part of the D-form or L-form sequence, or a racemate form thereof through a conventional peptide synthesis method or manufacturing method known to those skilled in the art.
  • the peptide is preferably synthesized using a solid state peptide synthesis method, but as described above, the peptide synthesis method and conditions are not limited thereto.
  • the present invention provides a polynucleotide encoding the peptide.
  • the polynucleotide may be in the form of RNA or DNA, and the DNA includes cDNA and synthetic DNA.
  • DNA can be single-stranded or double-stranded. If single-stranded, it may be the coding strand or the non-coding (antisense) strand, wherein the coding sequence encodes the same polypeptide, as a result of the degeneracy or redundancy of the genetic code.
  • the polynucleotides of the invention may also include variants of the polynucleotides described above, wherein the variants of the polynucleotide are naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide.
  • allelic variants are alternate forms of a polynucleotide sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded (encoded). It is well known in the art that a single amino acid can be encoded by more than one nucleotide codon and that the polynucleotide can be readily modified to produce alternating polynucleotides encoding the same peptide.
  • the present inventors have discovered cell-penetrating peptides having excellent cell-penetrating ability through previous prior studies, and in order to additionally discover peptides having excellent cell-penetrating ability, one or more amino acids are substituted based on the sequence and structure of the discovered peptides Various combinations of peptide candidates were designed and synthesized, and various cell-penetrating peptides having excellent cell-penetrating ability according to the present invention were discovered by analyzing their cell-penetrating ability.
  • 16 amino acids are divided into 4 regions (regions 1-4, 5-7, 8-12, and 13-16 from the N-terminus), and each region or Cell-permeable peptide candidates in which two or more regions were simultaneously substituted with one or more amino acids in various combinations were designed and synthesized. (See Examples 1 and 2).
  • the peptide according to the present invention has excellent cell permeability, and based on this, it can be used as a carrier that can effectively introduce any substance bound to the peptide into the cell.
  • the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.
  • the complex includes all those produced by simply mixing a peptide and a substance, a compound formed by mixing a peptide and a substance, or connecting or conjugating them by a chemical bond.
  • the complex may be linked by a physical bond, a chemical bond, a covalent bond, a non-covalent bond, self-assembly, or may be linked in an integrated or fused form using a mediator.
  • the complex may be a complex formed by expressing the peptide and the biologically active material in a fusion state with each other.
  • a gene expressing the peptide and a biologically active substance is inserted into one vector, and then an organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biologically active substance are fusion proteins (fusion protein) can be expressed.
  • fusion protein fusion protein
  • any linker may be included between the peptide and the biologically active material.
  • the cell-penetrating peptide may include both single or plural combined forms in order to efficiently deliver a biologically active substance into a cell, and the cell-penetrating peptide may be cell-permeable depending on the biologically active substance to be delivered.
  • the number of peptide bonds can be easily selected or adjusted by those skilled in the art.
  • a biologically active substance capable of forming a complex by binding to a cell-penetrating peptide preferably means 'a substance having biological or pharmaceutical activity', which penetrates into cells (in the cytoplasm or nucleus) to form a physiological It refers to a substance that is involved in activity regulation or can express pharmacological effects, or has biological activity in various parts of the body, such as cells, tissues, interstitial cells, and blood, which must be transported and acted upon.
  • a compound for example, but not limited to, a compound (chemical compound), protein, glycoprotein, peptide antibody (antibody), enzyme (enzyme), nuclease (nuclease), hormone, cytokine (cytokine), transcription factor (transcription factor) ), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorescence It may be one or more selected from the group consisting of dyes (fluorochromes).
  • the nucleases are CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-like effector nuclease (TALEN). It may be selected from the group consisting of, but is not limited thereto.
  • the nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid) ), and may be selected from the group consisting of morpholino, and may additionally include decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotide, or transfer RNA, but is not limited thereto. does not
  • the drug is a compound drug (chemical drug), bio drug (bio drug), nucleic acid drug (nucleic acid drug), peptide drug (peptide drug), protein drug (protein drug), natural product drug (natural product drug), It may be selected from the group consisting of a hormone, a contrast agent, and an antibody, but is not limited thereto.
  • bio-drug refers to various biopharmaceuticals such as (original) biologics, biogenerics, biobetters, and biosuperiors.
  • the bio-drug refers to any drug manufactured, secreted, or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.
  • the nanoparticles may be selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads, but is not limited thereto.
  • the present invention provides a composition for mass delivery comprising the complex as an active ingredient.
  • the composition for mass delivery may be used to deliver a biologically active material to a living tissue or blood or to promote cell permeation.
  • the composition may be delivered through cells constituting a living tissue or through cell-to-cell junctions, but there is no limitation on the delivery method.
  • the living tissue means one or more epithelial tissue, muscle tissue, nervous tissue, and connective tissue, and each organ may consist of one or more tissues, so mucosa, skin, brain, lung, liver, kidney, spleen, lung, heart, stomach , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genitalia, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsils, bronchi, alveoli However, it is not limited thereto.
  • the material having the biological activity is an extracellular partial protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or
  • a complex can be formed by binding to a monoclonal antibody (mAb) capable of specifically binding to these receptors or ligands and a modified form.
  • mAb monoclonal antibody
  • the binding between the peptide and the biologically active substance is by indirect linking by cloning technique using an expression vector at the nucleotide level, or by direct linking by chemical or physical covalent or non-covalent binding between the peptide and the biologically active substance. can do.
  • the composition including the complex when used as a pharmaceutical composition, the composition may include the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the composition.
  • composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
  • composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.
  • the pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added.
  • diluents such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and can act specifically on target organs.
  • a target organ-specific antibody or other ligand may be used in combination with the carrier.
  • it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's literature.
  • composition comprising the complex as an active ingredient is intravenous, intraperitoneal, intramuscular, intrathecal, intracerebroventricular, subcutaneous, intradermal. , intranasal (nasal), intramucosal (mucosal), inhalation (inhalation), it can be delivered into the living body by injecting orally (oral).
  • the dosage varies according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.
  • the present invention provides a method for transferring a mass into a cell, comprising the step of treating the cell with the composition for mass transfer.
  • the cell-penetrating peptide having a mass transfer function according to the present invention is a very small peptide, it is possible to minimize any biological interference with the active material that may occur.
  • the present inventors discovered a peptide consisting of 16 amino acids having excellent cell-penetrating ability as a result of previous studies (N-GHHERLKSDEWSVTSG-C). Furthermore, in order to additionally discover peptides with excellent cell-penetrating ability, the sequence and structure of the discovered peptides were analyzed, and 16 amino acids were divided into 4 regions (1 to 4, 5 from the N-terminus in consideration of stability and cell permeability). ⁇ 7, 8 ⁇ 12, and 13 ⁇ 16 position), and various combinations of cell-penetrating peptide candidate groups in which one or more amino acids were substituted for each region or two or more regions were designed and synthesized.
  • the present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 1-1.
  • the method is an organic synthesis method in which the C-terminus of an amino acid whose N-terminus is protected with F-moc is joined to the N-terminus of the resin one by one.
  • the solvent for all reactions was N,N -dimethylformamide ( N,N -dimethylformamide; DMF), and the amino acid coupling condition was 0.5M DIC ( N,N ′-Diisopropylcarbodiimide; DIC) in a 2M concentration amino acid solution.
  • a fluorescent substance including a carboxyl group (-COOH) at the N-terminus of the peptide may be linked by a chemical bonding method in order to later observe and quantify cell permeability.
  • the fluorescent material that can be used is 5-FAM (5-carboxylfluorecein), FITC (Fluorescein-5-isothiocyanate), cyanine 3 carboxylic acid, cyanine 5 carboxylic acid, cyanine 7 carboxylic acid ( Cyanine 7 carboxylic acid), and the like, and at least one of the above fluorescent materials may be used.
  • the present inventors first synthesized the last amino acid of the peptide synthesized on a solid resin, and then mixed well with 5-FAM: DIC: Oxyma: resin in a 2: 2.5: 4: 1 ratio, and then reacted in the resin. It proceeded at room temperature for 2 hours, but a magnetic stirrer was used. Next, when the color of the resin changed from yellow to dark yellow or orange during the synthesis process, a process of washing DMF and methylene chloride three times was alternately performed.
  • the lyophilized peptide was dissolved in distilled water or acetonitrile (ACN), and separated and purified using reverse phase high-performance liquid chromatography.
  • solvent A distilled water 99.9%, TFA 0.1%)
  • solvent B distilled water 9.9%, acetonitrile 90%, TFA 0.1%) were used as mobile phase solvents of the HPLC.
  • the HPLC mobile phase started with Solvent A 90% and Solvent B 10%, and the separation proceeded while increasing the solvent B to 1%/min gradient. Thereafter, the separated peptide solution was lyophilized to remove the solvent and then dissolved in a desired solvent to conduct the experiment.
  • Example 1 In order to evaluate the cell permeability of the peptide candidates synthesized in Example 1, the present inventors performed an in vitro permeability analysis on the blood-brain barrier cells.
  • hCMEC/D3 cells which are human blood-brain barrier cells, are seeded in a 96-well plate, and the epithelial cell growth medium is EGM (endothelial cell growth medium) until the cells reach 80-90% of the plate area at 37°C. , and incubated overnight under CO 2 conditions.
  • EGM endothelial cell growth medium
  • the FAM-linked candidate peptides prepared in Example 1-2 and the negative control FAM alone were diluted in the medium supplemented with the growth factor at 4uM to prepare an amount to be treated by 100 ⁇ L per well.
  • the culture solution was removed by suction from the cell culture plate, and the peptide was diluted and treated with a solution prepared in advance, and then cultured at 37° C.
  • the cells are partitioned with a size of 30 ⁇ M centered on DAPI, and only the cells of this size are selected for image reading of FAM fluorescence, and then the image is processed to mean -FAM value was obtained and a graph was created in which the FAM experimental group was corrected with a negative control group, and the blood-brain barrier cell permeability was calculated.
  • Statistics were performed by ONE-WAY ANOVA analysis, and multiple comparisons were obtained using the FAM value as a control and corrected by the Bonferroni method (95% confidence) (mean ⁇ SEM, *p ⁇ 0.1, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • Example 1-1 As designed in Example 1-1, various peptide candidates in which the amino acid region at positions 1 to 4 from the N-terminus were substituted were designed in a peptide composed of 16 amino acids discovered through the results of previous studies, and specific sequences Information is presented in Table 1 below.
  • FIG. 2A As a result of confirming cell permeability in the peptide group, As shown in FIG. 2A, it was confirmed that the peptides of the sequences #12 to #23 of Table 2 showed an excellent effect of influx into the blood-brain barrier cells, although there was a difference in degree. In contrast, as can be seen in FIG. 2B , it was confirmed that there was no significant difference in fluorescence intensity in the case of the peptides having sequences corresponding to #72 and #73 in Table 2 compared to the negative control FAM.
  • the amino acid at position 5 in the amino acid region at positions 5 to 7 from the N-terminus consists of R, I, V, L, N, K, Q, G or A, or the amino acid at position 7 is
  • K, R, H, N or Q it was found that excellent cell permeability was maintained, whereas when it was out of this, cell permeability was not maintained.
  • the novel cell-penetrating peptide according to the present invention has excellent cell-penetrating ability and mass-transfer effect, and is useful in the field of research, diagnosis or treatment of various diseases, etc. by effectively delivering substances having various biological activities into the living body such as cells and tissues It is expected to be usable.

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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une technologie d'administration intracellulaire pour l'administration de substances biologiquement actives dans des cellules et, plus particulièrement, un nouveau peptide de pénétration cellulaire ayant une excellente capacité de pénétration cellulaire, et son utilisation. Dans la présente invention, un nouveau peptide de pénétration cellulaire est synthétisé et son excellente capacité de pénétration cellulaire est identifiée. Par conséquent, un peptide de pénétration cellulaire selon la présente invention peut efficacement administrer des substances ayant une activité biologique dans le corps vivant telles que ses cellules et ses tissus, et devrait ainsi pouvoir être utilisé efficacement dans les domaines de la recherche basique, du diagnostic et du traitement de diverses maladies, et analogues.
PCT/KR2021/003945 2020-12-24 2021-03-30 Nouveau peptide de pénétration cellulaire et son utilisation WO2022139070A1 (fr)

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KR1020200183618A KR102274877B1 (ko) 2020-12-24 2020-12-24 신규한 세포 투과성 펩타이드 및 이의 용도

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150145132A (ko) * 2014-06-18 2015-12-29 한국과학기술연구원 신규 세포투과성 펩타이드 및 이의 용도
KR101647804B1 (ko) * 2015-06-24 2016-08-11 한국과학기술연구원 신규 세포투과 펩타이드 및 이의 용도
KR20170002475A (ko) * 2014-05-29 2017-01-06 주식회사 프로셀테라퓨틱스 신규한 세포투과성 펩타이드 및 이와 보툴리눔 독소 결합체 및 이들의 용도
KR20170015852A (ko) * 2015-07-31 2017-02-09 이화여자대학교 산학협력단 신규한 세포투과성 펩타이드
KR20200104524A (ko) * 2019-02-27 2020-09-04 주식회사 아임뉴런바이오사이언스 신규의 세포 투과성 펩타이드 및 이의 용도

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170002475A (ko) * 2014-05-29 2017-01-06 주식회사 프로셀테라퓨틱스 신규한 세포투과성 펩타이드 및 이와 보툴리눔 독소 결합체 및 이들의 용도
KR20150145132A (ko) * 2014-06-18 2015-12-29 한국과학기술연구원 신규 세포투과성 펩타이드 및 이의 용도
KR101647804B1 (ko) * 2015-06-24 2016-08-11 한국과학기술연구원 신규 세포투과 펩타이드 및 이의 용도
KR20170015852A (ko) * 2015-07-31 2017-02-09 이화여자대학교 산학협력단 신규한 세포투과성 펩타이드
KR20200104524A (ko) * 2019-02-27 2020-09-04 주식회사 아임뉴런바이오사이언스 신규의 세포 투과성 펩타이드 및 이의 용도

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