WO2013061818A1 - Nouveau complexe peptidique et complexe hybride de celui-ci, et utilisation dudit complexe hybride - Google Patents

Nouveau complexe peptidique et complexe hybride de celui-ci, et utilisation dudit complexe hybride Download PDF

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Publication number
WO2013061818A1
WO2013061818A1 PCT/JP2012/076654 JP2012076654W WO2013061818A1 WO 2013061818 A1 WO2013061818 A1 WO 2013061818A1 JP 2012076654 W JP2012076654 W JP 2012076654W WO 2013061818 A1 WO2013061818 A1 WO 2013061818A1
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peptide
leucine zipper
complex
tmr
egfp
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PCT/JP2012/076654
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English (en)
Japanese (ja)
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瑞生 北松
宏之 道上
飛霏 王
真実 中島
高史 大槻
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国立大学法人岡山大学
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Publication of WO2013061818A1 publication Critical patent/WO2013061818A1/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • the present invention relates to a method for introducing a protein or the like capable of expressing a predetermined function in a cell into the cell, a peptide complex used in the method, and the like.
  • CPP cell-penetrating peptide
  • the production efficiency of the CPP-fused protein used in the above-described technology varies considerably depending on the type of protein to which the CPP is linked, and it may be hardly produced.
  • the CPP may inhibit the function of the protein (CPP is generally positively charged, and this is a nucleic acid that is negatively charged. Adverse effects may be caused by the interaction with biological materials such as
  • Non-Patent Document 1 by the group of Dowdy et al. Introduces six histidines (so-called histidine tags) to the target protein, which are modified with a metal ligand. In addition, a method has been described in which intracellular transportability is provided by linking to a CPP via the metal (nickel) (without using a covalent bond).
  • Non-Patent Document 1 uses a histidine tag, which is generally widely used in protein purification, as a tag that gives intracellular transport ability. Concerns remain when applied to the intended intracellular delivery technology, the so-called drug delivery system (DDS).
  • DDS drug delivery system
  • Non-Patent Documents 2 to 5 by the group of Bosshard et al. Disclose mutant leucine zipper peptides that were artificially developed based on natural leucine zipper peptides.
  • Leucine zippers coiled coils
  • Each peptide that forms a dimer contains an ⁇ -helix of 7 amino acid residues, and leucine is arranged at the fourth (d) position, and the interaction between these hydrophobic amino acid residues. Is characterized by the formation of dimers.
  • the mutant leucine zipper peptide disclosed in the above non-patent document is designed to further include a basic amino acid (lysine) or an acidic amino acid (glutamic acid) at a predetermined position in the amino acid sequence, and is a natural leucine zipper peptide
  • the bond stability is improved more than that.
  • Non-Patent Documents 2 to 5 are only prepared for analysis of the thermodynamic behavior of leucine zipper peptides.
  • a desired substance (such as a peptide) can be efficiently introduced into a cell by linking a cell-invading peptide to one leucine zipper peptide of the dimer or using such a leucine zipper peptide. There is no description or suggestion of becoming.
  • An object of the present invention is to provide a means for delivering a substance to be transported into a cell, which can be exhibited without hindering the intended function later, and does not use a metal that may be affected by the living body.
  • a method may be considered in which this is formed and brought into contact with a cell.
  • the complex may move into the cell, but the complex does not come off after the transfer, and the target protein is not released within the cell. There is a possibility that the performance of a predetermined function is hindered.
  • the present inventors have found that it is preferable to use a pair of leucine zipper peptides (“orthogonal” leucine zipper tags) having a predetermined amino acid sequence, that is, one leucine zipper peptide.
  • a pair of leucine zipper peptides (“orthogonal” leucine zipper tags) having a predetermined amino acid sequence, that is, one leucine zipper peptide.
  • the present invention includes the following inventions.
  • a leucine zipper peptide [LZ (K)] consisting of the amino acid sequence of SEQ ID NO: 1 or a leucine zipper peptide [LZ (K) ′] consisting of an amino acid sequence having a mutation to 1 to 8 amino acids in the amino acid sequence
  • a leucine zipper peptide [LZ (E)] consisting of the amino acid sequence of SEQ ID NO: 2 or a leucine zipper peptide [LZ (E) ′] represented by an amino acid sequence having a mutation to 1 to 8 amino acids in the amino acid sequence
  • N′-EYQALKKKKVAQLKAKNQALKKKKVAQLKHK-C ′ SEQ ID NO: 1
  • N′-EYQALEKEVAQLEAENQALEKEVAQLEHE-C ′ SEQ ID NO: 2 ⁇ 2>
  • a peptide complex [B] comprising a second leucine zipper peptide capable of associating with the first leucine zipper peptide contained in the peptide complex [A], and a substance to be transported into the cells linked thereto;
  • a hybrid complex characterized in that it is formed from:
  • the first leucine zipper peptide is the leucine zipper peptide [LZ (K)] or [LZ (K) ′]
  • the second leucine zipper peptide is the leucine zipper peptide [LZ (E) Or [LZ (E) ′]
  • the first leucine zipper peptide is the leucine zipper peptide [LZ (E)] or [LZ (E) ′]
  • the second leucine zipper peptide is The leucine zipper peptide [LZ (K)] or [LZ (K) ′].
  • ⁇ 5> The hybrid complex according to ⁇ 4>, wherein the transported substance is a protein or a peptide.
  • ⁇ 6> A therapeutic or diagnostic agent containing the hybrid complex according to ⁇ 4> or ⁇ 5>.
  • the complex of the leucine zipper peptide and the transported substance such as protein and the complex of the leucine zipper peptide and the cell invasion peptide used in the present invention can be prepared relatively easily by a known method, There is no need to use metal.
  • the transported substance can be efficiently delivered into the cell, and after being delivered into the cell, the hybrid complex is again Since the substance is separated into two complexes, the transported substance (protein, etc.) linked to one peptide complex exhibits a predetermined function without being affected by the CPP linked to the other peptide complex. can do.
  • a hybrid complex can be expected, for example, as a full-scale intracellular protein delivery technique for the purpose of therapy and diagnosis.
  • Example 3 Schematic diagram of two leucine zipper peptides oriented in parallel (cited from Non-Patent Document 3).
  • the fluorescence spectrum when EGFP-LZ (K) and the peptide which modified various Tmr in Example 3 are mixed in an equivalent amount.
  • Example 3 the change in the fluorescence intensity derived from EGFP when Tmr-LZ (E) and Tmr-LZ (K) were gradually added dropwise to an aqueous solution containing 15 nM of EGFP-LZ (K).
  • On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph).
  • the center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph).
  • the right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • the observation image by the confocal laser microscope of the U251MG cell incubated with the mixed solution of EGFP-LZ (K) and Tmr-LZ (E) in Example 4-2.
  • On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph).
  • the center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph).
  • the right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph).
  • the center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph).
  • the right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph).
  • the center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph).
  • the right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • the observation image by the confocal laser microscope of the U251MG cell incubated with the mixed solution of EGFP-LZ (K) and Tmr-11R in Example 4-5 On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph). The center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph). The right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • the observation image by the confocal laser microscope of the U251MG cell incubated with the mixed solution of EGFP-LZ (K) and Tmr-LZ (K) in Example 4-6 On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph). The center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph). The right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • On the left is an image that captures the fluorescence of EGFP (displayed in green in a color photograph).
  • the center is an image that captures Tmr (modified with a peptide) (displayed in red in a color photograph).
  • the right side is an image in which the left and center images are superimposed (in the color photograph, the part where EGFP and Tmr overlap is displayed in yellow).
  • ⁇ Peptide complex ⁇ In the present invention, two types of peptide complexes are used in order to deliver the transported substance into the cell.
  • One includes a first leucine zipper peptide having a predetermined amino acid sequence and a cell invasion peptide linked to the first leucine zipper peptide, and further linked to the leucine zipper peptide and / or the cell invasion peptide as necessary.
  • a peptide complex that may contain a fluorescent dye referred to as “peptide complex [A]” for convenience).
  • peptide complex [B] a peptide complex
  • peptide complex [B] a second leucine zipper peptide having a predetermined amino acid sequence and a transported substance linked thereto.
  • the first and second leucine zipper peptides contained in the peptide complexes [A] and [B] only need to constitute a pair of leucine zipper peptides that can be associated, and either of the pair of leucine zipper peptides Either the first leucine zipper peptide may be linked to the cell-invading peptide in the peptide complex [A], or either may be linked to the transported substance in the peptide complex [B] as the second leucine zipper peptide.
  • the peptide complex [A] may be composed of only the above leucine zipper peptide and intracellular invading peptide, or only the above leucine zipper peptide, intracellular invading peptide and fluorescent dye, and inhibits the action and effect of the present invention. Other components may be included as long as they are not.
  • the peptide complex [B] may be composed only of the leucine zipper peptide and the substance to be transported, or may further contain other components within a range that does not inhibit the action and effect of the present invention. May be.
  • the terminal treatment of the peptide complexes [A] and [B] is not particularly limited.
  • the N-terminal side may be a primary amino group, and the C-terminal side (via ⁇ -alanine as necessary). ) Primary amide.
  • leucine zipper peptide constituting the peptide complexes [A] and [B] of the present invention
  • natural leucine zipper peptide or its derivatives (especially improved stability during complex formation between leucine zipper peptides) Can be used.
  • the leucine zipper peptide has a structure in which an ⁇ -helix composed of seven amino acid residues (abcdefg) is repeated several times (see FIG. 1).
  • the two leucine zipper peptides associate in a parallel orientation (aligned from the N-terminus to the C-terminus) with the side chains of the amino acids at positions a, d, e, and g facing inward, A composite (coiled coil) is formed.
  • the stability of this complex is considered to be particularly affected by the interaction between the side chains of amino acids at positions a, d, e and g. Therefore, in the present invention, it is preferable to use a leucine zipper peptide that can obtain high stability by interaction between amino acids at positions a, d, e, and g.
  • a leucine zipper peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 also referred to as “LZ (K)” in the present invention
  • a leucine zipper peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 may be expressed as a leucine zipper peptide in the present invention (LZ (K) and LZ (E)). It is preferred to use one as the first leucine zipper peptide for the peptide complex [A] and the other as the second leucine zipper peptide for the peptide complex [B].
  • the leucine zipper peptides having the amino acid sequences represented by SEQ ID NOs: 1 and 2 are described in Non-Patent Documents 2 and 3 described above.
  • a total of 8 amino acids at positions e and g with respect to the amino acid sequence of the peptide are all substituted with glutamic acid (E). Since the amino acids at positions e and g are oriented outward to some extent, lysine and glutamic acid are positively and negatively charged in aqueous solution, respectively. Therefore, a pair of leucine zipper peptides consisting of LZ (K) and LZ (E) are not only in interaction with leucine at the position d provided in the natural leucine zipper peptide, but also at the positions e and g. A complex can be stably formed by electrostatic interaction with lysine and glutamic acid.
  • leucine zipper peptide which consists of an amino acid sequence in which one or several (for example, 1 to 8) amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, It is also possible to use leucine zipper peptides LZ (K) ′ and LZ (E) ′ capable of forming a complex with L. As described above, the stability of leucine zipper peptide is considered to be greatly influenced by the interaction of amino acids at positions a, d, e and g. Therefore, the strength of the interaction of these amino acids is set within an appropriate range.
  • amino acid substitution for fluctuation, or to perform deletion, substitution or addition of other amino acids within a range that does not adversely affect the interaction of these amino acids.
  • the strength of amino acid interaction can be adjusted by those skilled in the art without undue trial and error.
  • Intracellular entry peptide The cell-penetrating peptide (CPP) constituting the peptide complex [A] of the present invention generally has an amino acid sequence rich in basic amino acids such as arginine and binds to the cell membrane to itself. It is known as a polypeptide having a function of being incorporated into cells, and is sometimes referred to as a cell membrane permeable peptide, a membrane transduction domain (PTD), or the like. Initially, CPP permeated through the cell membrane and thought to spontaneously enter the cell, so it was sometimes referred to as a “cell membrane permeable peptide”. It is thought that it is taken up into the cell through.
  • CPP Intracellular entry peptide
  • various CPPs can be used as long as they correspond to a target cell (usually a mammalian cell), and the kind thereof is not particularly limited.
  • the CPP may have a wild-type amino acid sequence, or may be substituted for the wild-type amino acid sequence as long as it has the ability to enter cells (preferably better than the wild-type). , May have a mutant amino acid sequence that has been deleted or added.
  • a polyarginine having an amino acid sequence consisting of only about 7 to 11 arginines for example, a polyarginine having an amino acid sequence represented by SEQ ID NO: 3 (denoted as “11R” in the present invention) May be included).
  • RRRRRRRRRRR SEQ ID NO: 3 a polymorphic amino acid sequence represented by YGRKKRRQRRRG contained in a trans-activator of transcription protein (Tat protein) derived from human immunodeficiency virus type I (HIV-1) having an amino acid sequence rich in arginine.
  • CPPs polypeptides comprising the amino acid sequence represented by RRRRNRTRRNNRRRVR derived from flockhouse virus (FHV), and CTP512 (Cytoplasmic Transduction Peptide) comprising the amino acid sequence represented by YGRRRRRRRRRRR are also mentioned as preferred CPPs in the present invention.
  • CTP512 Cytoplasmic Transduction Peptide
  • Rev peptide, feline herpesvirus Coat protein-derived peptide, and the like can also be used as the CPP in the present invention.
  • the order of arrangement of the leucine zipper peptide and the intracellular invading peptide is not particularly limited, and may be the order of leucine zipper peptide-intracellular invading peptide from the N-terminal side.
  • the order of invasion peptide-leucine zipper peptide may be used.
  • the cell invasion peptide and the leucine zipper peptide may be directly linked or may be linked via a linker (spacer) as necessary.
  • a linker it is possible to select a known linker used for linking general peptides or proteins (or compounds having a predetermined functional group) to each other as long as the effects of the present invention are not inhibited. it can.
  • a peptide composed of about 1 to 20 amino acids may be used as a linker so that the entire peptide complex [A] is composed of only amino acids, or a compound other than a peptide (amino acid), such as PEG (polyethylene glycol) ) Etc. can also be used as a linker.
  • the peptide complex [A] in which the intracellular invasion peptide and the leucine zipper peptide are linked can be produced by a known method, and the production method is not particularly limited.
  • the Fmoc-peptide solid-phase synthesis method commonly used as a peptide synthesis method is used to sequentially bind the amino acids constituting the peptide complex to form an amide bond between the intracellular entry peptide and the leucine zipper peptide.
  • Complexes linked by (peptide bonds) can be synthesized.
  • the linker can be used by a general method.
  • the transported substance linked to the peptide complex [B] is transported into the cell through the formation of a hybrid complex by the peptide complex [A] and the peptide complex [B].
  • a fluorescent dye may be linked to at least the peptide complex [A].
  • fluorescent dye various fluorescent dyes used for fluorescently labeling known peptides or proteins can be used, and are not particularly limited.
  • Fmoc-peptide solid phase synthesis It is possible to synthesize the peptide complex integrally by using the method, and the molecular size is sufficiently smaller than that of the fluorescent protein (for example, EGFP), so it is difficult to inhibit the formation of the hybrid complex.
  • fluorescent protein for example, EGFP
  • the “fluorescent amino acid” can be selected from known fluorescent amino acids.
  • fluorescent amino acids include 3- (9-oxo-9,10-dihydro-acridin-2-yl) alanine (Acd), N- ⁇ - (fluorescein-5,6-yl) carbonyl-lysine ( Fam), N- ⁇ - (tetramethylrhodamine-5,6-yl) carbonyl-lysine (Tmr), 3- (1-pyrenyl) alanine (Pyr), N- ⁇ - (10-oxo-2,3, 5,6-tetrahydro-1H, 4H, 10H-11-oxa-3-aza-benzo [de] anthracene-9-carbonyl) -ornithine (Cm3), N- ⁇ - (7-methoxy-coumarin-3-yl ) Carbonyl-lysine (Moc), N- ⁇ - (7-methoxy-coumarin-4-yl) acetyl-lysine (
  • N- ⁇ - (fluorescein-5,6-yl) carbonyl-lysine (Fam) and N- ⁇ - (tetramethylrhodamine-5,6-yl) carbonyl-lysine (Fam) used in Examples below Tmr) is an example of a preferred fluorescent amino acid in the present invention.
  • the generic names of such fluorescent dyes are named based on the main structures (skeletons) in the compounds or registered trademarks, and the scope of fluorescent dyes belonging to each can be appropriately understood by those skilled in the art. .
  • the fluorescent dye is based on a known peptide or protein and a method used for linking the fluorescent dye, and if necessary, using an appropriate linker, an amino group, a carboxyl group, a hydroxyl group, and a thiol possessed by an amino acid. It can be linked to the leucine zipper peptide via a functional group such as a group. From the viewpoint of not inhibiting the formation of the hybrid complex, a fluorescent dye is not present in the middle of the leucine zipper peptide (side chain of amino acid) but near the N-terminus or C-terminus, which does not affect the interaction of the leucine zipper peptide. It is preferable to connect.
  • the fluorescent amino acid can be placed at an arbitrary position by the Fmoc-peptide solid phase synthesis method, so that the fluorescent amino acid is bonded to the N-terminal or C-terminal of the leucine zipper peptide Can be easily manufactured.
  • a unique cysteine in the leucine zipper peptide is placed near the N-terminus or C-terminus (site that does not affect the interaction of the leucine zipper peptide), while the fluorescent dye is selected selectively from the thiol group of the cysteine.
  • the order of arrangement of the leucine zipper peptide, the cell invasion peptide and the fluorescent dye is not particularly limited.
  • the order of fluorescent dye-leucine zipper peptide-invading peptide may be used, or the order of entering intracellular penetrating peptide-fluorescent dye-leucine zipper peptide may be used.
  • the fluorescent dye (fluorescent amino acid, etc.) and the leucine zipper peptide and / or the intracellular invasion peptide may be directly linked or linked via a linker (spacer). It may be.
  • a linker a chemical structure or an appropriate length is selected from known ones used to link known peptides or proteins and fluorescent dyes as long as the effects of the present invention are not inhibited. Should be selected.
  • the transported material constituting the peptide complex [B] of the present invention can be introduced into cells by forming a hybrid complex according to the present invention, and can exhibit a predetermined function in the cells. If it is, it will not specifically limit, What is necessary is just to select in consideration of the use of the hybrid composite_body
  • a protein or peptide that exerts a physiological physiological activity in cells is a preferred transported substance in the present invention. Since the size (molecular weight) of proteins and the like may affect the efficiency of introduction into cells or the functioning of functions in cells, an appropriate range (generally, molecular weight 1,000 to 1,000,000) It is preferable to use a protein or the like having a size of
  • a known method can also be used for the method of preparing peptide complexes [A] and [B] in that case, that is, a method of linking a compound other than a protein such as a nucleic acid or a peptide to a leucine zipper peptide.
  • the arrangement order of the leucine zipper peptide and the transported substance is not particularly limited.
  • the order may be leucine zipper peptide-transported substance or the order of transported substance-leucine zipper peptide from the N-terminal side.
  • the transported substance and the leucine zipper peptide may be directly linked or may be linked via a linker (spacer) as necessary.
  • a linker it is possible to select a known linker used for linking general peptides or proteins (or compounds having a predetermined functional group) to each other as long as the effects of the present invention are not inhibited. it can.
  • a peptide composed of about 1 to 20 amino acids may be used as a linker so that the entire peptide complex [B] is composed of only amino acids, or a compound other than a peptide (amino acid), such as PEG (polyethylene glycol) ) Etc. can also be used as a linker.
  • the peptide complex [B] in which the transported substance and the leucine zipper peptide are linked can be produced by a known method, and the production method is not particularly limited.
  • a known genetic engineering technique such as an expression vector
  • the transported substance is a peptide having a relatively small size (number of amino acid residues)
  • the peptide complex is constructed using the Fmoc-peptide solid phase synthesis method commonly used as a peptide synthesis method.
  • the hybrid complex can be easily formed by mixing the peptide complex [A] prepared in advance and the peptide complex [B] in an appropriate solvent (usually in water or an aqueous solution such as a buffer). it can.
  • the mixed amount (molar ratio) of the peptide complexes [A] and [B] is also 1: 1. Or, if necessary, it may be adjusted in the vicinity thereof.
  • the above mixing ratio deviates from 1: 1
  • another peptide complex [A ] And / or [B] may be adsorbed nonspecifically to the hybrid complex, but such nonspecific adsorption may occur as long as the effect of the present invention is not inhibited. Permissible.
  • the hybrid complex can be introduced into the cell by bringing it into contact with a cell to which the transported substance is to be delivered.
  • a solution containing a hybrid complex is added to a cell, the hybrid complex is naturally taken up into the cell by endocytosis after a certain period of time, and then performs a predetermined function in the cell. become able to.
  • the cells are usually animal cells (including human cells) and may be in vivo or in vitro (cultured cells). That is, the method for delivering a substance to be transported into cells according to the present invention can be applied both in vivo and in vitro.
  • the association After being delivered into the cell, the association is naturally released and the hybrid complex can be separated again into peptide complexes [A] and [B].
  • the transported substance (protein or the like) linked to the peptide complex [B] has a predetermined function without being affected by the CPP (polyarginine: 11R or the like) linked to the peptide complex [A]. Can be achieved.
  • the peptide complex [B] forms a hybrid complex so that the transported substance is delivered into the cell so that the transported substance can more easily perform a predetermined function after being delivered into the cell.
  • a compound that can be cleaved in the intracellular environment for example, inside of lysosome, endosome, caveolae, etc.
  • a linker for linking leucine zipper peptide and transported substance May be.
  • a predetermined function can be exhibited even when the transported substance remains linked to the leucine zipper peptide without using such a specific linker.
  • the hybrid complex according to the present invention (or the peptide complex [A] and [B] for forming the hybrid complex), or the composition containing them, is treated / prevented according to the transported substance contained therein. It can be used as a pharmaceutical for diagnosis or diagnosis.
  • the present invention relates to a hybrid complex (or peptide complexes [A] and [B] for forming the hybrid complex) for the manufacture of a therapeutic (preventive) or diagnostic (composition) medicament.
  • the present invention relates to a use (method) for treatment / prevention or diagnosis comprising administering a hybrid complex (or peptide complexes [A] and [B] for forming the hybrid complex).
  • the subject of administration or application is a human or other animal (preferably a mammal), or a biological material such as a cell collected from them. it can.
  • the hybrid complex according to the present invention (or the peptide complex [A] and [ In addition to B]), it can be prepared as a pharmaceutical composition containing various other substances such as pharmaceutically acceptable carriers, excipients, wetting agents, emulsifiers, pH buffering agents and the like.
  • a microcapsule including a peptide complex [A] and [B] for forming the hybrid complex
  • other substances encapsulating or carrying other substances can be used.
  • liposomes can also be used.
  • pharmaceutical dosage forms can be solutions, suspensions, emulsions, tablets, pills, capsules, powders, excluding formulations and the like.
  • what is necessary is just to adjust suitably the administration method and dosage of the pharmaceutical of this invention according to the chemical
  • Example 1 Synthesis of orthogonal leucine zipper peptide to which fluorescent amino acid and intracellular invasion peptide are bound
  • Leucine zipper peptide having amino acid sequence represented by SEQ ID NO: 1 N'-EYQALKKKVAQLKAKNQALKKKVAQLKHK-C '): LZ (K)
  • a leucine zipper peptide having the amino acid sequence represented by SEQ ID NO: 2 N'-EYQALEKEVAQLEAENQALEKEVAQLEHE-C '): LZ (E)
  • fluorescent amino acid Fam N- ⁇ - (fluorescein-5,6-yl) carbonyl -Lysine
  • Tmr N- ⁇ - (tetramethylrhodamine-5,6-yl) carbonyl-lysine
  • the N-terminus is a primary amino group and the C-terminus is a primary amide.
  • bAla represents ⁇ -alanine.
  • Tmr-LZ (K): H-Tmr-EYQALKKKVAQLKAKNQALKKKVAQLKHK-bAla-NH 2 ([M + H] + calc 3972.33)
  • Tmr-LZ (E): H-Tmr-EYQALEKEVAQLEAENQALEKEVAQLEHE-bAla-NH 2 ([M + H] + calc 3979.91)
  • the synthesis of these peptide complexes was performed by the conventional Fmoc-peptide solid phase synthesis method. Specifically, it was synthesized as follows.
  • the resin used for the synthesis was Fmoc-NH-SAL PEG resin (0.24 mmol / g).
  • DMF dimethylformamide
  • a 20% piperidine DMF solution was used as a deprotection reagent
  • an HBTU / NMM DMF solution was used as a coupling solution.
  • the synthesis was performed manually, and the operation of washing the resin with DMF, deprotecting, and then coupling was defined as one cycle.
  • the peptide was extended on the resin surface by repeating this cycle.
  • the peptide solution cut out from the resin was air-dried with N 2 gas.
  • the synthesized peptide was 2 ⁇ mol scale, and after synthesis, it was dissolved in 100 ⁇ L of DMSO and stored in a freezer.
  • the entire 100 ⁇ L DMSO solution was subjected to HPLC, and the peptides were fractionated and purified.
  • the peptide after purification was confirmed with MALDI-TOF-Mass (FIG. 2).
  • the molar concentration of the finally obtained solution was calculated from the absorbance of Fam (500 nm) or Tmr (550 nm) using UV-vis spectrum of these purified solutions.
  • Example 2 Synthesis of an orthogonal leucine zipper to which EGFP is bound A peptide complex in which a His tag, EGFP, a linker and LZ (K) are bound by a genetic engineering technique which is a conventional method (see SEQ ID NO: 4 below. The elements are connected with a hyphen (-)). Specifically, a plasmid (pET28b) containing a gene having a continuous base sequence encoding each element is constructed, expressed in E. coli, and the resulting fusion protein is recovered with an affinity column, and then the His tag is cleaved with a protease. Thus, a peptide complex was obtained.
  • pET28b plasmid containing a gene having a continuous base sequence encoding each element
  • Example 3 Verification of interaction of peptide complex by fluorescence measurement Using the various peptide complexes modified with Tmr prepared in Example 1 and the peptide complex modified with EGFP prepared in 1-2, Whether these complexes interacted with each other was examined using FRET (EGFP is a donor and Tmr is
  • an EGFP-LZ (K) solution having a known concentration was diluted to 1 ⁇ M using a 70 mM HEPES (pH 7.0) buffer solution.
  • a Tmr-modified peptide solution instead of PBS Buffer for Group 1 below
  • the HEPES buffer solution the final concentrations of EGFP-LZ (K) and Tmr-modified peptide are respectively
  • the 15 nM solution was adjusted to 1000 ⁇ L. After incubating for about 30 minutes, measurement was performed at room temperature using a fluorescence spectrum measuring apparatus (FP-6600, manufactured by JASCO Corporation). At this time, the excitation wavelength was measured at 489 nm from which EGFP emitted light.
  • EGFP-LZ (K) and Tmr modified peptides can be combined in the following eight ways in Group 1-8 (Group 1 does not use Tmr modified peptides and is EGFP-LZ (K) alone). Group 1; EGFP-LZ (K) + PBS (Buffer) Group 2; EGFP-LZ (K) + TMR-LZ (E) Group 3; EGFP-LZ (K) + 11R-TMR-LZ (E) Group 4; EGFP-LZ (K) + TMR-LZ (E) -11R Group 5; EGFP-LZ (K) + TMR-11R Group 6; EGFP-LZ (K) + TMR-LZ (K) Group 7; EGFP-LZ (K) + 11R-TMR-LZ (K) Group 8; EGFP-LZ (K) + TMR-LZ (K) -11R
  • FIG. 3 shows the fluorescence spectrum of the mixed solution when EGFP-LZ (K) and a peptide modified with Tmr are added in an equivalent amount (Group 2-8).
  • the peptide concentration was 15 nM in all cases, and a 70 mM HEPES (pH 7.0) buffer solution was used as a solvent.
  • the fluorescence intensity was measured at room temperature at an excitation wavelength of 489 nm.
  • As a control the results of only EGFP-LZ (K) are shown (Group 1). The value of 508 nm of this control curve was taken as 1, and the fluorescence intensity of other curves was relatively shown.
  • EGFP-LZ (K) and 11R-Tmr-LZ (E) (Group 3), Tmr-LZ (E) -11R (Group 4) and Tmr-LZ are capable of interaction between leucine zippers.
  • E Group 2
  • a large decrease in Fam fluorescence intensity was observed. This is because the interaction between LZ (K) and LZ (E) has caused EGFP and Tmr linked to each other to approach each other, thereby causing energy transfer.
  • 11R does not affect these leucine zipper interactions.
  • EGFP-LZ (K) and Tmr-modified LZ (E) (with or without 11R) successfully form a hybrid due to specific interaction between leucine zippers. It is clear.
  • Fig. 4 shows the fluorescence titration curve of Tmr-LZ (E) against EGFP-LZ (K).
  • a buffer solution of 70 mM mM HEPES (pH 7.0) was used.
  • the fluorescence intensity was measured at 508 nm with an excitation wavelength of 489 nm at room temperature. It can be seen that the fluorescence corresponding to EGFP of EGFP-LZ (K) gradually decreases as Tmr-LZ (E) is added. This decrease indicates that EGFP and Tmr are close in distance due to hybridization between leucine zippers and occur in FRET.
  • the fluorescence intensity corresponding to EGFP did not depend on the added Tmr-LZ (K), and almost no decrease in fluorescence intensity was observed. This result indicates that EGFP did not cause Tmr energy transfer because a hybrid could not form between leucine zippers, as expected.
  • Example 4 Intracellular protein transport by CPP via peptide tag (confocal laser microscope) Method First, a glass bottom dish (mini 3 well) was coated with 50 ⁇ g / mL collagen type 1 in order to prepare an environment in which cells are easily adsorbed on the dish surface. Next, 1 ⁇ 10 4 cells / well of U251MG cells, which are a type of glioma, were seeded on the dish and incubated overnight for adhesion. On the other hand, 200 ⁇ M EGFP-LZ (K) and 200 ⁇ M of each solution were added in a microtube, and the mixed solution was incubated at room temperature for 1 hour.
  • K ⁇ M EGFP-LZ
  • This mixed solution was added to U251 cells in the dish to a final concentration of 10 ⁇ M, and further incubated at 37 ° C. for 4 hours. Finally, the dish was washed twice with PBS, replaced with a medium, and then observed with EGFP (green) and Tmr (red) modified with a peptide using a confocal laser microscope. A total of 8 combinations were observed. The observed combinations are shown below.
  • FIG. 5 is a confocal microscope image of U251MG cells incubated with EGFR-LZ (K) (corresponding to Group 1).
  • EGFP-LZ (K) contains some basic amino acid (lysine) in LZ (K) (10 out of 29 residues), so it may be intracellular (without LZ (E))
  • fluorescence green
  • fluorescence red
  • Tmr-modified peptide is not incubated, no fluorescence (red) is observed.
  • FIG. 6 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and Tmr-LZ (E) (corresponding to Group 2).
  • EGFP-LZ (K) and Tmr-LZ (E) form a hybrid between leucine zippers, but are not expected to be introduced into cells because CPP is not added.
  • CPP is not added.
  • EGFP-LZ (K) was not introduced into the cells.
  • FIG. 5 it can be seen that the cell is slightly adsorbed on the “edge”.
  • Tmr-LZ (E) can be seen adsorbing as if the thread was pulled throughout the dish, details are unknown.
  • FIG. 7 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and 11R-Tmr-LZ (E) (corresponding to Group III).
  • EGFP-LZ (K) and 11R-Tmr-LZ (E) form a hybrid between leucine zippers and are expected to be introduced into cells because CPP is added.
  • CPP is added.
  • EGFP-LZ (K) was successfully transported into the cell.
  • 11R-Tmr-LZ (E) is also transported into the cell at the same time, it is considered that EGFP-LZ (K) is transported into the cell via 11R-Tmr-LZ (E).
  • FIG. 8 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and Tmr-LZ (E) -11R (corresponding to Group IV4).
  • K EGFR-LZ
  • E Tmr-LZ
  • -11R Compared to the previous 11R-Tmr-LZ (E), Tmr-LZ (E) -11R differs in the position of 11R in the peptide.
  • FIG. 9 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and Tmr-11R (corresponding to Group 5). Since EGFP-LZ (K) and Tmr-11R cannot form a hybrid, it is expected that they will not be introduced into cells. As expected, EGFP-LZ (K) was not transported into the cell. On the other hand, since Tmr-11R is added with CPP, it can be seen that it is introduced into the cell alone. These results indicate that EGFP-LZ (K) cannot be introduced into cells simply by mixing with CPP.
  • FIG. 10 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and Tmr-LZ (K) (corresponding to Group 6).
  • EGFP-LZ (K) and Tmr-LZ (K) cannot form a hybrid between leucine zippers.
  • CPP since CPP is not added, it is expected not to be introduced into cells. As expected, EGFP-LZ (K) was not introduced into the cell and Tmr-LZ (K) was not introduced into the cell.
  • FIG. 11 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and 11R-Tmr-LZ (K) (corresponding to Group 7).
  • EGFP-LZ (K) and 11R-Tmr-LZ (K) cannot form a hybrid between leucine zippers.
  • 11R-Tmr-LZ (K) was transported into the cell, but EGFP-LZ (K) was not introduced into the cell.
  • FIG. 12 is a confocal microscope image of U251MG cells incubated with a mixed solution of EGFR-LZ (K) and Tmr-LZ (K) -11R. Compared with FIG. 11, the place where 11R was modified in Tmr-LZ (K) was different, but the results were the same.
  • EGFP-LZ (K) acquires the ability to introduce CPP into the cell and is transported into the cell only when it forms a specific hybrid with LZ (E). It was also clarified that the conjugate of LZ (E) and CPP, which is a protein carrier, can be transported into cells independently of the position of CPP in the conjugate. These can be achieved in the future by providing EGFP-LZ (K) to give intracellular transport ability to CPP by low-toxic peptide-peptide interaction without using metal bonds. It can be used as a safe and efficient protein drug delivery system.
  • Sequence number 1 Leucine zipper LZ (K) Sequence number 2: Leucine zipper LZ (E) Sequence number 3: Polyarginine Sequence number 4: Peptide conjugate

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Abstract

La présente invention concerne un moyen d'administration d'une substance d'intérêt à l'intérieur d'une cellule, qui, même lorsque la substance d'intérêt est une protéine, peut introduire la protéine à l'intérieur d'une cellule avec une efficacité élevée indépendamment du type de protéine, permet que la protéine exerce une fonction donnée sans inhiber la fonction après l'introduction de la protéine dans la cellule, et n'utilise pas de métal qui peut affecter un corps vivant. Un complexe peptidique [A] selon la présente invention est caractérisé en ce qu'il comprend un peptide à fermeture éclair à leucine [LZ(K)] comprenant la séquence d'acides aminés représentée par SEQ ID No.1 : N'-EYQALKKKVAQLKAKNQALKKKVAQLKHK-C' ou similaire, ou un peptide à fermeture éclair à leucine [LZ(E)] comprenant la séquence d'acides aminés représentée par SEQ ID No.2 : N'-EYQALEKEVAQLEAENQALEKEVAQLEHE-C' ou similaire, et un peptide d'invasion intracellulaire [CPP] relié au peptide à fermeture éclair à leucine.
PCT/JP2012/076654 2011-10-25 2012-10-16 Nouveau complexe peptidique et complexe hybride de celui-ci, et utilisation dudit complexe hybride WO2013061818A1 (fr)

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WO2011071279A2 (fr) * 2009-12-11 2011-06-16 광주과학기술원 Système de transfert de charge à base de liant peptidique bipode
WO2011071280A2 (fr) * 2009-12-11 2011-06-16 광주과학기술원 Liant peptidique bipode à ciblage intracellulaire

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BR112012011467A2 (pt) * 2009-10-30 2019-09-24 Otsuka Chemical Co Ltd forma glicosilada de análogo ao glp-1 antigênico.
CA2789432A1 (fr) * 2010-02-10 2011-08-18 Vera A. Semenova Correlats serologique de protection contre une infection par bacillus anthracis

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WO2011071279A2 (fr) * 2009-12-11 2011-06-16 광주과학기술원 Système de transfert de charge à base de liant peptidique bipode
WO2011071280A2 (fr) * 2009-12-11 2011-06-16 광주과학기술원 Liant peptidique bipode à ciblage intracellulaire

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