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WO2008015841A1 - Protéine de fusion inhibitrice de kinase et préparation pharmaceutique - Google Patents

Protéine de fusion inhibitrice de kinase et préparation pharmaceutique Download PDF

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Publication number
WO2008015841A1
WO2008015841A1 PCT/JP2007/061650 JP2007061650W WO2008015841A1 WO 2008015841 A1 WO2008015841 A1 WO 2008015841A1 JP 2007061650 W JP2007061650 W JP 2007061650W WO 2008015841 A1 WO2008015841 A1 WO 2008015841A1
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kinase
peptide
fusion protein
cells
src
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Japanese (ja)
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Yoshio Umezawa
Moritoshi Sato
Taro Hitosugi
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention depends on activation of kinases by inhibiting the activation of specific kinases (Src, Akt, PKC, etc.) in intracellular organelles (lipid rafts, mitochondria, Golgi, etc.)
  • the present invention relates to a kinase-inhibitory fusion protein that can inhibit or reduce cell function and a pharmaceutical composition containing the kinase-inhibitory fusion protein.
  • Non-patent Documents 1 and 2 Activation of tyrosine kinase Src is required for cell adhesion to integrin ligands and tumor cell functions such as mitogenesis induced by growth factors.
  • Non-patent Document 3 Is known to be closely related to tumorigenesis and metastasis. Therefore, a method for treating cancer by administering an inhibitor of tyrosine kinase Src has been proposed (for example, Patent Document 1).
  • Non-Patent Document 4 shows that various pseudo-substrate inhibitory peptides against Src (about 30 types of peptides such as MIYKYYF) can be incorporated into 3T3 cells transformed with v-Src. It has been reported that phosphorylation is not inhibited and does not cause morphological changes. This indicates that even when an Src inhibitor alone is administered to a cancer cell, it cannot be expected to effectively suppress the growth or metastasis of the cancer cell.
  • lipid raft one of the intracellular organelles, cholesterol raft, called lipid raft, is considered to function as a platform for intracellular protein signaling.
  • Force tyrosine kinase Src is known to be distributed in both lipid raft and non-raft regions in cell membranes (Non-Patent Documents 5 and 6).
  • Serine / threonine kinase Akt also promotes cell survival and promotes apoptosis. It is related to cell function control such as inhibition of cis, and it is known that disease cells are involved in canceration and arteriosclerosis. Furthermore, the present inventors have developed a new fluorescent probe and have revealed that Akt activation occurs in various organelles (Non-patent Document 7). However, it is unclear how Akt activation in each organelle plays a role in cell functions.
  • Patent Document 1 Special Table 2003-525862
  • Non-Patent Document 1 Playford, M.P. & Schaller, M.D. The interplay between Src and integrins in normal and tumor biology. Oncogene. 23, 7928-7946. (2004).
  • Patent Document 2 Bromann, P.A., Korkaya, H. & Courtneidge, S.A.The interplay bet we en Src family kinases and receptor tyrosine kinases.Oncogene. 23, 7957-7968. (200
  • Patent Document 3 Summy, J.M. & Gallick, G.E.Src family kinases in tumor progression and metastasis.Cancer Metastasis Rev. 22, 337-358. (2003).
  • Non-Patent Document 4 Kamath, JR, Liu, R., Enstrom, AM, Lou, Q. & Lam, KS Development and characterization of potent and specific peptide inhibitors of p60c_src prote in tyrosine kinase using pseudosubstrate-based inhibitor design approach. J. Pept. R es.62, 260-268, 2003
  • Non-patent literature 5 Sargiacomo, M., Sudol, M., Tang, Z. & Lisanti, MP.Signal transducing molecules and glycosy phosphatidylinositoHinked proteins form a caveolin-rich in soluble complex in MDCK cells.J. Cell Biol. 122 , 789-807. (1993).
  • Non-Patent Document 6 Liang, X. et al. Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction. J. Biol. Chem. 276, 30987-30994. (2001).
  • Non-Patent Document 7 Sasaki et al. J. Biol. Chem. 273 30945-30951 (2003).
  • the inventors of the present application have developed a new organelle-localized fluorescent indicator for detecting kinase activation sites in various organelles of living cells. It was found that the ability to inhibit or reduce cellular function (especially disease-related functions) by activating with organelles and inhibiting their local activation.
  • the present invention is based on the novel findings as described above, and is based on a novel disease whose mechanism of action is to specifically inhibit a kinase that is locally activated in an intracellular onionelle in the organelle.
  • the problem is to provide a means of treatment.
  • the present invention has a kinase inhibitory peptide characterized by having a kinase inhibitory peptide and an intracellular organelle localization peptide, and specifically inhibiting intracellular kinase activation.
  • a kinase inhibitory peptide characterized by having a kinase inhibitory peptide and an intracellular organelle localization peptide, and specifically inhibiting intracellular kinase activation.
  • this kinase-inhibitory fusion protein is that the kinase inhibitor peptide is a tyrosine kinase Src inhibitor peptide, and the intracellular organelle localization peptide is a lipid raft localization peptide. It is a fusion protein.
  • this kinase-inhibiting fusion protein against Src is sometimes referred to as “Src-inhibitory fusion protein” or “SIFP” (Src Inhibitory Fusion Protein).
  • SIFP is particularly described as “lipid raft localized SIFP”.
  • SIFP Single preferred embodiment of this SIFP is to use a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an inhibitory peptide of tyrosine kinase Src, and from the amino acid sequence of SEQ ID NO: 2 as a lipid raft localization peptide. Is to use the peptide
  • the SIFP has another preferred embodiment in which the lipid raft localization peptide is modified with a palmitoyl group.
  • the inhibitory peptide of the kinase is a serine / threonine kinase Akt inhibitory peptide
  • the intracellular organelle localization peptide is An inhibitory fusion protein where the peptide is a mitochondrial localization peptide.
  • Akt-inhibiting fusion protein A kinase-inhibiting fusion protein for Akt is sometimes referred to as “Akt-inhibiting fusion protein” or “AIFP” (Inhibitory Fusion Protein).
  • One preferred embodiment of the AIFP is to use a peptide consisting of the amino acid sequence of SEQ ID NO: 3 as an inhibitory peptide of serine Z threonine kinase Akt, and SEQ ID NO: 4 as a mitochondrial localization peptide.
  • a peptide consisting of the amino acid sequence of SEQ ID NO: 3 is an inhibitory peptide of serine Z threonine kinase Akt, and SEQ ID NO: 4 as a mitochondrial localization peptide.
  • the kinase-inhibiting fusion protein of the present invention is not limited to the Src-inhibiting fusion protein (SIFP) and the Akt-inhibiting fusion protein (AIFP), which are specific examples thereof.
  • SIFP Src-inhibiting fusion protein
  • AIFP Akt-inhibiting fusion protein
  • a fusion protein comprising a combination of an inhibitory peptide of various kinases and various organelle-localizing peptides was prepared, and the fusion protein was prepared according to the test method disclosed in the present invention.
  • the present invention also provides a cell membrane permeable kinase inhibitory fusion protein in which a cell membrane permeable peptide is linked to the N-terminal side of the kinase inhibitor fusion protein.
  • a specific embodiment of this cell membrane permeable kinase-inhibiting fusion protein is “cell membrane permeable SIFP” or “cell membrane permeable AIFP” in which a cell membrane permeable peptide is linked to the N-terminal side of SIFP or AIFP.
  • the cell membrane-permeable SIFP may or may not be modified with a palmitoyl group in its lipid raft localization peptide.
  • Palmitoyl-modified ones can be localized to lipid rafts as they are, and those that are not palmitoyl-modified are palmitoyl-modified by intracellular enzymes and are also localized to lipid rafts. To do.
  • the present invention provides a cancer cell membrane permeable kinase inhibitory fusion in which a cell membrane impermeable peptide and a cancer cell specific protease recognition sequence are linked to the N-terminal side of the cell membrane permeable kinase inhibitory fusion protein.
  • Provide protein A specific embodiment of the cancer cell membrane-permeable kinase-inhibiting fusion protein is “cancer cell membrane-permeable SIFP” or “cell membrane-permeable AIFP”.
  • the present invention provides an expression vector having a polynucleotide encoding the kinase-inhibitory fusion protein.
  • the present invention further provides a pharmaceutical composition comprising each of the expression vector, the cell membrane permeable kinase inhibitory fusion protein, and the cancer cell membrane permeable kinase inhibitory fusion protein.
  • the kinase-inhibiting fusion protein of the present invention specifically inhibits the activation of intracellular kinases in which the kinase is locally activated, thereby inhibiting its activation (particularly related to diseases). Function) can be inhibited or reduced.
  • the Src inhibitory fusion protein SIFP
  • SIFP Src inhibitory fusion protein
  • Akt inhibitory fusion protein AIFP
  • the protein itself is taken up into the cell, it can be applied to diseased tissues such as cancer tissue as a simpler and more effective drug form.
  • the cancer cell membrane-permeable kinase-inhibiting fusion protein is taken up only by cancer cells, for example, even when administered systemically, it has the ability to exert specific effects on cancer cells. S can.
  • FIG. 1 shows a fluorescence indicator (TM-S reus) for detecting Src activity in biological membranes.
  • TM-Srcus principle for visualizing Src activity in cell membranes. Upon activation of Src, tyrosine phosphorylation recognition (SH2) domain strength S phosphorylation Binding to Src substrate (Y314) domain causes structural change of TM-Srcus, which causes intramolecular FRET reaction Occurs.
  • B cDNA composition of TM-Srcus and TM-Srcus314A.
  • TM-Srcus is connected in series It is a fusion protein with seven parts: transmembrane domain, cyan fluorescent protein (CFP), Y314 containing a cystein phosphorylation site (red Y), flexible linker (Ln), SH2 domain, yellow fluorescent protein (YFP) ), Consisting of an extra-nuclear signal sequence (NES).
  • the Y314 domain is a mutant Src substrate domain that contains an alanine mutation site (red A).
  • Figure 2 shows Src activation in lipid rafts of MCF7 cells.
  • the left and center of (a) are pseudo-color images of the TM_Srcus CFP / YFP emission rate in the basal cell membrane of MCF 7 cells using a total reflection fluorescence microscope (TIRFM).
  • the area surrounded by a white line shows the basal cell membrane of a single MCF7 cell.
  • the target area (R0I1) includes a blue-shifted area indicating changes in the CFP / YFP emission rate of TM_Srcus.
  • the target area (R0I2) is located where TM-Srcus does not show any change in CFP / YFP emission rate.
  • the right side of (a) is a pseudo color image by TIRFM of the fluorescence intensity of lipid raft marker Alexa_647 CTXB in the basal cell membrane of a single MCF7 cell. Alexa-647 CTXB is concentrated in ROI 1. MCF7 cells expressing TM_Srcus were stained with Alexa-647 CTXB prior to E 2 stimulation.
  • (B) shows the time course of CFP / YFP luminescence rate for 1 ⁇ M E2 stimulation in ROI 1 and ROI 2 of the basal cell membrane of MCF7 cells expressing TM-Srcus.
  • C is a Western plot analysis of low and high density fractions of MCF7 cells expressing TM-Srcus (68 kDa).
  • FIG. 2 shows lipid raft or non-raft localized Src inhibitory fusion protein (SIFP) (a), lipid raft or non-raft localized SIFP, SIFP Y6A, and SIFP Del as YFP controls.
  • SIFP Src inhibitory fusion protein
  • Lipid rafts or non-raft localized SIFPs are YFPs, flag tags, Src inhibitory peptides containing tyrosine phosphorylation sites (red Y), and And localized sequences.
  • Lipid raft or non-raft localized SIFP Y6A has an alanine mutation-inhibiting peptide containing an alanine mutation site (red A).
  • red A lipid raft or non-raft localized SIFP Del
  • the inhibitory peptide against Src has been removed.
  • B is a fluorescence image of MCF7 cells expressing lipid raft or non-raft localized SIFP, using a confocal fluorescence microscope. YFP images in lipid raft or non-raft localized SIFP are shown in green. Staining with Alexa-647 CTXB is red. The merged image and transmission image are shown together.
  • FIG. 4 shows intracellular localization and inhibitory effect of lipid raft or non-raft localized SIFP in MCF7 cells analyzed using density gradient fraction. Lysates of MCF7 cells expressing lipid raft or non-raft localized SIFP were subjected to a density gradient to obtain low and high density fractions. Low density and high density fractions were immunoprecipitated with anti-flag antibody or anti-pTy antibody and immunoblotted with anti-GFP antibody. Treatment with the Src-specific inhibitor PP2 was performed at 10 for 10 hours. The results of (b) and (c) are representative examples of the results of three independent measurements. 4] Figure 4 shows the physiological effects of lipid raft or non-raft localized SIFP on cell function.
  • (A) shows the adhesion ability of MCF7 cells expressing lipid raft or non-raft localized SIFP.
  • Lipid raft localization type SIFP reduced the adhesion of MCF7 cells.
  • the adhesion index is the number of fluorescence-positive adherent cells divided by the number of fluorescence-positive cells initially plated. This result shows 6 or more independent measurements.
  • (B) shows the results of analysis of the cell cycle of MCF7 cells expressing lipid rafts or non-raft localized SIFP, SIFP Y6A, and YFP controls, respectively.
  • Flow cytometry using PI (propidium iodide) staining shows the number of YFP positive cells in the Gl, S and G2 / M phases of the cell cycle. Only lipid raft localization type SIFP induced cell cycle arrest of MCF7 cells.
  • the result of (b) shows a representative example of three independent measurement results.
  • FIG. 5 shows the structure of the Akt inhibitory fusion protein (AIFP) prepared in Example 2 (upper), and the structure of the control AIFP without the Akt inhibitor peptide (lower).
  • Fig. 6 is a microscopic image of AIFP-introduced breast cancer cells (MCF-7 cells) stained with mitochondrial markers and visualized AIFP GFP.
  • Upper left is a mitochondrial marker stained image
  • upper right is a GFP signal image (indicating AIFP position)
  • lower left is a transmitted light image
  • lower right is a mitochondrial marker stained image and a GFP signal image It is.
  • FIG. 7 shows the results of measuring the cell cycle profile of MCF-7 cells expressing AIFP by flow cytometry. The left is the result of AIFP-expressing cells, and the right is the result of control AIFP-expressing cells.
  • FIG. 8 shows the results of cell attachment assay for normal human cells (HEK293 cells) expressing lipid raft-localized SIFP.
  • the black bar is the result of lipid raft localized SIFP
  • the white bar is the result of lipid raft localized SIFP Del (control).
  • FIG. 9 shows the results of cell cycle assembly of normal human cells (HEK293 cells) expressing lipid raft-localized SIFP.
  • the left figure shows the results of lipid raft-localized SIFP Del (control), and the right figure shows the results of lipid raft-localized SIFP
  • FIG. 10 shows the structure of the cancer cell membrane-permeable SIFP prepared in Example 3.
  • Targeting is the lipid raft localization peptide and “inhibitory” is the Src inhibitory peptide.
  • HA is part of the hemagglutinin protein derived from influenza virus. The region indicated by the black frame is a cancer cell-specific protease recognition sequence.
  • FIG. 11 shows the results of Western blot analysis confirming that purified cancer cell membrane-permeable SIFP is cleaved by MMP treatment.
  • a cleaved band (arrow) was observed only with MMP treatment (MMP +).
  • FIG. 12 shows the result of immunostaining the cells after 3.5 hours after adding only cancer cell membrane-permeable SIFP to the MCF-7 cell culture medium.
  • the lower panel shows the result of immunostaining the cells 3.5 hours after adding cancer cell membrane-permeable SIFP and MMP to the medium of MCF-7 cells.
  • the left side shows an image stained with an anti-flag antibody, and the right side shows an image with transmitted light.
  • FIG. 13 shows the results of comparison of the degree of cell proliferation when MCF-7 cell culture medium is supplemented with cancer cell membrane-permeable SIFP with and without MMP.
  • the kinase-inhibitory fusion protein of the present invention is a protein in which a kinase inhibitory peptide and an intracellular Onoreganella localization peptide are fused.
  • the Src inhibitory fusion protein which is a specific example thereof, is a protein in which an Src inhibitory peptide and a lipid raft localization peptide are fused.
  • SIFP Src inhibitory fusion protein
  • An Src inhibitory peptide is a peptide that acts on Src and specifically inhibits its activity (phosphorylation of tyrosine residues), for example, a pseudo-substrate inhibitory peptide against Src (MIYKYYF: sequence) Number 1) (Non-Patent Document 4) can be used.
  • the lipid raft-localizing peptide is a peptide that specifically binds to intracellular lipid rafts.
  • it is derived from the 9 amino acids at the C-terminus of H-Ras protein (CMSCKCVLS: SEQ ID NO: 2). Can be used.
  • Proteins localized in lipid rafts such as H_Ras protein, have a structure in which palmitoyl acid is attached to the C-terminal cysteine residue by post-translational modification by an enzyme (palmitoyltransferase) in mammalian cells.
  • an enzyme palmitoyltransferase
  • Src inhibitory peptides and lipid raft localization peptides are not limited to these Src inhibitory peptides include FVGFLGFLG (and Ramdas, NU Obeyesekere, G. Sun, JS McMurray, RJ Budde, N-myristoylation of a peptide substrate for Src converts it i nto an apparent slow-binding bisubstrate-type inhibitor, J. Pept. Res. 53 (1999) 569 -577.), EFLYGVFF (T. Nishi, RJ Budde, JS McMurray, NU Obeyesekere , N. Saf dar, VA Levin, H.
  • FVGFLGFLG and Ramdas, NU Obeyesekere, G. Sun, JS McMurray, RJ Budde, N-myristoylation of a peptide substrate for Src converts it i nto an apparent slow-binding bisubstrate-type inhibitor, J. Pept. Res. 53
  • an Akt-inhibiting fusion protein is a protein in which an Akt-inhibiting peptide and a mitochondrial localization peptide are fused.
  • An Akt inhibitory peptide is a substance that acts on Akt and acts on it (phosphate of serine / threonine residues).
  • a pseudo-substrate inhibitory peptide for Akt (ARKRERTYSFGHHA: SEQ ID NO: 3) can be used.
  • the mitochondrial localization peptide is a peptide that specifically binds to the mitochondrion in the cell, and is composed of, for example, the amino acid sequence 1 to 35 of the mitochondrial protein Tom20 [ ⁇ MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPNFK: SEQ ID NO: 4) Peptides can be used.
  • sequence information is obtained from existing protein databases (for example, GenBank database), and various kinase-inhibiting fusion proteins are created. be able to.
  • the kinase-inhibiting peptide and the onoreganella localization peptide may be directly linked to each other, or a linker may be interposed between the two. You may let them.
  • the kinase-inhibiting fusion protein of the present invention can be produced, for example, by a genetic engineering method. That is, the target fusion protein can be obtained in vitro by preparing RNA by in vitro transcription from a vector harboring a polynucleotide encoding the fusion protein and performing in vitro translation using this as a cage.
  • a large amount of a kinase-inhibiting fusion protein can be obtained from prokaryotic cells such as Escherichia coli and Bacillus subtilis recombined by an expression vector and eukaryotic cells such as yeast, insect cells and mammalian cells. .
  • the pharmaceutical composition of the present invention contains the above-described kinase-inhibitory fusion protein as an active ingredient, and has a therapeutic effect or symptom-improving effect on a specific disease according to the effect of cell-localized kinase inhibition. is doing.
  • a kinase inhibitor fusion protein SIFP and AIFP which are specific examples of these, can be used as an active ingredient of an anticancer agent due to excellent actions such as stopping the cell cycle of cancer cells.
  • One method for using a kinase-inhibiting fusion protein as a pharmaceutical composition is to use an expression vector carrying a polynucleotide encoding these fusion proteins as a viral vector used in gene therapy. It is to build using.
  • the viral vector shall be derived from the genome of a virus selected from the family Baculovirus, Parvoviridae, Piconorenowinores, Herpesviridae, Boxviridae, Adenoviridae, or Picornavirus. be able to. Chimeric vectors that take advantage of the advantages of each parent vector can also be used (see, eg, Feng (1997) Nature Biotechnology 15: 866-870).
  • the vector is an adenovirus (eg, a replicating non-competent vector derived from the human adenovirus genome, see, eg, US Pat. Nos. 6,096,718; 6,110,458; 6, 113,913; 5,631,236); A vector derived from an associated virus and retrovirus genome.
  • Retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (For example, US Pat. Nos.
  • the polynucleotide encoding the fusion protein may be linked under the control of the disease gene promoter so that the kinase-inhibiting fusion protein is expressed specifically in the disease tissue. it can.
  • the kinase inhibitory fusion protein expressed in the cell by the recombinant virus vector as described above is obtained at a given site in the cell depending on the types of the kinase inhibitor peptide and the organelle localization peptide. Specifically inhibits the activity of specific kinases. For example, in the case of SIFP, lipid raft localization peptides are palmitoylated by post-translational modification by intracellular enzymes and selectively inhibit Src activation in lipid rafts. AIFP specifically inhibits Akt activity in mitochondria. According to this, SI Proliferation and metastasis of cancer cells expressing FP or AIFP are effectively prevented.
  • Another method for using the kinase-inhibiting fusion protein of the present invention as a pharmaceutical composition is a method of incorporating a kinase-inhibiting fusion protein itself into a cell. That is, it is a method of bringing the cell membrane permeable kinase-inhibiting fusion protein of the present invention into contact with cells of diseased tissue. Specifically, it is a method in which cell membrane-permeable SIFP or cell membrane-permeable AIFP is brought into contact with cancer cells.
  • a cell membrane permeable kinase-inhibiting fusion protein is produced by linking a cell membrane-permeable peptide to the N-terminal side of a kinase-inhibiting fusion protein.
  • a cell membrane-permeable peptide is a peptide consisting of about 10 amino acid sequences containing a large amount of arginine. Proteins added with this peptide are known to penetrate the cell membrane and enter the cell. Until now (This, f row erima, Hiv-tat (Schwarze, SR, Ho, A., Vocero— Akbani, A.
  • cell membrane permeable peptides can be directly fused with a kinase inhibitory fusion protein to produce a cell membrane permeable kinase inhibitory fusion protein.
  • kinase inhibitory activity prepared separately by genetic engineering and chemical synthesis methods
  • the fusion protein and the cell membrane permeable peptide can also be prepared by a method such as peptide bonding by a known method.
  • the cell membrane-permeable kinase-inhibiting fusion protein as described above is permeated through the cell membrane and taken into the cell, and then localized at a predetermined site in the cell according to the type of the organelle-localizing peptide. Thus, it inhibits the activity of certain kinases.
  • cell membrane-permeable SIFP in contact with cancer cells has a lipid raft-localized peptide cysteine, which is palmitoyl-modified by palmitoyl transferase, is localized to lipid raft, and lipid raft-specific activation of Src Is inhibited.
  • plasma membrane-permeable AIFP in contact with cancer cells is localized to mitochondrials and specifically inhibits Akt activation. This effectively prevents the growth and metastasis of cancer cells incorporating SIFP and AIFP.
  • the lipid raft-localized peptide cysteine can be incorporated into cells in a state of being palmitoylated in advance without depending on the action of palmitoyltransferase in the cell.
  • SFP inhibitory activity specific to lipid rafts can be imparted to SIFPs expressed or chemically synthesized in E. coli or the like by chemically modifying the C-terminal cysteine with palmitoyl groups.
  • a palmitoyl ester with a maleimide group is newly synthesized and reacted with SIFP. Since cysteine thiol groups and maleimide groups are known to bind selectively, this reaction allows the palmitoyl group to be artificially bonded to SIFP cysteine.
  • cancer cell membrane permeable kinase inhibitory fusion protein particularly SIFP or AIFP.
  • This cancer cell membrane permeable kinase-inhibiting fusion protein further comprises a cell membrane-impermeable peptide and a cancer cell-specific protease recognition sequence linked to the N-terminal side of the cell membrane-permeable kinase-inhibiting fusion protein.
  • a cell membrane impermeable peptide e.g., a cell membrane impermeable peptide
  • a peptide consisting of polyglutamine inactivates the inner cell membrane-permeable peptide and does not take it into normal cells. However, when they come into contact with cancer cells, the protease is expressed specifically by cancer cells (for example, MMP2). A specific recognition sequence (MMP2 cleavable site) is cleaved. As a result, the cell membrane permeable peptide is exposed at the N-terminus, and the cell membrane permeability activity is acquired.
  • SIFP and AIFP which are specific examples of the kinase-inhibiting fusion protein of the present invention, include, for example, esophageal cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, parotid cancer, head and neck cancer, bone * soft part.
  • lipid raft localized SIFP in order to distinguish it from control (non-raft localized SIFP).
  • ferfc trans domain derived from transmembrane phosphoprotein (Cbp, amino acids 1-5: Kawabuchi, M. et al. Transmembrane phosphop rotein Cbp regulates the activities of Src-family tyrosine kinases Nature 404, 999-1 003, 2000), Y314 domain derived from a phosphopeptide containing Cbp Tyr-314 fused to linker 1, Y314A domain fused to linker 1, S derived from the carboxyl terminus of Src kinase.
  • Cbp transmembrane phosphoprotein
  • H2 domain (Csk, amino acids 80-162: Takeuchi, S., Takayama, ⁇ , Ogawa, A., Tamura, K. & Okada,. Fransmembrane phosphoprotein Cbp positively regulates the activit y of the carboxylate terminal Src kinase, Csk J. Biol. Chem. 275, 29183—29186, 2000), CFP, protein derived from human immunodeficiency virus (Ullman, KS, Powers, MA & Forbes, DJ Nuclear export receptors: from importin to exportin. Cell 90 , 9 NES derived from “—970, 1 997) Each cDNA fragment of YFP was prepared by PCR and cloned into pBluescript SK (+).
  • lipid raft localization peptide C-terminal H_Ras, CMSCKCVLS
  • non-raft localized peptide fused with linker C-terminal Rho_A, GCLVL
  • Src inhibitory peptide fused with flag tag MIYKYYF
  • Src inhibitory peptide fused with flag tag Alanine mutant MIYKYAF
  • YFP cDNA fragments were prepared by PCR and cloned into pBlueScript SK (+).
  • MCF-7 Breast cancer cells (MCF-7) cells that express TM_Srcus are treated with steroid-free medium (2% activated charcoal-treated eagle fetal serum-free Eagle red-free Eagle's minimum essential medium) for 12 hours. Starved and washed twice using Hank's equilibrated medium (HBSS) (Sigma). CCD camera CoolSnap ES (Roper Scientific) for total reflection fluorescence imaging The cells were observed under a total reflection fluorescence microscope IX70 (Olympus) controlled by MetaFluor (Universal Imaging). The wavelength of the excitation light was 440 ⁇ 10 and the exposure time was 300 ms.
  • Fluorescence images of CFP and YFP were obtained using a 60 X oil immersion objective PlanApo60 (Olympus) through filters at 480 ⁇ 15 nm and 535 ⁇ 12.5 nm.
  • cells are pretreated with PP2 (Calbiochem), stimulated with 17 / 3_estradiol (E2) (Sigma), and cooled CCD camera MicroMax (Roper Scientific) And imaging at room temperature on a Carl Zeiss Axiovert 135 microscope (Carl Zeiss) controlled by MetaFluor.
  • the wavelength of the excitation light was 440 ⁇ 10 nm and the exposure time was 200 ms.
  • a 40 X oil immersion objective Carl Zeiss
  • fluorescent images were obtained through filters at 480 ⁇ 15 nm and 535 ⁇ 12.5 nm.
  • MCF-7 cells in 3 10cm diameter dishes expressing TM-Srcus and lipid raft and non-lipid raft localized SIFP were scraped into ice-cold HBSS, 2,000 mm, 4 ° Centrifuge and precipitate at C, and use 180 ⁇ 1 TNE (10 mM Tris-HC1, pH 7.6, 500 mM NaCl, ImM EDTA), 1% TritonX, 10% sucrose, 2 mM orthovanadate. Complete dissolution at 4 ° C by pipetting through a 200 ⁇ l yellow tip and incubating on ice for 20 minutes. 360 ⁇ of cold 0% Optiprep TM (Axis-Shield PoC AS) was added to the extract and incubated on ice for 10 minutes.
  • the low and high density fractions were diluted with equal amounts of TNE, 1% TritonX, 10% sucrose, 2 mM orthovanadate.
  • the diluted sample was immunoprecipitated using an anti-flag antibody (Sigma) or an anti-phosphotyrosine antibody (PY20, Santa Cruz Biotechnology).
  • the sump nore is 10% acrylic. Separation was performed by electrophoresis on a medium SDS gel, and the electrophoresed protein was transferred to a nitrocellulose membrane.
  • the membrane was probed with 1% dry milk or 3% BSA in TBST solution, and the primary antibody; anti-phosphotyrosine antibody (PY20, Santa Cruz Biotechnology), anti-GFP antibody (Clontech), anti-force beolin 1 (BD Transduction Laboratories) antibody and anti-Src antibody (GD11, Upstate Biotechnology). Bands were visualized using horse radish peroxidase conjugate anti-rabbit or anti-mouse IgG (Amersham Life Science).
  • MCF-7 cells at 37 ° C under 5% CO in minimal essential medium (Sigma) supplemented with 10% fetal bovine serum, 1% penicillin Z streptomycin, and O.lmM non-essential amino acid Cultured.
  • Cells were transfected using LipofectAMINE 2000 (Invitrogen). Cells were plated 24-36 hours before transfection on glass bottom dishes for fluorescent imaging or on plastic dishes for immunoblot analysis.
  • MCF-7 cells expressing TM-Srcus were fixed with 4% paraformaldehyde. Fixed cells were stained with a primary antibody against Src (scl8, Santa Cruz Biotechnology) followed by a Cy-5 conjugate anti-IgG secondary antibody. Under the confocal laser microscope LSM 510 (Carl Zeiss), the cells on the cover slide were observed at room temperature.
  • MCF-7 cells expressing TM-Srcus and lipid raft and non-raft localized SIFP are washed with HBSS and 0.01% with 2 ⁇ g / ml Alexa647-CTXB (Molecular probes) Incubation was carried out at 37 ° C for 1 hour in a minimal essential medium supplemented with BSA, 1% penicillin Z streptomycin, 25 mM HEPES and O.lmM non-essential amino acids. After staining, the cells were washed twice with HBSS and observed at room temperature under TIRFM or confocal microscopy. The exposure time was 633 ⁇ 10 nm and the excitation was 200 ms. A fluorescence image of Alexa647_CTXB was obtained through TIRFM or confocal microscope using the 60 X oil immersion objective lens, PlanApo 60 and 100 X oil immersion objective, respectively. 8.Cell adhesion assembly
  • MCF-7 cells expressing lipid raft and non-raft localized SIFP were washed with PBS, trypsinized, and resuspended in PBS. This cell suspension (10 6 cells / ml) was spread on a 24 ⁇ 24 mm micro cover glass (Matsunami) and on the bottom of a 6 well plate (Nunc).
  • the / o confluent cells were each transformed with DNA constructed by encoding SIFP and incubated at 37 ° C for 24 hours.
  • the transfected cells on the cover slides were washed with HBSS and fixed with methanol at -20 ° C for 20 minutes. Fluorescent positive cells on the cover slide were counted directly under a fluorescence microscope (X 40). To count adherent fluorescence-positive cells, the transfected cells on the bottom of the well were trypsinized and resuspended in media.
  • the whole cell suspension (10 6 cells / ml) was placed on a cover slide coated at 4 ° C with 33 ⁇ g / ml fibronectin PBS solution and incubated at 37 ° C for 4 hours.
  • the cells adhering to the fibronectin-coated cover slide were washed with HBSS, and fixed with methanol at -20 ° C for 20 minutes.
  • Adherent fluorescence positive cells were counted directly under a fluorescence microscope (X 40).
  • the adhesion index is the number of fluorescent positive cells adhered divided by the number of fluorescent positive cells initially plated.
  • MCF-7 cells (1.5 ⁇ 10 6 cells) expressing lipid raft and non-raft localized SIFP were washed with PBS, trypsinized, and resuspended in ice-cold PBS. This cell suspension was fixed with 70% ethanol for 30 minutes at 4 ° C. The fixed cells were washed twice with PBS, incubated with 100 ⁇ g / ml RNase A (Qiagen) in PBS for 30 minutes at room temperature, and PI staining (50 ⁇ g at 4 ° C for 30 minutes). / ml). PI stained cells were analyzed by FACS (Beckman Coulter).
  • TM-Srcus is a fusion protein comprising an Src substrate domain, a phosphorylation recognition domain, a linker-ligated lj, and two GFP variants.
  • the adjacent phosphorylation recognition site specifically binds to this phosphorylated tyrosine.
  • the emission ratio is the fluorescence intensity of CFP divided by the YFP tendency intensity.
  • FRET intramolecular fluorescence resonance energy transfer
  • TM-Srcus has a transmembrane domain with high affinity for cell membranes to directly detect Src activation in all regions of biological membranes such as cell membranes and intracellular membranes. This domain localizes this indicator to biological membranes, together with a lipidated signal sequence consisting of palmitoylated cysteine residues and hydrophobic transmembrane sequences. Therefore, the indicator 1 can monitor the activation of Src throughout the biological membrane (Fig. La).
  • TIRFM total reflection fluorescence microscope
  • the pseudo-color image representing the CFP / YFP emission ratio showed a local blue shift on the basal cell membrane in response to stimulation by ⁇ 2 ( Figure 2a left and middle). This indicates that Src activation occurs in a spatially limited region of the cytoplasmic membrane.
  • lipid raft marker Alexa 647-cholera toxin B subunit (CTXB 2 ) (Simons,
  • MCF-7 cells were stained using K. & ⁇ re, D. Lipid rafts ana signal transduction. Nat. Rev. Mol. Cell Biol. 1, 1, ⁇ -39, 2000).
  • a blue-shifted area representing Src activation The region is located in the region of interest l (ROIl) where lipid raft markers are concentrated.
  • ROIl region of interest l
  • R0I2 target region 2
  • FIG. 2b shows that, when stimulated with E2, Src activation is induced in spatially limited lipid rafts in the cell membrane.
  • EGFR and ER in addition to Src itself were found to migrate into lipid rafts when MCF-7 cells were stimulated by E2 (Fig. 2d and thus lipids). It can be concluded that raft-specific Src activation is due to the formation of the EGFR / ER / Src complex at that location.
  • a lipid raft-localized SIFP was created as a molecule that inhibits lipid raft-specific Src activation, and its effect was confirmed.
  • SIFP prepared in this example consists of YFP, flag tag, Src inhibitory peptide, lipid raft localization peptide (C-terminal 9 amino acids of H-Ras: CMSCKCVLS).
  • a fusion protein in which Rho-A C-terminal 5-amino acid (GCLVL) was linked to the C-terminus of the Src inhibitory peptide was prepared as a molecule to be localized in the non-lipid raft of the biological membrane (upper part of Fig. 3a).
  • a fusion protein having an alanine variant of the Src inhibitory peptide (middle panel in Fig. 3a) and a fusion protein having no Src inhibitory peptide (lower panel in Fig. 3a) were prepared.
  • lipid raft localized SIFP is a force localized with lipid raft marker, Alexa-647CTXB, and SIFP localized to non-lipid raft (hereinafter referred to as non-raft).
  • Loft-localized SIFP was not localized with this raft marker (Fig. 3b).
  • the intracellular localization of these SIFPs was further confirmed using density gradient fractionation.
  • the low-density fraction and the high-density fraction are subjected to immunoprecipitation using an anti-flag antibody, followed by anti-GFP antibody (Gagnoux-Palacios, L. et al.
  • lipid raft and non-raft localized SIFP are phosphorylated by Src in MCF-7 cells.
  • Low-density fraction and high-density fraction derived from MCF-7 cells that express lipid raft or non-raft-localized SIFP are immunoprecipitated using anti-pTyr antibody and purified using anti-GFP antibody. Muno blotting. Lipid raft-localized SIFP was only phosphorylated in lipid rafts of MCF-7 cells, and this phosphorylation in lipid rafts was blocked by the specific Src family kinase inhibitor PP2 (Fig. 3c).
  • lipid raft-localized SIFP specifically inhibits Src kinase activity only in lipid rafts.
  • Non-raft-localized SIFP is not phosphorylated in the non-raft region of the cell membrane (Fig. 3c), indicating that Src activation does not occur in the non-raft region of the cell membrane.
  • lipid rafts or non-lipid raft localized SIFPs were expressed and examined for cell attachment of MCF-7 cells.
  • the degree of adhesion of MCF-7 cells transfected with SIFP was measured and expressed as an adhesion index obtained by dividing the number of fluorescent positive cells adhering by the number of fluorescent positive cells plated first.
  • Lipid raft-localized SIFP significantly reduced the number of cells that adhere to fibronectin, one of the integrin ligands.
  • lipid raft-localized SIFP Del without the inhibitory peptide did not impair cell adhesion to fibronectin (FIG. 4a).
  • Lipid raft-localized SIFP Y6A (middle of Fig.
  • lipid rafts and non-raft localized SIFPs affect the cell cycle of MCF-7 cells.
  • the DNA content of MCF-7 cells transfected with lipid raft or non-raft localized SIFP was assessed by flow cytometry using Propidium iodide (PI) staining.
  • Cell cycle profiles of YFP positive cells expressing SIFP were obtained using this assay (Fig. 4b).
  • Lipid raft and non-raft localized YFP cell cycle profiles were used as controls for cell cycle progression in MCF-7 cells expressing lipid raft and non-raft localized SIFP, respectively.
  • Lipid raft localized type SIFP is a force that reduces the ratio of cells in G / M phase to cells in G phase S, lipid raft localized type
  • SIFP Y6A did not decrease this ratio (top of Figure 4b).
  • the cell cycle profile of YFP-positive cells expressing non-raft-localized SIFP is similar to the cell cycle profile of YFP-positive cells expressing non-raft-localized YFP (bottom of Fig. 4b).
  • Non-raft localized SIFP Y6A showed a cell cycle profile similar to non-raft localized YFP (lower part of Fig. 4b).
  • the effect of lipid raft-localized SI FP on the cell cycle of human normal cells (HEK293 cells) is similar to that of lipid raft-localized SIFP De 1 (control) without inhibitory peptides, as shown in Fig. 9 Met.
  • lipid raft-localized SIFP was able to inhibit Src activity in lipid rafts, thereby preventing cell cycle progression and cell adhesion.
  • lipid raft-localized SIFP had no effect on the cell cycle and cell adhesion of human normal cells (HEK293 cells). This indicates that lipid raft-localized SIFP is promising as an anticancer drug component with few side effects.
  • the non-palmitoril integrin ⁇ 6 ⁇ 4 modified so that the Src substrate integrin ⁇ 6; 3 4 is not localized in lipid rafts reduces ERK phosphorylation and inhibits mitotic cleavage.
  • the ability of the integrin 6/34 or the upstream kinase such as Src to control the initial steps of this lipid raft-specific integrin signaling cascade depends on endogenous Src and integrin.
  • Src activity is associated with cell adhesion of metastatic cells from the Filder model of colorectal tumor metastasis (Jones, RJ et al. Elevated c-Src is linked to altered cell-matrix adhesion rather than prolileration in KM12C human col orectal cancer Br. J. Cancer. 87, 1128-1135, 2002), Src-dependent cell cycle progression promotes tumor cell growth (Summy, JM & Gallick, GE Src family kinases in tumor progression and metastasis. Cancer Metastasis Rev. 22, 337-358, 2003) is known.
  • the spatially limited inhibition of Src activity provided by the present invention makes it possible to more effectively prevent cancer cell proliferation and metastasis.
  • an expression vector for AIFP (upper part of FIG. 5) containing an mitochondrial localization sequence (SEQ ID NO: 4) and an Akt inhibitory peptide (SEQ ID NO: 3), and an Akt inhibitory peptide
  • the expression vector of control AIFP bottom of Fig. 5) that does not contain Each was built.
  • AIFP was introduced into MCF-7 cells, stained with mitochondrial markers, and AIFP GFP was visualized. The results are as shown in FIG. It was confirmed that AIFP is localized and expressed in mitochondria.
  • SIFP a lipid raft localized type and cancer cell membrane permeable
  • a cell membrane-permeable peptide (a peptide consisting of 11 arginines), a cancer cell-specific protease recognition sequence (PLGLAG: SEQ ID NO: 5)
  • a cell membrane impermeable peptide (a peptide consisting of 11 gnoretamines) was constructed.
  • Cancer cell membrane permeability SIFP cleavage by cancer cell specific protease Cancer cell membrane permeability SIFP expression vector is introduced into Escherichia coli and cancer cell membrane permeability SIFP expressed from the vector is generated on His-tag column did. The cancer cell membrane-permeable SIFP was supplemented with MMP and then analyzed by Western plotting using an anti-flag antibody.
  • cancer cell membrane-permeable SIFP recognizes MMP when MMP is present. It was confirmed that the sequence was cleaved and the N-terminal polyglutamine sequence was cleaved.
  • Cancer cell membrane-permeable SIFP (1 ⁇ ) expressed in E. coli and purified was added to the culture medium of cultured MCF-7 cells. Then, uptake of cancer cell membrane permeable SIFP into MCF-7 cells with or without the addition of protease MMP to this medium was observed by immunostaining using anti-flag antibody.
  • cancer cell membrane-permeable SIFP was efficiently taken into MCF-7 cells by releasing the N-terminal polyglutamine sequence (cell membrane-impermeable peptide) by MMP and exposing the cell membrane-permeable peptide. It was confirmed that
  • Purified cancer cell membrane-permeable SIFP (1 ⁇ ), or cancer cell membrane-permeable SIFP (1 ⁇ ) and MMP were added to the medium of MCF-7 cells, and each cell was grown.
  • FIG. 13 is a graph in which the value obtained by dividing the number of cells on the fourth day from the start of culture by the number of cells at the start of culture is drafted.
  • MCF-7 cells cultured under conditions of cancer cell membrane-permeable SIFP + MMP hardly proliferated even after 4 days of culture.

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Abstract

L'invention porte sur une protéine de fusion inhibitrice de kinase comprenant un peptide inhibiteur de kinase et un peptide situé dans une organelle intracellulaire. Ladite protéine inhibe l'activité de la kinase d'une manière spécifique à une organelle intracellulaire, et son mécanisme d'action est tel qu'elle inhibe spécifiquement une kinase pouvant être localement activée à l'intérieur d'une organelle intracellulaire.
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