WO2002015925A1 - Technique de regulation de l'apoptose et polypeptide de regulation d'apoptose - Google Patents

Technique de regulation de l'apoptose et polypeptide de regulation d'apoptose Download PDF

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WO2002015925A1
WO2002015925A1 PCT/JP2001/007179 JP0107179W WO0215925A1 WO 2002015925 A1 WO2002015925 A1 WO 2002015925A1 JP 0107179 W JP0107179 W JP 0107179W WO 0215925 A1 WO0215925 A1 WO 0215925A1
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amino acid
polypeptide
acid sequence
seq
activity
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PCT/JP2001/007179
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Takashi Tsuruo
Naoya Fujita
Saori Sato
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU2001280105A priority Critical patent/AU2001280105A1/en
Priority to JP2002520846A priority patent/JPWO2002015925A1/ja
Publication of WO2002015925A1 publication Critical patent/WO2002015925A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to a method for controlling apoptosis, which comprises a step of controlling Akt activity in a cell, in particular, a step of controlling binding between Hsp90 and Akt, a polypeptide having an activity of controlling Akt activity in a cell,
  • the present invention relates to a DNA encoding the polypeptide, an antibody recognizing the polypeptide, a method for producing the polypeptide, a medicament containing the polypeptide or the antibody, and a method for using them.
  • Apoptosis originally means cell death actively caused by cells themselves under physiological conditions, and is deeply involved in morphology, tissue formation, homeostasis maintenance, biological defense during development, and cell division ( Proliferation) plays an important role in maintaining the life of an individual as two sides of the same coin.
  • Apoptosis is triggered by various physiological or pathological signals from outside or inside the cell. Inherited or acquired abnormalities in the process of death controlled by a gene are thought to induce various diseases by over-inducing or suppressing apoptosis.
  • Diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage. Conversely, diseases related to the onset of apoptosis suppression include cancer and autoimmune diseases.
  • cancer treatment and immunosuppressive therapy are being carried out, using the fact that radiation and drugs cause apoptosis.
  • protecting cells that play an important role in the living body by suppressing apoptosis from death is the key to suppressing cell death during ischemia in the brain and heart, and from normal cell death during anticancer drugs and radiation therapy. It can be used for medical treatment purposes such as protection of stomach.
  • Akt is phosphoinositide 3-kinase (PI3K) It is known as a serine / threonine kinase that functions in downstream signaling pathways.
  • PI3K phosphoinositide 3-kinase
  • three isoforms Aktl / PKB and Akt2 / PKB? Akt3 / PBa
  • Akt2 and Akt3 are about 82% identical in amino acid to Aktl, and the threonine and serine residues required for phosphorylation and activation are conserved. The division of functions among subtypes has not been disclosed at this time.
  • PI3K is activated in cells by stimulation with growth factors, such as cytokines, and converts PI 4,5 bisphosphonate [PI 4,5 bisphosphonate (PIP2)] to PI 3,4,5 triphosphonic acid [PI 3,4 , 5 triphosphonate (PIP3)].
  • PIP3 acts on Akt as a second messenger, and Akt activity is increased by phosphorylation of Aktl threonine residues 308 and 473 at Aktl.
  • Known substrates of activated Akt include Bad, caspase 9 and IkB kinase, and phosphorylation of these substrates inactivates kt to suppress cell apoptosis.
  • Akt activity may contribute to immortalization of cancer cells and apoptosis resistance.
  • Studies on the expression of Akt in cancer showed that Aktl was colorectal cancer (20% of gastric adenocarcinoma), Akt2 was ovarian cancer (15%) ⁇ knee cancer (12%), breast cancer (3%), and Akt3 was estrogen receptor. It has been reported that such genes are highly expressed in breast cancers lacking Yuichi and androgen-independent prostate cancer [Biochem. J., 1-13 (1998), J. Biol. Chem., 274, 21528]. -21532 (1999)] 0 also, brain, endometrial, prostate cancer, PTEN [Science is a tumor suppressor gene mutations in breast cancer and the like are known, 275.
  • PTEN has been shown to have the activity to dephosphorylate PIP3, which is subsequently generated by PI3K, and to suppress the signal from PI3K to Akt (Cell, 05, 29-39 (1998) ].
  • Loss of PTEN activity in cells is thought to cause Akt homeostatic activation and suppress apoptosis to trigger cancer. Therefore, substances that suppress Akt activity induce apoptosis in cancer cells. It is considered to be useful as a therapeutic drug or a drug that enhances the sensitivity of cancer cells to apoptosis induced by anticancer drugs currently used for cancer treatment
  • Aktl activity is regulated by the balance between phosphorylation and dephosphorylation. It has been shown that protein phosphatase 2A (PP2A) is involved in Aktl dephosphorylation [Proc. Natl. Acad. Sci. USA, 5699-5704 (1996)].
  • P2A protein phosphatase 2A
  • Akt activation regulates glucose metabolism 6-phosphofructose 2-force rice [6-p osphofmctose 2 -kinase (PFK2)] activation caused by insulin stimulation, glucose metabolism enhancement, glucose transporter 4 involved in intracellular glucose uptake [glucose transporter 4] (GLUT4)] and PI3K-mediated suppression of glycogen synthesis due to increased glucose uptake by membrane localization and inactivation of glycogen synthase kinase-3 (GSK3). It has been reported that Akt activation is involved [Biochem. J., 3, 1-13 (1998)]. Therefore, it is considered that Akt-activating substances may be effective for diabetes and the like caused by abnormal glucose metabolism.
  • Heat shock protein is a group of proteins that are expressed in cells when cells are exposed to stress environments such as heat shock, and can be classified into several families according to their molecular weight (Hsp90, Hsp70, Hsp27 etc.). These proteins are It is also called a chaperone, and its main functions are generally considered to be protein folding, membrane permeation, association, and suppression of aggregation.
  • Hsp90 is a heat shock protein belonging to the Hsp90 family composed of a molecular chaperone with a molecular weight of about 90 kDa.
  • Hsp90, Hsp90 ?, Grp94, Hsp75 / TRAP1, etc. have been identified as eukaryotic Hsp90 families, but these Hsp90 family proteins are expressed in cells even in an environment without extracellular stress.
  • Hsp90 specifically forms complexes with various signal transduction factors involved in cell proliferation and canceration and contributes to the cell cycle and cell proliferation signals.
  • Hsp90 Signaling factors that are thought to require binding to the Hsp90 family to maintain intracellular function and stability include steroid hormone receptors (estrogens receptor, progesterone receptors, glucocorticoids). Drecept Yuichi), non-receptor tyrosine kinases (eg, v-src), receptor tyrosine kinases (eg, EGFR, ErbB2), serine threonine kinase (Raf-1, Cdk4, etc.). Therefore, it is thought that by controlling the function of Hsp90, intracellular signal transduction involving an intracellular signaling factor that specifically binds to Hsp90 can be varied.
  • Hsp90 family protein is increased in various cancer cell lines, and the correlation between Hsp90 expression level and prognosis in breast cancer patients has been reported. It is thought that it may play an important role in disease [Pharmacology & Therapeutics, 2, 129-168 (1997), Biochem. Pharmacol., 675-682 (1998), Investigational New Drugs, 11, 361 -373 (1999)].
  • Examples of compounds that bind to Hsp90 and inhibit its function include geldanamycin, herbimycin A and other ansamycin-based compounds and their derivatives, radicicol and its derivatives, and novobiocin. [Investigational New Drugs, 11, 361-373 (1999), J. Natl. Cancer Inst., 92, 242-248 (2000)].
  • Geldanamycin ⁇ ⁇ radicicol has been shown to bind to the ATP / ADP binding region located in the N-terminal region of Hsp90, which is contained in the amino acid sequence from position 221 to position 221). [Cell, m, 239-250 (1997), J. Biol.
  • Hsp90 functions together with co-chaperone molecules such as p50 / Cdc37 and p23 to form a molecular complex with the above-mentioned specific signaling factors, but low-molecular-weight compounds such as geldanamycin ⁇ radicicol
  • co-chaperone molecules such as p50 / Cdc37 and p23
  • low-molecular-weight compounds such as geldanamycin ⁇ radicicol
  • the present invention identifies functional sites on Hsp90 that are involved in the inhibition of apoptosis and the control of Akt activity involved in glucose metabolism, and is used to identify gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer caused by abnormal apoptosis control and glucose metabolism control signals.
  • Therapeutic, prophylactic and diagnostic agents for malignant tumors such as cancer, diabetes, etc., cell death inhibitors during ischemia of the brain, heart, etc., anticancer agents and agents to protect normal cells from cell death during radiation therapy Or to provide agents that enhance the effects of existing therapeutic agents.
  • the present inventors have found for the first time that Hsp90 binds to Akt in a cell, and that the binding of Hsp90 to Akt is important for maintaining Akt activity (phosphorylation). Further, the present invention has been completed by identifying a functional site on Hsp90 involved in the control of Akt activity involved in apoptosis suppression and glucose metabolism, and an Akt site binding to the functional site.
  • the present invention relates to the following inventions (1) to (40).
  • a method for controlling apoptosis which comprises a step of controlling Akt activity in a cell.
  • the control of Akt activity in a cell is carried out by a polypeptide consisting of the 327th to 340th amino acid sequence of SEQ ID NO: 1 or the polypeptide consisting of 335 to 348th amino acid sequence of SEQ ID NO: 3; The method according to any one of 1) to (4).
  • one or more amino acids are deleted or substituted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3.
  • the method according to any one of (1) to (4) above, wherein the method is performed using a polypeptide comprising an inserted or added amino acid sequence and having an activity of controlling Akt activity in a cell.
  • the control of intracellular Akt activity is at least 60% homologous to the amino acid sequence at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. Carried out by a polypeptide comprising an amino acid sequence having a property and having an activity of controlling intracellular Akt activity.
  • the control of Akt activity in the cell is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or The method according to the above (2) or (4), which is carried out using a polypeptide consisting of the amino acids 227 to 306 of the amino acid sequence of SEQ ID NO: 9.
  • the control of Akt activity in the cell is from the 289th to the 309th of the amino acid sequence of SEQ ID NO: 5, from the 290th to the 310th of the amino acid sequence of SEQ ID NO: 7, or The method according to (2) or (4), which is performed using a polypeptide consisting of positions 286 to 306 of the amino acid sequence of SEQ ID NO: 9.
  • the control of Akt activity in the cell is caused by the amino acid sequence of SEQ ID NO: 5 from the 229th position to the 309th position, the amino acid sequence of SEQ ID NO: 7 from the 23rd position to the 310th position, Alternatively, it has an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added at positions 227 to 306 of the amino acid sequence of SEQ ID NO: 9, and has an activity of controlling Akt activity in a cell.
  • the regulation of intracellular Akt activity is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or A polyamino acid sequence comprising an amino acid sequence having at least 60% homology with the amino acid sequence at positions 227 to 306 of the amino acid sequence set forth in SEQ ID NO: 9, and having an activity of controlling intracellular Akt activity.
  • the control of intracellular Akt activity is carried out by a compound that fluctuates the activity of the polypeptide described in any one of the above (5) to (15), which regulates intracellular Akt activity, The method according to (1) or (2) above.
  • One or more amino acids are deleted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3.
  • a polypeptide comprising an amino acid sequence substituted, inserted or added, and having an activity of controlling Akt activity in a cell.
  • a polypeptide comprising a sequence and having an activity of controlling Akt activity in a cell.
  • polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added at the 7th to 30th positions, and which has an activity of controlling Akt activity in a cell.
  • polypeptide consisting of an amino acid sequence having 60% or more homology with the amino acid sequence from the 7th to the 36th amino acid and having an activity to control intracellular Akt activity.
  • (27) A DNA that hybridizes with the DNA of (26) under stringent conditions and encodes a polypeptide having an activity of controlling intracellular Akt activity.
  • the transformant according to (29) is cultured in a medium, a polypeptide capable of controlling Akt activity in cells is produced and accumulated in the culture, and the polypeptide is separated from the culture.
  • (19) - (2 5) contains a step of contacting the cell with a test sample which expresses a polypeptide according to any one of the control of Akt activity in cells of said polypeptide A method for screening a compound that changes the activity of the compound.
  • a pharmaceutical composition comprising the antibody or antibody fragment according to 33) and an anticancer agent as active ingredients.
  • Anticancer drugs include psulfan, cyclophosphamide, ifosfamide, melphalan, nitrosoperrea, amsacrine, carboplatin, cisplatin, dacarbazine, azacytidine, cladribine, sieverabine, fludarabine, fluorouracil, hydroxyperia, leuco At least one selected from methotrexate, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, mitoxantrone, etoposide, paclitaxel, teniposide, vinblastine, vincristine, vindesine, geldanamycin and peracidicol
  • the pharmaceutical composition according to any one of the above (37) to (39), wherein In cells, when Akt is phosphorylated, Akt becomes stable and apoptosis is suppressed. When Akt is dephosphorylated, Akt becomes unstable and a
  • phosphorylated Akt is stabilized by binding to Hsp90. Furthermore, the fact that phosphorylated Akt binds to Hsp90 and maintains a stable state is due to the involvement of the phosphatase action site on Akt and the binding region between Hsp90 and Akt. was also found. From the above, it is possible to control apoptosis by controlling the intracellular Akt activity, particularly by controlling the binding between Hsp90 and Akt, thereby controlling the intracellular Akt activity.
  • “controlling intracellular Akt activity” includes increasing or decreasing intracellular Akt activity, for example, controlling the binding between Hsp90 and Akt, stabilizing phosphorylated Akt, It is performed by dephosphorylation of Akt.
  • Controlling the binding between Hsp90 and Akt means to stabilize or inhibit (particularly competitive inhibition) the binding between Hsp90 and Akt, and to stabilize or promote the binding between Hsp90 and Akt. Including. “Controlling apoptosis” also includes inducing or suppressing apoptosis.
  • the method for controlling the binding between Hsp90 and Akt is not particularly limited, but preferably includes Hsp90 or a partial polypeptide thereof, a partial polypeptide of Akt, a compound, a peptide, an antibody and an antibody fragment that inhibits the binding of Hsp90 to Akt. It is controlled by adding.
  • Hsp90 is not particularly limited, and any protein may be used as long as it belongs to the Hsp90 family, but Hsp903 or Hsp90 is preferably used.
  • Hsp90 5 and Hsp90 are proteins whose amino acid sequences are represented by SEQ ID NOS: 1 and 3, respectively, and whose base sequences are represented by SEQ ID NOs: 2 and 4, respectively.
  • the partial polypeptide of Hsp90 that controls apoptosis includes a polypeptide consisting of positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or a polypeptide consisting of positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3 (
  • a partial Hsp90 peptide having an activity of controlling Akt activity in cells is abbreviated as “Hsp90 polypeptide of the present invention”.
  • Akt is phosphorylated and apoptosis is suppressed.
  • Hsp90 polypeptide of the present invention binds to Akt but a dephosphorylating enzyme acts on Akt, Akt is dephosphorylated and apoptosis is induced.
  • the binding of Hsp90 to Akt can be controlled by a partial polypeptide of Akt. Is more preferable.
  • Akt is not particularly limited, and may be any protein as long as it belongs to the Akt family.
  • Aktl, Akt2 or Akt3 is used.
  • Aktl, Akt2 and Akt3 are proteins whose amino acid sequences are represented by SEQ ID NOS: 5, 7 and 9, respectively, and whose nucleotide sequences are represented by SEQ ID NOs: 6, 8 and 10, respectively.
  • the partial polypeptide of Akt that controls apoptosis includes amino acids 229 to 309 of the amino acid sequence described in SEQ ID NO: 5, 231 to 310 of the amino acid sequence described in SEQ ID NO: 7, or SEQ ID NO:
  • a polypeptide consisting of the amino acid sequence from position 227 to position 306 of the amino acid sequence 9 is preferred, and in particular, from position 289 to position 309 of the amino acid sequence of sequence number 5, from position 290 to position 310 of the amino acid sequence of sequence number 7, or Amino acid sequence described in SEQ ID NO: 9
  • Polypeptide consisting of positions 286 to 306 hereinafter, a partial Akt polypeptide having an activity of controlling intracellular Akt activity is abbreviated as “Akt polypeptide of the present
  • Akt polypeptide of the present invention As a result of binding of the Akt polypeptide of the present invention to Hsp90, the amount of Akt bound to Hsp90 decreases. Akt not bound to Hsp90 is induced by a phosphatase, and then apoptosis is induced.
  • Hsp90 polypeptide and Akt polypeptide of the present invention are abbreviated as “polypeptide of the present invention”
  • Polypeptides comprising a substituted, inserted or added amino acid sequence and having an activity to control intracellular Akt activity are disclosed in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols', 'molecular' biology), Nuc. Acid. Res., 10, 6487 (1982), Proc. Natl. Acad.
  • Deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence constituting the polypeptide of the present invention means that any one or more amino acid sequences in the same sequence Means that there is a deletion, substitution, insertion or addition of one or more amino acid residues at the position, the deletion, substitution, insertion or addition may occur simultaneously, Amino acids can be either natural or non-natural.
  • Natural amino acids include L-alanine, L-asparagine, L-asparaginic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine , L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
  • amino acid residues that can be substituted for each other.
  • Amino acid residues included in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, norin, norvaline, alanine, 2-aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group D lysine, arginine, ordinine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E Proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • Group G fenirualanine, tyrosine
  • polypeptide of the present invention In order for the polypeptide of the present invention to have an activity of controlling Akt activity in a cell, When calculated using the amino acid sequence of the polypeptide of the present invention and BLAST [J. Mol. Biol., 2, 403 (1990)] or FASTA [Methods Enzymol., 1M, 63-98 (1990)], etc. It has a homology of at least 60% or more, usually 80% or more, preferably 90% or more, and particularly preferably 95% or more.
  • the present invention also relates to a DNA encoding the polypeptide of the present invention, and a DNA that hybridizes with the DNA under stringent conditions and has an activity of controlling Akt activity in a cell.
  • DNA hereinafter abbreviated as DNAj of the present invention.
  • the DNA encoding the polypeptide of the present invention includes the nucleotides 979 to 1020 of the nucleotide sequence of SEQ ID NO: 2, the nucleotides 1003 to 1044 of the nucleotide sequence of SEQ ID NO: 4, and the nucleotide of SEQ ID NO: 6. 685 to 927, preferably 865 to 927 of the sequence, 691 to 930, preferably 868 to 930 of the salt tomb sequence described in SEQ ID NO: 8, and 679 of the base sequence described in SEQ ID NO: 10 And a DNA having a base sequence from 856th to 918th, preferably from 856th to 918th.
  • a large number of DNAs having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention other than those described above are all included in the present invention.
  • DNA capable of hybridizing under stringent conditions refers to a DNA having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention as a probe, a colony hybridization method, a plaque hybridization method. Or DNA obtained by using the Southern blot hybridization method or the like. Specifically, using a filter on which DNA derived from colonies or plaques is immobilized, 0.7 to 1.0 mol / After performing the hybridization at 65 ° C in the presence of sodium chloride in step 1, the SSC solution from 0 :! to 2x concentration (1x concentration of SSC solution is 150 mmol / 1 sodium chloride, (consisting of 1 mmol / sodium citrate) and washing the filter under 65 ° C conditions to obtain DNA that can be identified.
  • DNAs having a homology of preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more can be mentioned.
  • the binding between Hsp90 and Akt is measured by the following method. That is, after adding DNA encoding the tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. Identification of substances in immunoprecipitates by Western-blotting allows Hsp90 and Akt.
  • the state of binding to Akt can be measured.
  • Immunoprecipitation refers to a reaction in which an antigen-antibody complex is formed by reacting a protein with an antibody thereto.
  • Western blotting means that proteins are separated by polyacrylamide gel electrophoresis, and the proteins on the gel are transferred to a nitrocellulose membrane, polyvinylidene 'difluoride membrane, etc., and the proteins are detected using antibodies against the proteins. Refers to the method.
  • the immunoprecipitate of the monoclonal antibody against the tag contains a complex of the Hsp90 partial protein and the Akt protein. Will be obtained. Therefore, after subjecting the complex to polyacrylamide gel electrophoresis, the gel was transferred to a nitrocellulose membrane, and then subjected to an antibody against Hsp90 or a tag attached to Hsp90. By using such an antibody, it can be confirmed that Hsp90 has bound to the Akt protein.
  • the tag may be any tag such as a FLAG tag, V5 tag, HA tag, and the like.
  • a method for measuring Akt activity in cells includes a method for measuring phosphorylation of Akt. That is, after adding DNA encoding a tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. The substance in the immunoprecipitate is identified by Western plotting, and the phosphorylation of Akt can be measured by using an antibody against phosphorylated Akt.
  • Methods for measuring apoptosis include a method of extracting chromosomal DNA in cells and analyzing the chromosomal DNA by electrophoresis, a method of staining cells and analyzing them by morphological observation, and a method of apoptosis. Examples include a method of measuring protease, which is essential for induction.
  • DAPI 6-diamino-2-phenylindole
  • Methods for measuring protease which is essential for apoptosis induction, include a method for measuring caspase [Oncogene, 11, 1295 (1998)]. Specifically, cells are lysed in a lysis solution containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS. After reacting the cell lysate with acetyl-L-aspartyl-L-glucamyl-L-valyl-L-aswald-7-amino-4-methylcoumarin (DEVD-AMC) in ICE buffer, absorbance Caspase activity can be measured by measuring with a meter.
  • DEVD-AMC acetyl-L-aspartyl-L-glucamyl-L-valyl-L-aswald-7-amino-4-methylcoumarin
  • absorbance Caspase activity can be measured by measuring with a meter.
  • Examples of the method for producing the polypeptide of the present invention include a method using chemical synthesis and a method using a genetic method. This will be described in detail below.
  • polypeptides of the present invention are described, for example, in "Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki: Maruzen), “Fourth Edition Experimental Chemistry Course 22—Organic Synthetic IV Acids” -Amino acids and peptides "(Saburo Aimoto, Shoichi Kusumoto, Kuniaki Tatsuta, Yoshihiro Hayakawa, Keizo Yamamoto, Tateaki Wakamiya: Maruzen),” In Yuichi National Journal of Applied and Fudo " Mouth Tin 'Resina (International Journal oi Peptide Protein Research),, 161-214 (1990)', 'Solid-Phase Peptide Synthesis', Method's 'In' Enzymology I Vol.
  • Specific synthesis methods include the azide method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DCC method, the active ester method, the carboimidazole method, and the redox method.
  • both the solid phase synthesis method and the liquid phase synthesis method can be applied. That is, the desired polypeptide is synthesized by condensing the amino acid constituting the polypeptide of the present invention with the remaining portion and, if the product has a protecting group, removing the protecting group.
  • the amino acid residue constituting the polypeptide of the present invention is chemically modified or protected on the side chain and / or the peptide amino terminal and / or the peptide carboxy terminal
  • the peptide is chemically modified after synthesis.
  • a method known in the field of peptide synthesis chemistry such as synthesis of peptides using chemically modified amino acids or appropriate selection of reaction conditions for final deprotection of peptide synthesis (“Peptide synthesis”).
  • the polypeptide of the present invention can also be synthesized using an automatic peptide synthesizer.
  • Peptide synthesis using a peptide synthesizer is performed on a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation, or a peptide synthesizer manufactured by Advanced ChemTech Inc. (USA).
  • a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation, or a peptide synthesizer manufactured by Advanced ChemTech Inc. (USA).
  • N-Fmoc-amino acid or N-Boc-amino acid with appropriately protected side chains the synthesis can be carried out according to the respective synthesis programs.
  • polypeptide of the present invention can be purified by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the polypeptide of the present invention was prepared by the method described in Molecular Biology, Molecular Cloning Second Edition or Current Protocol, Inc. Molecular Biology, etc.
  • a DNA fragment of an appropriate length containing a portion encoding the polypeptide is prepared.
  • a DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide of the present invention so that the nucleotide sequence becomes an optimal codon for expression in a host cell.
  • the DNA is useful for efficient production of the polypeptide of the present invention.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of a promoter in an appropriate expression vector.
  • the recombinant vector is introduced into a host cell compatible with the expression vector.
  • any cells that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • the expression vector contains a promoter at a position where it can replicate autonomously in the host cell or can be integrated into the chromosome, and can transcribe DNA encoding the polypeptide of the present invention. Things are used.
  • the recombinant vector containing the DNA encoding the polypeptide of the present invention is capable of autonomous replication in the prokaryote and at the same time, is promoted. It is preferable that the vector be composed of a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. A gene that controls a promoter may be included.
  • expression vectors include pBrp2, pBTacl, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 669 (1984)], pLSAl [Agric. Biol. Chem., 277 ( 1989)], GELl [Proc. Natl. Acad. Sci.
  • the promoter may be any promoter that functions in the host cell.
  • ⁇ E _ promoter evening one P trp;
  • la promoter P L promoter
  • P R promoter Isseki one T7 promoter Isseki one such, be given promo one evening one derived from Escherichia coli or phage, etc.
  • the promoter evening one obtained by two series of P trp (P trp X 2) , tac promoter Isseki one, LaeT7 promoter Isseki one, k ⁇ I artificially designed modified promoter Isseki one as a promoter Etc. can also be used.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosomal binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • a transcription termination sequence is not always necessary for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
  • Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., such as Escherichia coli XLa-Blue, Escherichia coli XL2-Blue Escherichia coli DH1, Escherichia coli MC I 000, Escherichia coli KY3276 N Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli ⁇ ⁇ o.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia Escherichia coli GI6 coli ficaria ⁇ Serratia fonticoia ⁇ Serratia liquefaciens ⁇ Serratia marcescens.Bacillus subtilis, Bacillus am loliquefacines
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, fifi, 2110 (1972)], the protoplast method (Sho 63-248394), or the methods described in Gene, 11, 107 (1982) and Molecular & General Genetics, Hyundai, 111 (1979).
  • examples of expression vectors include YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, and pHS15.
  • any promoter can be used as long as it can be expressed in yeast strains.
  • promoters for glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP Promo One Night, ADH Promo One, gal 1 Promo One, Gal 10 Promo One, Heat Shock Polypeptide Motor, MFI Promoter, CUP One Promo One, etc.
  • any method can be used as long as it is a method for introducing DNA into yeast.
  • the method include an elect-portion method [Methods Enzymol., 1Q4, 182 (1990)] and a spheroplast method. [Proc. Natl. Acad. Sci. USA, 15., 1929 (1978)], lithium acetate method [J. BacterioL, 1, 163 (1983)], Proc. Natl. Acad. Sci. USA, IS, 1929 ( 1978)].
  • any promoter can be used as long as it functions in animal cells.
  • the promoter of the cytomegalovirus (CMV) IE (immediate early) gene and the early promoter of SV40 can be used.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • host cells examples include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 (63-299).
  • any method for introducing a recombinant vector into animal cells any method can be used as long as DNA can be introduced into animal cells.
  • electroporation method Cytotechnology, 133 (1990)
  • calcium phosphate method Japanese Patent Application Laid-Open No. 2-227075
  • lipofection method Proc. Natl. Acad. Sci. USA, S4, 7413 (1987), Virology, 52, 456 (1973)] and the like.
  • an insect cell When an insect cell is used as a host, for example, the current protocol 'In' Morefu-Noroshi, Bacxuovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology , fi, 47 (1988), etc., to express the polypeptide. That is, a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected into insect cells to express the polypeptide. be able to.
  • Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (all manufactured by Invitoi'ogen) and the like.
  • Baculoviruses are, for example, viruses that infect night roth moths Art Grapher's Force Reformi Force ⁇ Nuclea ⁇ Polyhemdosis ⁇ Virus
  • Methods for co-transferring the above-described recombinant gene into insect cells and the above baculovirus to prepare a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [ Proc. Natl. Acad. Sci. USA, M, 7413 (1987)].
  • examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the 35S promoter of cauliflower mosaic virus (CaMV), the inactin 1 promoter, and the like.
  • Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfa-alpha, rice, wheat, and wheat.
  • any method for introducing DNA into plant cells can be used.
  • Agrobacterium Agrobacterium ⁇ (Japanese Patent Application No. 59-140885, Japanese Patent Application Laid-Open No. 70080, WO 94/00977), the electoral poration method (JP-A-60-251887), a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like.
  • a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition.
  • polypeptide with a sugar chain added thereto When expressed by yeast, animal cells, insect cells or plant cells, a polypeptide with a sugar chain added thereto can be obtained.
  • the polypeptide of the present invention is produced by culturing the transformant of the present invention obtained as described above in a medium, producing and accumulating the polypeptide of the present invention in the culture, and collecting from the culture. can do.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • the transformant of the present invention is a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host
  • the transformant is used as a medium for culturing the transformant.
  • Either a natural medium or a synthetic medium may be used as long as the medium contains an assimilable carbon source, a nitrogen source, inorganic salts, and the like, and can efficiently culture the transformant.
  • the carbon source may be any as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digests thereof can be used.
  • potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culturing is preferably maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, and the like.
  • an antibiotic such as ambicillin or tetracycline may be added to the medium during the culture.
  • Transform with a recombinant vector using an inducible promoter as a promoter When culturing the isolated microorganism, if necessary, an inducer may be added to the medium. For example, when culturing a microorganism transformed with a recombinant vector using a promoter, isoforms such as isopropyl-/?-D-thiogalactovyranoside are transformed with a recombinant vector using the trp promoter. When culturing the transformed microorganism, indoleacrylic acid or the like may be added to the medium.
  • RPMI1640 medium As a medium for culturing transformants obtained using animal cells as a host, commonly used RPMI1640 medium [The Journal of the American Medical Association, 122., 519 (1967)], Eagle's MEM medium [Science , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, l, 1 (1950)] or fetal bovine serum etc. And the like can be used.
  • the cultivation is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C; and in the presence of 5% CO 2 .
  • antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
  • Culture media for transformants obtained using insect cells as a host include commonly used ⁇ - ⁇ media (Pharmingen), Sf-900 II SFM medium (Life Technologies), Ex Cell 400 And Ex Cell 405 (both manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 1, 788 (1962)] and the like. Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7 and 25 to 30 ° C.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
  • a culture medium for culturing the transformant a commonly used Murashige and Skog (MS) medium, a white (White) medium, or a plant formone such as auxin or cytokinin added to these mediums is used. Media or the like can be used.
  • Cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days. If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
  • a transformant derived from a microorganism, an animal cell, or a plant cell having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method, and the polypeptide is obtained. Is produced and accumulated, and the polypeptide is collected from the culture, whereby the polypeptide can be produced.
  • a method for expressing the gene, secretory production, fusion polypeptide expression, and the like can be performed according to the method described in Molecular 'Cloning 2nd edition, in addition to direct expression.
  • the polypeptide of the present invention can be produced in a host cell, secreted outside the host cell, or produced on the host cell outer membrane.
  • the host cell to be used or the polypeptide to be produced can be used.
  • the method can be selected by changing the structure of the peptide.
  • polypeptide of the present invention When the polypeptide of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 2 ⁇ 4, 17619 (1989)] and the method of Lowe et al. [Pro Natl. Acad. Sci. USA, M, 8227 (1989), Genes Develop., 4, 1288 (1990) ⁇ , or the method described in JP-A-5-336963, WO 94/23021, etc.
  • the polypeptide can be actively secreted out of the host cell.
  • the polypeptide of the present invention is expressed in a form in which a signal peptide is added in front of the polypeptide containing the active site of the polypeptide of the present invention using a gene recombination technique, whereby the polypeptide of the present invention can be expressed in host cells. It can be actively secreted out. Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, an animal into which the gene has been introduced (transgenic non-human animal) or a plant (transgenic plant) can be created. The polypeptide of the present invention can also be produced using an individual.
  • the polypeptide can be produced by producing or accumulating the polypeptide, and collecting the polypeptide from the animal or plant individual.
  • a method for producing the polypeptide of the present invention using an animal individual for example, known methods [American Journal of Clinical Nutrition, fiS, 639S (1996), American Journal of Clinical Nutrition, ⁇ 3, 627S (1996), Bio / Technology, S, 830 (1991)] to produce the polypeptide of the present invention in an animal constructed by introducing a gene.
  • a transgenic non-human animal into which DNA encoding the polypeptide of the present invention has been introduced is bred, and the polypeptide is produced and accumulated in the animal.
  • the polypeptide By collecting the polypeptide, the polypeptide can be produced.
  • the place of production and accumulation in the animal include milk (eg, JP-A-63-309192) and eggs of the animal.
  • the promoter used at this time any promoter that can be expressed in an animal can be used.
  • a promoter specific to mammary gland cells, a casein promoter, a casein promoter, A lactoglobulin promoter and a whey acid protein promoter are preferably used.
  • a transgenic plant into which DNA encoding the polypeptide of the present invention has been introduced can be prepared by a known method [tissue culture, 20 (1994)]. Tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)], producing and accumulating the polypeptide in the plant, and collecting the polypeptide from the plant. By doing so, a method for producing the polypeptide can be mentioned.
  • a general enzyme isolation and purification method can be used.
  • the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton.
  • the cells are disrupted using a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, normal enzyme isolation and purification
  • the cells When the polypeptide is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the polypeptide as a precipitate fraction. .
  • the insoluble form of the recovered polypeptide is solubilized with a protein denaturant.
  • the polypeptide is returned to a normal tertiary structure by diluting or dialyzing the lysate and reducing the concentration of the protein denaturant in the lysate. After this operation, a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
  • the polypeptide of the present invention or a derivative such as a polypeptide having a sugar chain added to the polypeptide is secreted extracellularly, the polypeptide or the polypeptide is added to the culture supernatant.
  • Derivatives of the peptide can be recovered. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. You can get it.
  • polypeptide examples include, for example, amino acids 327 to 340 of the amino acid sequence of SEQ ID NO: 1, amino acids 335 to 348 of the amino acid sequence of SEQ ID NO: 3, and amino acids of SEQ ID NO: 5. 229 to 309 and 289 to 309 of the sequence; 231 to 310 and 290 to 310 of the amino acid sequence described in SEQ ID NO: 7, and the amino acid sequence 227 to 306 of SEQ ID NO: 9 It is better to give polypeptides consisting of 286th to 306th. 2.
  • the DNA of the present invention can be prepared by using the method described in Molecular 'Cloning Second Edition or Current Protocols' in Molecular Biology, etc. Alternatively, it can be prepared from cells.
  • the human or non-human animal tissue or cell may be any tissue or cell in which Akt or Hsp90 is expressed, and examples include brain, testis, heart, lung and the like.
  • Total RNA or mRNA is prepared from tissues or cells of a human or non-human animal. Prepare a cDNA library from the prepared total RNA or mRNA.
  • a gene fragment which encodes the polypeptide of the present invention by PCR using the prepared cDNA library as a ⁇ type by preparing a degiene primer based on the amino acid sequence of the polypeptide of the present invention To get.
  • the cDNA library can be screened to obtain the DNA of the present invention encoding the peptide of the present invention.
  • the mRNA of human or non-human animal tissues or cells may be a commercially available one (for example, manufactured by Clontech), or may be prepared from human or non-human animal tissues or cells as described above. .
  • guanidine thiocyanate-cesium trifluoroacetate method As methods for preparing total RNA from tissues or cells of human or non-human animals, guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, l, 3 (1987)], guanidine acid thiocyanate, phenol-clo Oral form (AGPC) method [Analytical Biochemistry, 1Q2, 156 (1987), Experimental Medicine, S, 1937 (1991)].
  • oligo (dT) -immobilized cellulose column method Molecular 'Cloning 2nd edition
  • mRNA can be prepared by using a kit such as the Fast Track mRNA Isolation Kit (Invitrogen) or the Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. Methods for preparing a cDNA library include methods described in Molecular 'Clothing Second Edition, Current Protocols' In 'Molecular' Biology, A Laboratory Manual, 2nd Ed. (1989), or commercially available. Examples of such kits include a method using the is: Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and a ZAP-cDNA Synthesis Kit (STRATAGENE).
  • any phage vector, plasmid vector or the like can be used as long as it can replicate autonomously in E. coli K12 strain.
  • ZAP Express [Stratagene, Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 11, 9494 (1989)]
  • Lambda ZAP II (Stratagene), gtl0, A gtll [DNA cloning, A Practical Approach, 1, 49 (1985)]
  • human riplEx (Clontech), ⁇ ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mol. Cell. Biol) ., S, 280 (1983)]
  • pUC18 Gene,, 103 (1985)].
  • Escherichia coli As the Escherichia coli to which the vector into which the cDNA is incorporated is introduced, any microorganisms belonging to Escherichia coli can be used. Specifically, Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, SS, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 ( 1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Kschericliia coli K802 [J. Mol. Biol., 16, 118 (1966)] and Escherichia coli JM105 [Gene,, 275 (1985)].
  • This cDNA library may be used as it is for subsequent analyses, but in order to reduce the proportion of incomplete-length cDNA and obtain full-length cDNA as efficiently as possible, the oligocap method [Gene, Michi'171 (1994) , Gene, Fiber, 149 (1997), Protein Nucleic Acid Enzyme, 603 (1996), Experimental Medicine, 11, 2491 (1993), cDNA Cloning, Yodosha (1996), Gene Ripplery, Yodosha ( 1994)] may be used for the following analyses. Based on the amino acid sequence of the polypeptide of the present invention, a degenerative primer specific to the nucleotide sequence at the 5 'end and 3' end of the nucleotide sequence predicted to encode the amino acid sequence is prepared.
  • a gene fragment encoding the polypeptide of the present invention can be obtained by amplifying DNA using the prepared cDNA library type I as a type II and PG method [PCR Protocols, Academic Press (1990)]. That the obtained gene fragment is a DNA encoding the polypeptide of the present invention can be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 ( 1977)] or by using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (manufactured by PE Biosystems).
  • cDNA or cDNA libraries synthesized from mRNAs contained in tissues or cells of human or non-human animals are subjected to colony hybridization / plaque hybridization (Molecular Cloning). Inning (2nd edition), the DNA of the present invention can be obtained.
  • a cDNA or cDNA library synthesized from mRNA contained in tissues or cells of a human or non-human animal can also be obtained by performing screening using the method.
  • the nucleotide sequence of the obtained DNA of the present invention is analyzed from the end by a nucleotide sequence analysis method usually used, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 (1977)] or ABIPRISM377.
  • the base sequence of the DNA is determined by analysis using a base sequence analyzer such as a DNA sequencer (manufactured by PE Biosystems).
  • the present invention By searching the base sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST based on the determined base sequence of the cDNA, the present invention
  • the gene encoding the polypeptide can be determined.
  • the DNA of the present invention is obtained by chemically synthesizing with a DNA synthesizer such as Perkin 'Elma's DNA synthesizer Model 392 using the phosphoramidite method. You can also.
  • a purified antibody of the polypeptide of the present invention or a partial fragment of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen to obtain a polyclonal antibody.
  • Antibodies that recognize the polypeptide of the present invention such as monoclonal antibodies (hereinafter, abbreviated as “antibodies of the present invention”) can be produced.
  • the antibody of the present invention also includes those antibody fragments, and may be a recombinant antibody such as a humanized antibody and an antibody fragment thereof.
  • a purified product of the polypeptide of the present invention or a partial polypeptide of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen and administered to animals.
  • a polyclonal antibody can be prepared.
  • Animals to be administered include egrets, goats, rats, mice, hamsters, and the like.
  • the dose of the antigen is preferably 50 to 100 g per animal.
  • a peptide obtained by covalently binding a peptide to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin.
  • the peptide serving as the antigen can be synthesized using a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen used for immunization.
  • Enzyme-linked immunosorbent assay [Enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin (1976 ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] Confirm.
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)], DEAE-Sepharose column, There is a method in which a chromatographic method using an anion exchange column, a protein A or G-column, a gel filtration column, or the like is used alone or in combination.
  • Rats whose serum shows a sufficient antibody titer against the partial polypeptide of the polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
  • the spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • MEM medium manufactured by Nissui Pharmaceutical
  • the spleen cells in the obtained precipitate fraction are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, washed three times with MEM medium, and the obtained spleen cells are separated from antibody-producing cells. Used as
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., M, 1 (1978), Europ. J. Immunol., Fi, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 27fi, 269 (1978)], P3-X63-Ag8653 (653) [J.
  • HAT medium Hypoxanthin (10 ⁇ 4 inol / l), thymidine (1.5 X 10- 5 mol / l) and amino Noputerin (4 X 10 ⁇ 7 medium mol / 1) was added] are suspended in 100 ml.
  • the suspension is dispensed into 96-well culture plates at 100 ⁇ / well, and cultured at 37 ° C. overnight in a 5% CO 2 incubator for 7 to 14 days.
  • the following methods can be given as specific examples of the enzyme immunoassay.
  • a partial fragment polypeptide of the polypeptide of the present invention used as the antigen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below was used as the primary antibody.
  • reacting with an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a second antibody followed by a reaction according to the labeling substance, and the polypeptide of the present invention.
  • Those that specifically react with are selected as hybridomas producing the monoclonal antibody of the present invention.
  • Cloning was repeated twice by the limiting dilution method using the hybridoma [the first time using HT medium (medium obtained by removing aminopterin from HAT medium), and the second time using normal medium].
  • the antibody with a strong antibody titer is selected as a hybridoma strain producing the monoclonal antibody of the present invention.
  • Pristane treatment [2,6,10,14-Tetramethylpenedecane (Pristane) 0.5 ml was intraperitoneally administered and bred for 2 weeks. 8 ⁇ : For 10-week old mice or nude mice, (c) 5 to 20 ⁇ 10 6 cells / animal of the polypeptide monoclonal antibody-producing polypeptide of the present invention obtained in the above, are injected intraperitoneally. In 10 to 21 days, Hypridoma becomes ascites cancer.
  • the ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solid components.
  • a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
  • the subclass of the antibody is determined using a mouse monoclonal antibody evening kit or a rat monoclonal antibody typing kit.
  • the amount of polypeptide is calculated by the Lowry method or from the absorbance at 280 nm.
  • a method for screening a substance using the polypeptide of the present invention Using the polypeptide of the present invention, a compound that changes the activity of the polypeptide that regulates intracellular Akt activity can be screened. Specifically, cells expressing the polypeptide of the present invention are brought into contact with a test sample, and qualitatively or quantitatively examined for the binding of Hsp90 to Akt and for the induction or suppression of apoptosis. A compound that changes the activity of controlling the Akt activity can be selected.
  • the test substance is not particularly limited as long as it can be added to the culture system of the cells expressing the polypeptide of the present invention. Examples of the test substance include a low molecular compound, a polymer compound, an organic compound, an inorganic compound, a protein, Genes, viruses, cells, etc. are listed. The test substance excluding the gene may be added directly to the culture medium.
  • Examples of a method for efficiently introducing a gene into cells include a method in which the gene is placed on a virus vector such as retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, etc., and added to a culture system.
  • a virus vector such as retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, etc.
  • a method of encapsulating the vesicles in an artificial vesicle structure such as ribosome and adding to a culture system may be used.
  • a report on gene analysis using a recombinant virus vector [Pro Natl. Acad. Sci. USA, 22, 6733 (1995); Nucleic Acids Res., 1 &, 3587, 1990; Nucleic Acids Res., 2 ⁇ , 3816 (1995)].
  • Hsp90 and Akt The binding between Hsp90 and Akt and the induction or suppression of apoptosis can be measured by the methods described above.
  • the polypeptide of the present invention a compound that inhibits the binding between the polypeptide and a protein that binds to the polypeptide, and the antibody of the present invention can be used to activate or activate a protein that binds to the polypeptide of the present invention. It can be used as an active ingredient (hereinafter abbreviated as “active ingredient of the present invention”) of a pharmaceutical preparation such as a therapeutic drug, a preventive drug, or a diagnostic drug for a disease caused by reduction.
  • active ingredient of the present invention a pharmaceutical preparation such as a therapeutic drug, a preventive drug, or a diagnostic drug for a disease caused by reduction.
  • the polypeptide and a protein that binds to the polypeptide are Examples of the compound that inhibits the binding to white matter include a compound obtained by the above screening method.
  • the diseases caused by the activation or reduction of the activity of the protein binding to the polypeptide of the present invention include the following diseases.
  • diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage.
  • Diseases associated with suppression of apoptosis include, for example, malignant tumors such as stomach cancer, ovarian cancer, breast cancer, knee cancer, and prostate cancer, and autoimmune diseases.
  • diseases caused by abnormal glucose metabolism control signals include diabetes and the like.
  • the active ingredient of the present invention is effective in inhibiting the effects of existing therapeutic agents such as a cell death inhibitor during ischemia of the brain and heart, an anticancer agent, a protective agent against cell death of normal cells during radiotherapy, and an anticancer agent. Used as a potentiating agent.
  • anticancer drugs include alkylating agents, antimetabolites, antitumor antibiotics, and plant alloids. More specifically, busulfan, cyclophosphamide, cyclophosphamide, and sulfad , Ifosphamiae melphafun (melp alan) N nitrosourea, amsacrine, amsacrine ⁇ carplatin, cisplatin, cisplatin, dacarbazine N azacitidine, cladribine Bottles (cytarabine), fludarabine (fludarabine), funooleurouracil (fluorouracil) N -hydroxyperea (hydroxyurea) lipocoholin (leucovorin) methotrexate (methotrexate ⁇ actinomycin D), bleomycin ), Daunorubicin (danorubicin), doxorubicin (doxorubicin), my toma Shin C (mit
  • a recombinant vector prepared for gene therapy into which a gene encoding a polypeptide as an active ingredient is inserted is introduced into cells taken out of the patient, and then the cells are introduced into a living body.
  • an artificial vehicle structure such as ribosome A peptide fragment that enhances the efficiency of introduction into cells as an active ingredient, such as the TAT peptide described in Examples [Science, 2, 1569-1572 (1999)] Fusion and administration to living organisms.
  • the administration ratio and administration schedule of the anticancer agent and the active ingredient of the present invention are determined in consideration of the age and weight of the patient, and the nature or severity of the condition to be treated.
  • the active ingredients of the present invention can be contained as pharmaceutical preparations, alone or as a mixture with any other active ingredient for treatment.
  • these pharmaceutical preparations are prepared by mixing the active ingredient with one or more pharmacologically acceptable carriers and by any method well-known in the technical field of pharmaceuticals.
  • the most effective route for treatment can be oral or parenteral, for example, intravenous.
  • Dosage forms include tablets, powders, granules, syrups, and injections.
  • Liquid preparations suitable for oral administration include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil It can be produced by using oils such as, for example, preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Tablets, powders, granules, etc.
  • excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, and polyvinyl alcohol. , Hydroxypropylcellulose, gelatin and other binders, fatty acid esters And a plasticizer such as glycerin.
  • Formulations suitable for parenteral administration comprise a sterile aqueous preparation containing the active compound which is preferably isotonic with the blood of the recipient.
  • a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of saline and a putose sugar solution.
  • parenteral preparations are also selected from the diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc. exemplified for the oral preparations.
  • auxiliary components can also be added.
  • the dose and frequency of administration of the active ingredient of the present invention will vary depending on the mode of administration, the age and weight of the patient, the nature or severity of the condition to be treated, but usually 0.01 mg to lg per adult for oral administration. Preferably, 0.05 to 50 mg is administered once or several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg, preferably 0.01 to: 10 mg per adult is administered once or several times a day. However, the dose and the number of administrations vary depending on the various conditions described above.
  • FIG. 1 shows the results of an experiment in which binding between Hsp90 and Akt in BALB / 3T3 and 293T cells was detected by Western blotting using an anti-Hsp90 antibody after immunoprecipitation with an anti-Akt antibody.
  • FIG. 2 shows the results of an experiment in which a region essential for binding to Hsp90? was analyzed using an Aktl deletion mutant.
  • FIG. 3 shows the results of an experiment in which a region essential for binding to Aktl was analyzed using an Hsp90-deletion mutant.
  • the c Figure 5 shows the results of an experiment analyzing the inhibition and inhibition of phosphorylation of Aktl the binding of Hsp90 ⁇ and Aktl in a cell, Aktl missing
  • the results of an experiment analyzing changes in Akt activity when a mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in cells were shown.
  • FIG. 6 shows that Aktl deletion mutant gene was introduced into cells and Hsp90?
  • FIG. 4 shows the results of an experiment in which the induction of apoptosis of cells when the binding of A was inhibited was analyzed by nuclear staining with DAPI.
  • Figure ⁇ ⁇ shows the results of an experiment in which the induction of cell apoptosis was analyzed by measuring caspase 3-like activity when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in the cells. .
  • FIG. 8 shows that when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 ⁇ to Aktl in the cells, VP-16 increased the sensitivity to apoptosis-inducing stimulus by the caspase substrate PARP.
  • the experimental result analyzed by the intracellular cleavage activity is shown.
  • Fig. 9 shows the results of an analysis of the decrease in sensitivity of cells to apoptosis-inducing stimuli when transfected with a peptide containing an Akt-binding site on Hsp90?
  • a caspase substrate Show.
  • WB is Western 'printing
  • a-Hsp90 is an anti-Hsp90 antibody
  • hi-Akt is an anti-Akt antibody
  • Cont. IgG is control IgG
  • IP is immunoprecipitated
  • pFLAG-akL is FLAG. Is the full-length Aktl gene with pcg, pcDNA3.1-Hsp90?
  • -FLAG is anti-FLAG tag M2 monoclonal antibody
  • -P-Akt Thr 3 ° 8
  • HI-V5 is an anti-V5 tag monoclonal antibody
  • HI-HA is an anti-HA tag monoclonal antibody
  • FLAG-Akt is a full-length Aktl protein with FLAG ligature
  • c-Akt Is the endogenous Aktl full-length protein
  • 1-309 aki is the Aktl partial sequence (1-309) with FLAG fragment.
  • DMEM medium containing 10% fetal bovine serum (FBS), 37 ° C, 5% C0 2 and cultured in the presence, mouse fibroblast BALB / 3T3 (ATCC CCL-163 ) and human embryonic kidney cells 293T cells [Pro Natl. Acad. Sci.
  • the full-length human Aktl gene was obtained from human osteosarcoma Saos-2 cells (ATCC HTB-85) [J. Cell. Physiol., Phoenix 290 (2000)] by reverse transcription PGR (RT-PCR).
  • RT-PCR reverse transcription PGR
  • various fragments of the Aktl gene (1-433 (AC), 120-480 ( ⁇ ), 120-433 (ANC), -175, 1-228, 1-331, 205-480, 340-480, 1-246, 1-267, 1-288, deletion mutants having amino acid sequences corresponding to positions 1-309 Gene fragment).
  • the above full-length Aktl gene and deletion mutant were subcloned into the pFLAG-CMV-2 vector to obtain a vector for cell expression.
  • Hsp90? and Hsp90a genes (manufactured by Invitrogen) subcloned into the pcDNA3.1 / GS vector and the expression vector containing the full-length or deletion mutant of Aktl described above were combined with Superfect transfection reagent (Qiagen).
  • Superfect transfection reagent Qiagen
  • Aktl protein or its deletion mutant protein was immunoprecipitated from the obtained cell lysate using an anti-FLAG tag M2 monoclonal antibody (manufactured by Sigma), and Hsp90? Or Hsp90 in the immunoprecipitate was used.
  • the Hsp90-binding region of Aktl is a region having the amino acid sequence at positions 229 to 309 of the amino acid sequence of Aktl described in SEQ ID NO: 5, and among these, the region having the amino acid sequence at positions 289 to 309 is particularly preferable. It proved to be necessary for strong binding.
  • Example 3
  • Hsp90 gene The full-length human Hsp90 gene (WT) was obtained from Invitrogen. Also,;? Various Hsp90 gene fragment by PCR (SEQ ID NO: 1 1-301 of Hsp90 amino acid sequence set forth 1-530 (Mid), 1-635, 534-724 ( ⁇ ) ⁇ 534-680, 1-315, 1-326, 1-340,
  • Aktl was immunoprecipitated from the obtained cell lysate using an anti- (His) 6 tag antibody, and the presence or absence of Hsp90? Or its deletion mutant in the immunoprecipitate was determined by using an anti-FLAG tag M2 monoclonal antibody (Sigma) ) Was detected by Western blotting.
  • Figure 3 As a result, it was revealed that the Akt-binding region of Hsp90? Is a region having the 327th to 340th amino acid sequences of the amino acid sequence of Hsp90? Described in SEQ ID NO: 1.
  • Aktl deletion mutant gene with HA fragment using pHM6 vector.
  • SEQ ID NO: 5 A gene encoding the amino acid sequence corresponding to positions 1-309, 1-228, and 1-288 of the amino acid sequence described), a full-length Hsp90-tagged gene with a V5 tag (pcDNA3.1-Hsp90-), and The FLAG-tagged Aktl full length gene (pFLAG-Aktl) was introduced into 293T cells. Thereafter, the cells were cultured for 24 hours under serum-free conditions, and a cell lysate was prepared by the method of Example 1. Aktl in the obtained cell lysate was immunoprecipitated with an anti-FLAG tag M2 monoclonal antibody (Sigma).
  • Akt activity of the immunoprecipitate was measured using an Akt kinase assay kit (manufactured by Upstate Biotechnology) using a partial peptide at the phosphorylation site of GSK3, one of the substrates.
  • Fig. 5 shows the results.
  • the partial sequence of Aktl (amino acid sequence 1 to 309) revealed that Akt activity was reduced when the binding between Aktl and Hsp90 was inhibited.
  • caspase-3 like protease activity in the above cells was measured by the method described in Oncogene, 1295-1304 (1998). Specifically, the collected cells were lysed in a lysate containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS, and the resulting cell lysate was equivalent to 5 g protein and DEVD-AMC ⁇ acetyl -JJ-asp artyl-L-glutamyl-L-valyl-L-aspart-7-amino-4-methylcoumariii) (Peptide Laboratories) in ICE buffer [10 mM HEPES (pH 7.4) / 10% glycerol / 2 ⁇ dithiothreitol] at 37 ° C for 1 hour.
  • ICE buffer 10 mM HEPES (pH 7.4) / 10% glycerol / 2 ⁇ dithiothreitol
  • Akt deletion mutant enhances sensitivity to apoptosis stimulation by VP-16 i
  • Aktl Inhibition of the binding of Aktl to Hsp90 ⁇ by the partial sequence of Aktl (amino acid sequence at position 309 to 309) shown in Examples 1 to 4 and the resulting decrease in Akt activity are associated with apoptosis stimulation by anticancer drugs.
  • VP-16 was used to verify that it increased the sensitivity.
  • the pFLAG-CMV-2 vector is subcloned with an Aktl deletion mutant gene (corresponding to the gene encoding the amino acid sequence of amino acids 1 to 309 of Aktl) or its vector control.
  • HSP327-TAT peptide (LEFRALLFIPRRAP-GYGRKKRRQKRHG) in which the TAT peptide [Science, 2, 1569-1572 (1999)] was conjugated to the C-terminus of Hsp90?
  • HSP287-TAT peptide (PIWTRNPDDITQEE-GYGRKKRRQRRRG), which is a TAT-binding peptide having the amino acid sequence at positions 287-300 of the amino acid sequence shown in SEQ ID NO: 1 that is not the Akt-binding region, and apoptosis.
  • human fibroblast sarcoma cells HT1080 (ATCC CCL-121) were not treated with peptide, and 0.1 mol / L or 1 mol / L of HSP327-TAT or HSP287-TAT peptide was added. And cultured in serum-free DMEM medium for 24 hours.
  • a functional site on Hsp90 involved in apoptosis suppression and regulation of Akt activity involved in glucose metabolism and a functional site on Akt binding to the Hsp90 were identified.
  • Prophylactic and therapeutic drugs against malignant tumors such as gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer, and prostate cancer, and diseases such as diabetes, cell death inhibitors during ischemia in the brain and heart, normal in anticancer drugs and radiation therapy Drugs that protect cells from cell death or enhance the effects of existing therapies Agent is provided,

Abstract

On a recensé des besoins en médicaments, en méthodes de prévention et de diagnostic de maladies causées par des erreurs de régulation de l'apoptose ou par des erreurs de signaux de régulation du métabolisme des glucides, notamment des tumeurs malignes telles que le cancer de l'estomac, le cancer de l'ovaire, le cancer du sein, le cancer du pancréas et le cancer de la prostate. Cette invention a trait à des inhibiteurs de la mort cellulaire dans l'ischémie cérébrale, cardiaque etc., à des agents protégeant les cellules normales de la mort cellulaire dans des traitements faisant appel à des agents anticancéreux ou à des rayons, ou à des médicaments qui potentialisent les effets de médicaments existants. Cette invention concerne aussi une technique de régulation de l'apoptose qui consiste à réguler l'activité Akt dans les cellules, et en particulier à réguler la liaison de Hsp avec Akt, et un moyen qui convient pour le traitement ou le diagnostic de maladies causées par des erreurs de régulation de l'apoptose ou par des erreurs de signaux de régulation des métabolismes des glucides par utilisation de la régulation de l'apoptose.
PCT/JP2001/007179 2000-08-22 2001-08-22 Technique de regulation de l'apoptose et polypeptide de regulation d'apoptose WO2002015925A1 (fr)

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JP2013213055A (ja) * 2002-07-18 2013-10-17 Helix Biopharma Corp 癌細胞増殖を阻害するためのウレアーゼの使用
JP2006501196A (ja) * 2002-07-18 2006-01-12 へリックス バイオファーマ コーポレイション 癌細胞増殖を阻害するためのウレアーゼの使用
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AU2005221470B2 (en) * 2003-03-12 2010-05-20 Tufts University Inhibitors of extracellular hsp90
US7959915B2 (en) 2003-03-12 2011-06-14 Tufts University Inhibitors of extracellular Hsp90
US9068987B2 (en) 2003-03-12 2015-06-30 Trustees Of Tufts College Inhibitors of extracellular HSP90
US7691838B2 (en) 2003-05-30 2010-04-06 Kosan Biosciences Incorporated Method for treating diseases using HSP90-inhibiting agents in combination with antimitotics
EP1628657A4 (fr) * 2003-05-30 2007-04-25 Kosan Biosciences Inc Methode de traitement de maladies mettant en oeuvre des agents inhibant hsp90 conjointement avec des complexes de coordination du platine
EP1628667A4 (fr) * 2003-05-30 2007-04-04 Kosan Biosciences Inc Methode permettant de traiter des maladies a l'aide d'agents d'inhibition hsp90 combines a des antimitotiques
WO2008032876A1 (fr) * 2006-09-15 2008-03-20 Tokai University Médicament préventif et curatif pour le cancer du sein er négatif ou her2 négatif et méthode de criblage
US8071766B2 (en) 2008-02-01 2011-12-06 Takeda Pharmaceutical Company Limited HSP90 inhibitors
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