WO2011158863A1 - Novel substrate peptide of protein kinase - Google Patents

Novel substrate peptide of protein kinase Download PDF

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Publication number
WO2011158863A1
WO2011158863A1 PCT/JP2011/063699 JP2011063699W WO2011158863A1 WO 2011158863 A1 WO2011158863 A1 WO 2011158863A1 JP 2011063699 W JP2011063699 W JP 2011063699W WO 2011158863 A1 WO2011158863 A1 WO 2011158863A1
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peptide
amino acid
phosphorylated
seq
substrate
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PCT/JP2011/063699
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French (fr)
Japanese (ja)
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佳樹 片山
琢郎 新留
健 森
暁明 韓
彬斗 秦
由洋 矢山
隆 下村
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国立大学法人九州大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • the present invention relates to a substrate peptide that is phosphorylated by various protein kinases and uses thereof.
  • Protein kinases are a group of enzymes responsible for post-translational modifications that form the core of cell signaling. Protein kinase assays are extremely important in drug discovery and diagnosis. Several substrate peptides have already been reported for various kinases and are commercially available.
  • An object of the present invention is to provide a protein kinase substrate peptide having an excellent function and suitable for preparing a peptide array.
  • the present inventors screened substrate peptides for each of the three types of protein kinases from substrate peptides having a phosphorylation site sequence by protein kinases, and are shown in Tables 1 to 3 A substrate peptide was found and the present invention was completed.
  • the present invention is as follows.
  • the peptide according to (1) which is composed of an added or inserted amino acid sequence and is phosphorylated by an epidermal growth factor receptor (2) labeled with a labeling substance.
  • step (a) A method for measuring epidermal growth factor receptor activity, (a) reacting the peptide according to (1) or (2) with a test sample; (b) quantifying the peptide phosphorylated in step (a).
  • a method for detecting a disease associated with epidermal growth factor receptor activity (a) reacting the peptide according to (1) or (2) with a test sample isolated from a mammal; (b) quantifying the peptide phosphorylated in step (a).
  • a reagent for measuring epidermal growth factor receptor activity comprising the peptide according to (1) or (2).
  • a diagnostic agent for a disease associated with epidermal growth factor receptor activity comprising the peptide according to (1) or (2).
  • a peptide array for detecting epidermal growth factor receptor activity wherein the peptide according to (1) or (2) is immobilized on a substrate.
  • a method for measuring MET receptor tyrosine kinase activity (a) reacting the peptide according to (9) or (10) with a test sample; (b) quantifying the peptide phosphorylated in step (a).
  • a method for detecting a disease associated with MET receptor tyrosine kinase activity (a) reacting the peptide according to (9) or (10) with a test sample isolated from a mammal; (b) quantifying the peptide phosphorylated in step (a).
  • a reagent for measuring MET receptor tyrosine kinase activity comprising the peptide according to (9) or (10).
  • a diagnostic agent for a disease associated with MET receptor tyrosine kinase activity comprising the peptide according to (9) or (10).
  • a peptide array for detecting MET receptor tyrosine kinase activity wherein the peptide according to (9) or (10) is immobilized on a substrate.
  • the peptide according to (17) which comprises an added or inserted amino acid sequence and is labeled with a peptide (18) that is phosphorylated by an undifferentiated lymphoma kinase (18).
  • a method for measuring anaplastic lymphoma kinase activity (a) reacting the peptide according to (10) or (11) with a test sample; (b) quantifying the peptide phosphorylated in step (a).
  • a method for detecting a disease associated with anaplastic lymphoma kinase activity (a) reacting the peptide according to (17) or (18) with a test sample isolated from a mammal; (b) quantifying the peptide phosphorylated in step (a).
  • a reagent for measuring anaplastic lymphoma kinase activity comprising the peptide according to (17) or (18).
  • a diagnostic agent for a disease associated with anaplastic lymphoma kinase activity comprising the peptide according to (17) or (18).
  • a peptide array for detecting anaplastic lymphoma kinase activity wherein the peptide according to (17) or (18) is immobilized on a substrate.
  • a substrate peptide for protein kinase is provided.
  • the activity of a protein kinase using the peptide as a substrate can be measured. Since the substrate peptide of the present invention has been screened based on the phosphorylation reaction on the peptide array, it is suitable for use in a peptide array for detecting the activity of protein kinase.
  • the kinase-specific substrate peptide of the present invention includes a substrate peptide for an epidermal growth factor receptor (hereinafter also simply referred to as “EGFR”), a substrate peptide for a MET receptor tyrosine kinase (hereinafter also simply referred to as “MET”), and It is a substrate peptide for anaplastic lymphoma kinase (hereinafter also simply referred to as “ALK”).
  • EGFR epidermal growth factor receptor
  • MET MET receptor tyrosine kinase
  • ALK anaplastic lymphoma kinase
  • a specific substrate that can be used for measuring the activity of phosphorylase is useful not only for drug screening but also for disease diagnosis and pre-drug diagnosis. However, it is not easy to search for a substrate that selectively reacts with a specific one of these phosphorylases existing in a living body of more than 500 types.
  • ALK anaplastic lymphoma kinase
  • MET MET receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • the present invention relates to a substrate peptide that is phosphorylated by an epidermal growth factor receptor (EGFR) and uses thereof.
  • EGFR is a tyrosine kinase type receptor that recognizes epidermal growth factor (EGF) that controls cell proliferation or growth and performs signal transduction. Its structure is a glycoprotein with a molecular weight of 170 kDa (kilodalton) and exists through the cell membrane.
  • Examples of substrate peptides that are phosphorylated by EGFR include those having the following amino acid sequences (Table 1).
  • the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by EGFR Also included is a peptide.
  • “1 or several” is usually “1 to 5”, more typically “1 to 3”, and most preferably “1 to 2”. The smaller the better.
  • the introduction of mutations such as the above-mentioned deletion, substitution, addition and the like can be carried out using a mutation-introducing kit using site-directed mutagenesis, such as GeneTailor TM Site-Directed Mutagenesis System (Invitrogen) and TaKaRa Site-Directed Mutagenesis System.
  • Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by EGFR In general, the larger the homology value, the better.
  • peptide means a peptide composed of at least two or more amino acids linked by peptide bonds, and includes oligopeptides, polypeptides and the like. Furthermore, a polypeptide in which a certain three-dimensional structure is formed is called a protein. In the present invention, such a protein is also included in the “peptide”. Therefore, the peptide of the present invention means any of oligopeptides, polypeptides, and proteins.
  • the above definition can be applied to the term “peptide” for MET and ALK.
  • Substrate peptide for MET receptor tyrosine kinase (MET) MET receptor tyrosine kinase (MET) forms a heterodimer having a disulfide bond.
  • the C-terminal intracellular region contains a multifunctional binding site that binds various signaling molecules.
  • the ligand for MET is hepatocyte growth factor (HGF), which stimulates abnormal growth of epithelial cells.
  • HGF hepatocyte growth factor
  • the signaling pathway activated by the interaction between HGF and MET is involved in cell adhesion, tumor invasive growth, and the like. Examples of such MET substrates include the following (Table 2).
  • the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by MET
  • a peptide is included in addition to the peptide consisting of the amino acid sequence shown in SEQ ID NOs: 27 to 56.
  • the meaning of “one or several” is the same as described above, and the mutation introduction kit using the site-directed mutagenesis method is the same as described above for the method of introducing mutation such as deletion, substitution, addition, etc. Etc. can be performed.
  • the method for confirming the introduction of mutation can also be confirmed by using the above-mentioned various amino acid sequencing methods and the structural analysis methods such as X-ray and NMR.
  • Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by MET In general, the larger the homology value, the better.
  • amino acid sequences of the peptides shown in Table 2 are MET substrate motif sequences.
  • X is a naturally occurring amino acid such as 20 kinds of ⁇ -amino acids (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro , Ser, Thr, Trp, Tyr, Val).
  • Substrate peptide for anaplastic lymphoma kinase is one of receptor-type tyrosine kinases, and its intracellular region has kinase activity. When a ligand binds to the extracellular region of ALK, the cell is activated and works for proliferation.
  • ALK is known to fuse with EML4 (Echinoderm microtubule-associated protein like protein 4) resulting from an inversion in the short arm of chromosome 2 (2p) (the fusion gene is called “EML4-ALK”) ). This fusion gene was obtained from analysis of Japanese lung cancer cases and was found to be found in about 10% of lung cancer cases.
  • ALK plays an important role in the developmental process of the brain and is known to affect specific neurons in the nervous system. It was revealed that ALK gene mutation was involved in the development of neuroblastoma, one of the children's solid tumors. It was confirmed that there was. Therefore, ALK gene mutation is considered to cause canceration in malignant lymphoma, lung cancer, and neuroblastoma in children. Examples of such ALK substrates include the following peptides (Table 3).
  • the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by ALK Also included is a peptide.
  • the meaning of “one or several” is the same as described above, and the mutation introduction kit using the site-directed mutagenesis method is the same as described above for the method of introducing mutation such as deletion, substitution, addition, etc. Etc. can be performed.
  • the method for confirming the introduction of mutation can also be confirmed by using the above-mentioned various amino acid sequencing methods and the structural analysis methods such as X-ray and NMR.
  • Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by ALK In general, the larger the homology value, the better.
  • the substrate peptide can be obtained by a screening method using a peptide microarray, a screening method using a glass peptide array called “PepStar” by JPT peptide technologies (referred to as JPT method), or a micro flow by LC Science. It is selected from a library or the like by a method such as a screening method using an array using a route (referred to as LC method).
  • a method using thiazoline ring formation is employed.
  • glutaraldehyde is bound to a substrate such as a high-density aminated glass substrate (Matsunami Glass), an AE substrate or a GC substrate (Sumitomo Bakelite Development), and a substrate peptide having an N-terminal Cys is bound thereto.
  • a substrate such as a high-density aminated glass substrate (Matsunami Glass), an AE substrate or a GC substrate (Sumitomo Bakelite Development), and a substrate peptide having an N-terminal Cys is bound thereto.
  • a substrate peptide having an N-terminal Cys is bound thereto.
  • an anti-phosphorylated amino acid antibody is prepared and fluorescently labeled, and detection is performed by reacting the labeled antibody with a phosphorylated peptide.
  • a peptide having high fluorescence intensity is selected as a substrate peptide.
  • a peptide sequence is designed based on peripheral peptide sequence information including a phosphorylation site of a substrate protein or information obtained from known literature, and this is spotted on a glass peptide array. After making kinase act on this, a phosphorylation site
  • a peptide sequence is designed based on peripheral peptide sequence information including a phosphorylation site of a substrate protein, or information obtained from known literature. It is also possible to redesign the sequence based on the screening result by the JPT method.
  • a peptide having the designed sequence is synthesized and added to the microchannel to detect phosphorylation. Then, the peptide having a high detection result is selected as the substrate peptide of the present invention.
  • the substrate peptides screened in this way are shown in Tables 1 to 3 above.
  • the substrate peptide of the present invention is obtained by the screening method, it is obtained by expressing the peptide by a peptide synthesis method described later, or by genetic engineering using a nucleotide sequence encoding the peptide. be able to.
  • the peptide of the present invention can be produced by a known peptide synthesis method, for example, a method using a fully automatic peptide synthesizer.
  • the peptide may be a peptide derived from a natural product, or may be obtained by artificial chemical synthesis, and is not limited. Chemically synthesized peptides can be obtained using known peptide synthesis methods.
  • Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method.
  • the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis.
  • a commercially available peptide synthesizer may be used. After the synthesis reaction, the peptide can be purified by combining known purification methods such as chromatography.
  • the peptide of the present invention can be used for measuring the activity of protein kinase.
  • the kinase activity is measured by reacting each kinase substrate peptide with a test sample suspected of having kinase activity in an appropriate buffer or the like so that the substrate peptide is phosphorylated.
  • the phosphorylated peptide is detected and quantified by a method that detects phosphorylation (ie, detects the presence of phosphate groups).
  • the method for detecting and quantifying the phosphorylated peptide is not limited.
  • a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or a radioisotope element (radio)
  • a method using an isotope for example, a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or a radioisotope element (radio)
  • a method using an isotope for example, a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or a radioisotope element (radio)
  • a method using an isotope for example, a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or a radioisotope element (radio)
  • the activity can be measured with higher accuracy if a plurality of substrate peptides are used in combination.
  • the activity measurement can be performed using 5 or more, preferably 10 or more, most preferably all of the peptides each containing the amino acid sequence represented by SEQ ID NOs: 1 to 26. preferable.
  • activity measurement is performed using 5 or more, preferably 10 or more, and most preferably all types of peptides including the amino acid sequences represented by SEQ ID NOs: 27 to 56, respectively. It is preferable.
  • activity measurement is performed using, for example, 5 or more, preferably 10 or more, and most preferably all types of peptides including the amino acid sequences represented by SEQ ID NOs: 57 to 72, respectively. It is preferable.
  • the quantification of the phosphorylated peptide can be performed by a conventional method. For example, it is possible to quantify by an immunological method using an antibody that binds to a phosphorylated peptide.
  • the peptide of the present invention may be labeled with a labeling substance for the purpose of facilitating the quantification of the phosphorylated peptide.
  • Any labeling substance can be used without particular limitation as long as it can be used in a usual immunological measurement method.
  • an enzyme, biotin, a fluorescent substance, a radioactive substance, etc. can be used alone or in combination. Examples of the enzyme include peroxidase, ⁇ -D-galactosidase, alkaline phosphatase, and microperoxidase.
  • Examples of the fluorescent substance include fluorescein isothiocyanate, phycobiliprotein, and phycoerythrin.
  • Examples of the luminescent substance include isolucinol and lucigenin.
  • Examples of radioactive substances include 125 I, 131 I, 14 C, and 3 H.
  • enzyme-labeled avidin As the enzyme of the enzyme-labeled avidin, the same enzyme as that used when the antibody is labeled can be used.
  • a reagent that binds to a phosphorylation site examples include an anti-phosphorylated serine / threonine antibody, an anti-phosphorylated tyrosine antibody, and a Phos-tag.
  • the phos tag is a dinuclear complex of zinc and binds to the phosphate group itself.
  • Phostag has a biotin group in the molecule, and avidin and its analogs, or anti-biotin antibody is labeled with a detection group similar to that described above, and is used for detection by binding to the biotin site.
  • the substrate peptide group of the present invention can be used in any other method for evaluating kinase activity.
  • it can be used for activation analysis such as autoradiography using ATP having radioactive 32 P, or an assay utilizing change in the aggregation state of gold colloid whose surface is negatively charged.
  • activation analysis such as autoradiography using ATP having radioactive 32 P, or an assay utilizing change in the aggregation state of gold colloid whose surface is negatively charged.
  • an assay using colloidal gold negative charge on the colloidal gold surface and aggregation due to electrostatic interaction of the positively charged substrate peptide are suppressed by a decrease in aggregation ability due to a decrease in positive charge on the peptide side accompanying peptide phosphorylation. Use the color change that accompanies it.
  • the peptide of the present invention may be provided as a reagent for measuring EGFR, MET, or ALK activity in combination with any component such as a carrier or excipient that is acceptable for measurement applications.
  • the measurement reagent can also be provided as a measurement kit in combination with a detection antibody or the like.
  • a peptide array in which the peptide of the present invention is immobilized on a substrate can be used.
  • the peptide is immobilized on a substrate such as glass, polydimethylsiloxane, or gold through chemical modification.
  • the shape of the substrate can be applied to any of the substrates reported so far, such as a flat plate, a pillar array, and a well array.
  • glass is coated with a silane coupling agent, or the surface is coated with a polysaccharide, and polydimethylsiloxane is coated with a surface such as a polysaccharide, or the surface is modified with plasma or the like.
  • the substrate peptide can be immobilized by using a cross-linking agent or the like for this, converting the terminal carboxy group into an active ester, or introducing a formyl group or a maleimide group and then reacting the peptide terminal as a cysteine residue.
  • an array on which various types of substrates are immobilized is treated with an enzyme solution or a cell lysate to phosphorylate the substrate immobilized on the surface, thereby antiphosphorylated serine / threonine antibody or antiphosphorylated tyrosine. It is possible to simultaneously evaluate the degree of phosphorylation of many substrates by the same detection means as described above after binding a reagent that binds to a phosphate group such as an antibody or phostag.
  • Methods for detecting diseases associated with protein kinase activity Components (eg, blood, urine, isolated from mammals (eg, humans, rats, hamsters, rabbits, cats, dogs, cows, horses) using the peptides of the present invention. Lysate from any mammalian tissue (brain, liver, heart, muscle, skin, etc.), mammalian normal or cancer cell culture lysates, and cancer tissue lysates that arise in mammals
  • a disease associated with the protein kinase activity can be diagnosed.
  • Disease related to EGFR activity refers to a disease in which the EGFR activity in the sample differs between the affected patient and a healthy subject. Examples of such diseases include kidney cancer, non-small cell lung cancer, prostate cancer, head and neck cancer, ovarian cancer, stomach cancer, colon cancer, breast cancer, brain tumor (glioma), prostate cancer, oral cancer and the like. Is not limited.
  • Disease related to MET activity refers to a disease in which the MET activity in the sample differs between affected patients and healthy subjects. Examples of such diseases include colon cancer, renal cancer, non-small cell lung cancer, prostate cancer, head and neck cancer, ovarian cancer, gastric cancer, colon cancer, breast cancer, brain tumor (glioma), prostate cancer, oral cancer and the like. However, it is not limited to these.
  • the “disease related to ALK activity” refers to a disease in which ALK activity in the sample is different between affected patients and healthy subjects.
  • kidney cancer non-small cell lung cancer
  • prostate cancer head and neck cancer
  • ovarian cancer stomach cancer
  • colon cancer breast cancer
  • brain tumor glioma
  • neuroblastoma prostate cancer
  • oral cancer oral cancer
  • the disease of the patient when the EGFR activity is different between the patient-derived sample and the healthy subject-derived sample, the disease of the patient is an indicator that there is a possibility of a disease related to EGFR activity. .
  • the MET activity differs between the patient-derived sample and the healthy subject-derived sample
  • the disease of the patient is an indicator that there is a possibility of a disease related to the MET activity.
  • the ALK activity differs between the patient-derived sample and the healthy subject-derived sample
  • the disease of the patient is an indicator that there is a possibility of a disease related to the ALK activity.
  • the diagnostic method of the present invention can be applied to various diagnoses such as pre-medication diagnosis, intraoperative diagnosis, and clinical diagnosis.
  • the method for measuring protein kinase activity using the peptide of the present invention is the same as described above.
  • the peptide of the present invention can be provided as a diagnostic agent for diseases associated with protein kinase activity in combination with any component such as a carrier or excipient that is acceptable for diagnostic use.
  • the diagnostic agent may be provided as a diagnostic kit in combination with a detection antibody or the like.
  • the peptide arrays for detecting various protein kinase activities of the present invention can be used for diagnosis of the above-mentioned diseases related to protein kinase activity.
  • Nucleic acid The present invention also relates to a nucleic acid encoding the above substrate peptide.
  • the nucleic acid include DNA or RNA, and DNA is preferable.
  • the nucleic acid can be introduced into a vector and the substrate peptide of the present invention can be expressed in vivo.
  • a nucleic acid sequence fused with the nucleic acid and a nucleic acid encoding a marker molecule is incorporated into a vector, and the substrate peptide of the present invention and the marker molecule are incorporated in vivo.
  • a marker molecule for example, a marker molecule detectable using an antigen-antibody reaction
  • the designed substrate was commissioned and synthesized by JPTpeptide technologies, and this was chemically bonded to the glass substrate at the N-terminus. Then, 300 ⁇ L of each kinase reaction solution [ATP (+) and ATP ( ⁇ )] was added on the substrate and incubated at 37 ° C. for 2 hours for phosphorylation. After the reaction, the operation of immersing in 10 ⁇ TBS solution and washing for 5 minutes was performed 5 times. Then, a blocking operation was performed by applying a solution obtained by diluting Blocking One-P 5 times with TBST solution to the substrate at room temperature for 1 hour.
  • Cultured HCC827 (human lung cancer cells) was incubated in serum-free medium for 18 hours. Thereafter, the cells were washed with D-PBS ( ⁇ ), and the cells were peeled off using a cell scraper, and the cells were collected. After centrifugation, the supernatant was removed. Lysis buffer was added thereto and sonication was performed. After ultracentrifugation, the supernatant (cytoplasmic component) was collected. Lysis buffer containing a surfactant was added to the remaining pellets, and sonication was performed. After ultracentrifugation, cell membrane components were collected as the supernatant. Subsequently, a peptide array was prepared.
  • a solution prepared by mixing a substrate peptide (peptide No. 109: SEQ ID No. 25) and TCEP in 50 mM carbonate buffer (pH 9.5) so as to have a concentration of 0.1 mM is Genex Arrayer (manufactured by Kaken Genex, pin tip diameter 150 ⁇ m) ) was spotted on a maleimide-modified GC substrate and incubated overnight at room temperature to immobilize the peptide. After incubation, the substrate was immersed in a TBS-T solution and washed for 3 minutes three times to remove unreacted peptides.
  • EGFR pep CGGEQEDEPEDYFEWLEP (SEQ ID NO: 73)
  • FIG. 1 shows the phosphorylation of each peptide by HCC827 lysate, and the enzyme activity in HCC827 cells known to be highly activated by EGFR could be evaluated by the peptide of the present invention.
  • Cultured HCC827 (human lung cancer cells) was incubated in serum-free medium for 18 hours. Next, the medium was discarded and replaced with a serum-free medium supplemented with Iressa (Gefitinib, Astra Zeneca), an EGFR inhibitor, and incubated for 2 hours. The serum-free medium containing Iressa was discarded and washed with D-PBS ( ⁇ ). Thereafter, D-PBS (-) was added, and the cells were peeled off using a cell scraper, and the cells were collected. After centrifugation, the supernatant was removed. Lysis buffer was added thereto and sonication was performed. After ultracentrifugation, the supernatant (cytoplasmic component) was collected.
  • a lysis buffer containing a surfactant was added and sonication was performed. After ultracentrifugation, cell membrane components were collected as the supernatant. Preparation of a peptide array and evaluation of enzyme activity using the array were performed in the same procedure as in Example 2.
  • FIG. 2 shows the phosphorylation rate of each peptide when HCC827 is treated with Iressa, an inhibitor of EGFR, and the inhibitory activity of Iressa is increased by the peptide of the present invention (peptide number 109: SEQ ID NO: 25). It was shown that evaluation is possible.
  • substrate peptides for EGFR, MET and ALK are provided.
  • the substrate peptide of the present invention is used in a phosphorylase activity measurement kit, a disease diagnostic kit, a pre-medication diagnostic kit, a screening method for a phosphorylase inhibitor, and the like.
  • SEQ ID NO: 1-73 Synthetic peptide SEQ ID NO: 49: Xaa represents Asp or Glu (location: 1-2) SEQ ID NO: 49: Xaa represents any naturally occurring amino acid (location: 3) SEQ ID NO: 49: Xaa represents any naturally occurring amino acid (location: 6) SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 1-2) SEQ ID NO: 50: Xaa represents a naturally occurring arbitrary amino acid (location: 5) SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 8) SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 10) SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 1-2) SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 5) SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 8) SEQ ID NO: 51: 51: Xaa represents any naturally

Abstract

Disclosed is a substrate peptide that is phosphorylated by various protein kinases. The peptide: comprises an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted in (a) a peptide comprising any one of the amino acid sequences represented by (a) sequence numbers 1 to 72, or (b) any one of the amino acid sequences represented by sequence number 1 to 72; and is phosphorylated by EGFR, MET kinase or ALK.

Description

タンパク質キナーゼの新規基質ペプチドNovel substrate peptides for protein kinases
　本発明は、各種タンパク質キナーゼによりリン酸化される基質ペプチドおよびその用途に関する。 The present invention relates to a substrate peptide that is phosphorylated by various protein kinases and uses thereof.
  タンパク質キナーゼは細胞情報伝達の中核をなす、翻訳後修飾を担当する酵素群である。タンパク質キナーゼのアッセイは創薬、診断において極めて重要なものである。既に種々のキナーゼに関して幾つかの基質ペプチドが報告されており、市販もされている。 Protein kinases are a group of enzymes responsible for post-translational modifications that form the core of cell signaling. Protein kinase assays are extremely important in drug discovery and diagnosis. Several substrate peptides have already been reported for various kinases and are commercially available.
  しかしながら、タンパク質キナーゼの既存の基質ペプチドは必ずしも満足できるものではない。たとえば、基質を実際の細胞内キナーゼの計測に用いることは、診断や創薬に極めて有用であるが、細胞内キナーゼの量や活性は、細胞の種類や目的とする状態によって様々であるため、よりリン酸化されやすい基質のほうが良いというものではない。実際に目的の系に存在するキナーゼの量に適したリン酸化されやすさが必要である。すなわち、一つの酵素に対して一種類の基質が存在すればよい訳ではなく、リン酸化されやすい多くの基質を用意することが重要である。この点、従来、各種タンパク質キナーゼの基質として、多くのペプチド配列が示されている（特許文献１）。
  しかし、これらの基質ペプチド以外にも、発がん、癌の増殖、浸潤、転移などと密接な関係のあるキナーゼに反応するペプチドの開発が望まれる。 However, existing substrate peptides for protein kinases are not always satisfactory. For example, the use of a substrate for actual measurement of intracellular kinases is extremely useful for diagnosis and drug discovery, but the amount and activity of intracellular kinases vary depending on the type of cells and the intended state. A substrate that is more easily phosphorylated is not better. In fact, it is necessary to be easily phosphorylated in accordance with the amount of kinase present in the target system. That is, it is not necessary that one kind of substrate exists for one enzyme, but it is important to prepare many substrates that are easily phosphorylated. Conventionally, many peptide sequences have been shown as substrates for various protein kinases (Patent Document 1).
However, in addition to these substrate peptides, it is desired to develop peptides that react with kinases that are closely related to carcinogenesis, cancer growth, invasion, metastasis, and the like.
特開２００８－２８９３７４号公報JP 2008-289374 A
　本発明は、優れた機能を有し、ペプチドアレイの作製に適したタンパク質キナーゼの基質ペプチドを提供することを目的とする。 An object of the present invention is to provide a protein kinase substrate peptide having an excellent function and suitable for preparing a peptide array.
  本発明者は、上記課題を解決するため鋭意研究を行った結果、タンパク質キナーゼによるリン酸化部位配列を持つ基質ペプチドから、３種のタンパク質キナーゼそれぞれに対する基質ペプチドをスクリーニングし、表１～３に示す基質ペプチドを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors screened substrate peptides for each of the three types of protein kinases from substrate peptides having a phosphorylation site sequence by protein kinases, and are shown in Tables 1 to 3 A substrate peptide was found and the present invention was completed.
　すなわち、本発明は以下の通りである。
（１） 以下の(a)又は(b)のペプチド。
  (a) 配列番号１～２６で表されるいずれかのアミノ酸配列からなるペプチド
  (b) 配列番号１～２６で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、上皮細胞成長因子受容体によりリン酸化されるペプチド
（２）標識物質により標識化されている（１）に記載のペプチド。 That is, the present invention is as follows.
(1) The following peptide (a) or (b).
(a) a peptide consisting of any one of the amino acid sequences represented by SEQ ID NOs: 1 to 26 (b) one or several amino acids deleted or substituted in any amino acid sequence represented by SEQ ID NOs: 1 to 26 The peptide according to (1), which is composed of an added or inserted amino acid sequence and is phosphorylated by an epidermal growth factor receptor (2) labeled with a labeling substance.
（３）上皮細胞成長因子受容体活性の測定方法であって、
(a) （１）又は（２）に記載のペプチドと被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (3) A method for measuring epidermal growth factor receptor activity,
(a) reacting the peptide according to (1) or (2) with a test sample;
(b) quantifying the peptide phosphorylated in step (a).
（４）上皮細胞成長因子受容体活性に関連する疾患の検出方法であって、
(a) （１）又は（２）に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (4) A method for detecting a disease associated with epidermal growth factor receptor activity,
(a) reacting the peptide according to (1) or (2) with a test sample isolated from a mammal;
(b) quantifying the peptide phosphorylated in step (a).
（５）（１）又は（２）に記載のペプチドを含む、上皮細胞成長因子受容体活性の測定用試薬。
（６）（１）又は（２）に記載のペプチドを含む、上皮細胞成長因子受容体活性に関連する疾患の診断薬。
（７） 基板上に（１）又は（２）に記載のペプチドが固定化された、上皮細胞成長因子受容体活性検出用ペプチドアレイ。
（８）（１）に記載のペプチドをコードする核酸。 (5) A reagent for measuring epidermal growth factor receptor activity, comprising the peptide according to (1) or (2).
(6) A diagnostic agent for a disease associated with epidermal growth factor receptor activity, comprising the peptide according to (1) or (2).
(7) A peptide array for detecting epidermal growth factor receptor activity, wherein the peptide according to (1) or (2) is immobilized on a substrate.
(8) A nucleic acid encoding the peptide according to (1).
（９）以下の(a)又は(b)のペプチド。
  (a) 配列番号２７～５６で表されるいずれかのアミノ酸配列からなるペプチド
  (b) 配列番号２７～５６で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、MET受容体チロシンキナーゼによりリン酸化されるペプチド
（１０）標識物質により標識化されている（９）に記載のペプチド。 (9) The following peptide (a) or (b).
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 27 to 56 (b) one or several amino acids deleted or substituted in any one of the amino acid sequences represented by SEQ ID NOs: 27 to 56 The peptide according to (9), which is composed of an added or inserted amino acid sequence and is labeled with a peptide (10) that is phosphorylated by a MET receptor tyrosine kinase.
（１１）MET受容体チロシンキナーゼ活性の測定方法であって、
(a) （９）又は（１０）に記載のペプチドと被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (11) A method for measuring MET receptor tyrosine kinase activity,
(a) reacting the peptide according to (9) or (10) with a test sample;
(b) quantifying the peptide phosphorylated in step (a).
（１２）MET受容体チロシンキナーゼ活性に関連する疾患の検出方法であって、
(a) （９）又は（１０）に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (12) A method for detecting a disease associated with MET receptor tyrosine kinase activity,
(a) reacting the peptide according to (9) or (10) with a test sample isolated from a mammal;
(b) quantifying the peptide phosphorylated in step (a).
（１３）（９）又は（１０）に記載のペプチドを含む、MET受容体チロシンキナーゼ活性の測定用試薬。
（１４）（９）又は（１０）に記載のペプチドを含む、MET受容体チロシンキナーゼ活性に関連する疾患の診断薬。
（１５）基板上に（９）又は（１０）に記載のペプチドが固定化された、MET受容体チロシンキナーゼ活性検出用ペプチドアレイ。
（１６）（９）に記載のペプチドをコードする核酸。 (13) A reagent for measuring MET receptor tyrosine kinase activity, comprising the peptide according to (9) or (10).
(14) A diagnostic agent for a disease associated with MET receptor tyrosine kinase activity, comprising the peptide according to (9) or (10).
(15) A peptide array for detecting MET receptor tyrosine kinase activity, wherein the peptide according to (9) or (10) is immobilized on a substrate.
(16) A nucleic acid encoding the peptide according to (9).
（１７）以下の(a)又は(b)のペプチド。
  (a) 配列番号５７～７２で表されるいずれかのアミノ酸配列からなるペプチド
  (b) 配列番号５７～７２で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、未分化リンパ腫キナーゼによりリン酸化されるペプチド
（１８）標識物質により標識化されている（１７）に記載のペプチド。 (17) The following peptide (a) or (b):
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 57 to 72 (b) one or several amino acids deleted or substituted in any amino acid sequence represented by SEQ ID NOs: 57 to 72 The peptide according to (17), which comprises an added or inserted amino acid sequence and is labeled with a peptide (18) that is phosphorylated by an undifferentiated lymphoma kinase (18).
（１９）未分化リンパ腫キナーゼ活性の測定方法であって、
(a) （１０）又は（１１）に記載のペプチドと被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (19) A method for measuring anaplastic lymphoma kinase activity,
(a) reacting the peptide according to (10) or (11) with a test sample;
(b) quantifying the peptide phosphorylated in step (a).
（２０）未分化リンパ腫キナーゼ活性に関連する疾患の検出方法であって、
(a) （１７）又は（１８）に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
(b) 工程(a)でリン酸化されたペプチドを定量する工程と
を含む前記方法。 (20) A method for detecting a disease associated with anaplastic lymphoma kinase activity,
(a) reacting the peptide according to (17) or (18) with a test sample isolated from a mammal;
(b) quantifying the peptide phosphorylated in step (a).
（２１）（１７）又は（１８）に記載のペプチドを含む、未分化リンパ腫キナーゼ活性の測定用試薬。
（２２）（１７）又は（１８）に記載のペプチドを含む、未分化リンパ腫キナーゼ活性に関連する疾患の診断薬。
（２３）基板上に（１７）又は（１８）に記載のペプチドが固定化された、未分化リンパ腫キナーゼ活性検出用ペプチドアレイ。
（２４）（１７）に記載のペプチドをコードする核酸。 (21) A reagent for measuring anaplastic lymphoma kinase activity, comprising the peptide according to (17) or (18).
(22) A diagnostic agent for a disease associated with anaplastic lymphoma kinase activity, comprising the peptide according to (17) or (18).
(23) A peptide array for detecting anaplastic lymphoma kinase activity, wherein the peptide according to (17) or (18) is immobilized on a substrate.
(24) A nucleic acid encoding the peptide according to (17).
  本発明により、タンパク質キナーゼの基質ペプチドが提供される。本発明の基質ペプチドの使用により、該ペプチドを基質とするタンパク質キナーゼの活性測定を行うことができる。本発明の基質ペプチドは、ペプチドアレイ上でのリン酸化反応に基づいてスクリーニングされたものであることから、タンパク質キナーゼの活性検出用ペプチドアレイでの使用に適している。 According to the present invention, a substrate peptide for protein kinase is provided. By using the substrate peptide of the present invention, the activity of a protein kinase using the peptide as a substrate can be measured. Since the substrate peptide of the present invention has been screened based on the phosphorylation reaction on the peptide array, it is suitable for use in a peptide array for detecting the activity of protein kinase.
HCC827細胞ライセートを用いてキナーゼ活性を検出した結果を示す図である。It is a figure which shows the result of having detected kinase activity using HCC827 cell lysate. HCC827細胞をEGFR活性阻害剤で処理したときのリン酸化を試験した結果を示す図である。It is a figure which shows the result of having tested the phosphorylation when HCC827 cell was processed with the EGFR activity inhibitor.
　以下、本発明を詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。なお、本明細書は、本願優先権主張の基礎となる特願2010-137453号明細書（2010年6月16日出願）の全体を包含する。また、本明細書において引用された全ての刊行物、例えば先行技術文献、及び公開公報、特許公報その他の特許文献は、参照として本明細書に組み込まれる。 Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. Note that this specification includes the entirety of Japanese Patent Application No. 2010-137453 (filed on June 16, 2010), which is the basis for claiming priority of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications, and other patent documents are incorporated herein by reference.
  本発明のキナーゼ特異的基質ペプチドは、上皮細胞成長因子受容体(以下、単に「EGFR」ともいう)に対する基質ペプチド、MET受容体チロシンキナーゼ（以下、単に「MET」ともいう）に対する基質ペプチド、及び未分化リンパ腫キナーゼ(以下、単に「ALK」ともいう)に対する基質ペプチドである。
 リン酸化酵素は、癌をはじめ各種疾患の発症と密接に関係していることが報告されている。実際に、リン酸化酵素の阻害剤は医薬品ターゲットとなっており、抗癌剤として開発されている例もある。したがって、リン酸化酵素の活性測定に用いることのできる特異的な基質は、医薬品スクリーニングのみならず、疾病診断、投薬前診断に有用である。しかし、生体内に500種以上も存在するこれらリン酸化酵素のうちの特定のものに選択的に反応する基質の探索は容易ではない。 The kinase-specific substrate peptide of the present invention includes a substrate peptide for an epidermal growth factor receptor (hereinafter also simply referred to as “EGFR”), a substrate peptide for a MET receptor tyrosine kinase (hereinafter also simply referred to as “MET”), and It is a substrate peptide for anaplastic lymphoma kinase (hereinafter also simply referred to as “ALK”).
It has been reported that phosphorylase is closely related to the onset of various diseases including cancer. In fact, inhibitors of phosphorylase have become pharmaceutical targets, and in some cases have been developed as anticancer agents. Therefore, a specific substrate that can be used for measuring the activity of phosphorylase is useful not only for drug screening but also for disease diagnosis and pre-drug diagnosis. However, it is not easy to search for a substrate that selectively reacts with a specific one of these phosphorylases existing in a living body of more than 500 types.
  本発明においては、癌関連のキナーゼである未分化リンパ腫キナーゼ（ALK）、MET受容体チロシンキナーゼ（MET）、及び上皮細胞成長因子受容体（EGFR）キナーゼに対し特異的な基質ペプチドを見出した。これらのペプチドはこれまでに報告されていないものである。 In the present invention, a substrate peptide specific to anaplastic lymphoma kinase (ALK), MET receptor tyrosine kinase (MET), and epidermal growth factor receptor (EGFR) kinase, which are cancer-related kinases, was found. These peptides have not been reported so far.
１．キナーゼ特異的基質ペプチド
（１）上皮細胞成長因子受容体（EGFR）に対する基質ペプチド
  本発明は、上皮細胞成長因子受容体（EGFR）によりリン酸化を受ける基質ペプチド及びその用途に関する。
  EGFRは、細胞の増殖又は成長を制御する上皮成長因子(EGF)を認識し、シグナル伝達を行うチロシンキナーゼ型受容体である。その構造は分子量170 kDa（キロダルトン）の糖タンパク質であり、細胞膜を貫通して存在する。
　EGFRによりリン酸化を受ける基質ペプチドとしては、以下のアミノ酸配列を有するものが挙げられる（表１）。 1. TECHNICAL FIELD The present invention relates to a substrate peptide that is phosphorylated by an epidermal growth factor receptor (EGFR) and uses thereof.
EGFR is a tyrosine kinase type receptor that recognizes epidermal growth factor (EGF) that controls cell proliferation or growth and performs signal transduction. Its structure is a glycoprotein with a molecular weight of 170 kDa (kilodalton) and exists through the cell membrane.
Examples of substrate peptides that are phosphorylated by EGFR include those having the following amino acid sequences (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
  あるいは、上記配列番号１～２６で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列において、1又は数個のアミノ酸が欠失、付加、挿入または置換されたアミノ酸配列を含み、EGFRによりリン酸化されるペプチドも挙げられる。ここで「1又は数個」とは、通常は「1～5個」であり、より典型的には「1～3個」であり、最も好ましくは「1～2個」であり、一般的には小さい程好ましい。上記欠失、置換、付加等の変異の導入は、部位特異的突然変異誘発法を利用した変異導入用キット、例えば、GeneTailorTM Site-Directed Mutagenesis System（インビトロジェン社）、及びTaKaRa Site-Directed Mutagenesis System（Mutan-K、Mutan-Super Express Km等：タカラバイオ社製）等を用いて行うことができる。また、上記欠失、置換又は付加の変異が導入されたペプチドであるかどうかは、各種アミノ酸配列決定法、並びにX線及びNMR等による構造解析法などを用いて確認することができる。 Alternatively, in addition to the peptide consisting of the amino acid sequence shown by SEQ ID NOs: 1 to 26, the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by EGFR Also included is a peptide. Here, “1 or several” is usually “1 to 5”, more typically “1 to 3”, and most preferably “1 to 2”. The smaller the better. The introduction of mutations such as the above-mentioned deletion, substitution, addition and the like can be carried out using a mutation-introducing kit using site-directed mutagenesis, such as GeneTailor ™ Site-Directed Mutagenesis System (Invitrogen) and TaKaRa Site-Directed Mutagenesis System. (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like can be used. In addition, whether or not the peptide has a deletion, substitution or addition mutation introduced therein can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray and NMR.
　さらに、上記配列番号１～２６で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列に対して、約80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上又は99％以上の相同性を有するアミノ酸配列を含み、EGFRによりリン酸化されるペプチドも挙げられる。上記相同性の数値は一般的に大きい程好ましい。 Furthermore, in addition to the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 1 to 26, about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more with respect to the amino acid sequence , Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by EGFR. In general, the larger the homology value, the better.
  本発明において、「ペプチド」とは、少なくとも２個以上のアミノ酸がペプチド結合によって結合して構成されたものを意味し、オリゴペプチド、ポリペプチドなどが含まれる。さらに、ポリペプチドが一定の立体構造を形成したものはタンパク質と呼ばれるが、本発明においては、このようなタンパク質も上記「ペプチド」に含まれるものとする。従って、本発明のペプチドは、オリゴペプチド、ポリペプチド、タンパク質のいずれをも意味するものである。以下、MET、ALKについても、「ペプチド」の用語は上記定義を適用することができる。 In the present invention, “peptide” means a peptide composed of at least two or more amino acids linked by peptide bonds, and includes oligopeptides, polypeptides and the like. Furthermore, a polypeptide in which a certain three-dimensional structure is formed is called a protein. In the present invention, such a protein is also included in the “peptide”. Therefore, the peptide of the present invention means any of oligopeptides, polypeptides, and proteins. Hereinafter, the above definition can be applied to the term “peptide” for MET and ALK.
（２）MET受容体チロシンキナーゼ（MET）に対する基質ペプチド
  MET受容体チロシンキナーゼ（MET）は、ジスルフィド結合を有するヘテロダイマーを形成する。そのＣ末端の細胞内領域は、種々のシグナリング分子と結合する多機能結合部位を含んでいる。METのリガンドは、上皮細胞の異常な増殖を刺激する肝細胞増殖因子（HGF）である。HGFとMETとの相互作用によって活性化されるシグナリング経路は、細胞接着、腫瘍の浸潤性増殖等に関与する。
  このようなMETの基質としては、以下のものが挙げられる（表２）。 (2) Substrate peptide for MET receptor tyrosine kinase (MET) MET receptor tyrosine kinase (MET) forms a heterodimer having a disulfide bond. The C-terminal intracellular region contains a multifunctional binding site that binds various signaling molecules. The ligand for MET is hepatocyte growth factor (HGF), which stimulates abnormal growth of epithelial cells. The signaling pathway activated by the interaction between HGF and MET is involved in cell adhesion, tumor invasive growth, and the like.
Examples of such MET substrates include the following (Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
  あるいは、上記配列番号２７～５６で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列において、1又は数個のアミノ酸が欠失、付加、挿入または置換されたアミノ酸配列を含み、METによりリン酸化されるペプチドも挙げられる。ここで「1又は数個」の意味は前記と同様であり、上記欠失、置換、付加等の変異の導入法も、前記と同様、部位特異的突然変異誘発法を利用した変異導入用キット等により行なうことができる。また、変異導入の確認法も、上記各種アミノ酸配列決定法、並びにX線及びNMR等による構造解析法などを用いて確認することができる。 Alternatively, in addition to the peptide consisting of the amino acid sequence shown in SEQ ID NOs: 27 to 56, the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by MET Also included is a peptide. Here, the meaning of “one or several” is the same as described above, and the mutation introduction kit using the site-directed mutagenesis method is the same as described above for the method of introducing mutation such as deletion, substitution, addition, etc. Etc. can be performed. In addition, the method for confirming the introduction of mutation can also be confirmed by using the above-mentioned various amino acid sequencing methods and the structural analysis methods such as X-ray and NMR.
　さらに、上記配列番号２７～５６で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列に対して、約80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上又は99％以上の相同性を有するアミノ酸配列を含み、METによりリン酸化されるペプチドも挙げられる。上記相同性の数値は一般的に大きい程好ましい。 Further, in addition to the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 27 to 56, about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more with respect to the amino acid sequence , Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by MET. In general, the larger the homology value, the better.
  ここで、表２に示すペプチドのアミノ酸配列において、配列番号49～56に示すアミノ酸配列はMETの基質のモチーフ配列である。Xは、天然に存在する任意のアミノ酸であり、例えば20種類のα-アミノ酸（Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val)のいずれかを表す。 に お い て Here, in the amino acid sequences of the peptides shown in Table 2, the amino acid sequences shown in SEQ ID NOs: 49 to 56 are MET substrate motif sequences. X is a naturally occurring amino acid such as 20 kinds of α-amino acids (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro , Ser, Thr, Trp, Tyr, Val).
（３）未分化リンパ腫キナーゼ（ALK）に対する基質ペプチド
  未分化リンパ腫キナーゼ（ALK：anaplastic lymphoma kinase）は、受容体型チロシンキナーゼの一つであり、細胞内領域がキナーゼ活性を持つ。ALKの細胞外領域にリガンドが結合すると、細胞は活性化されて増殖に働く。
  また、ALKは、2番染色体短腕(2p)内の逆位の結果生じるEML4(Echinoderm microtubule-associated protein like protein 4)と融合することが知られている（融合遺伝子を「EML4-ALK」という）。この融合遺伝子は、日本人の肺がん症例の解析から得られたもので、肺がん症例の約1割に見出されることが判明した。 (3) Substrate peptide for anaplastic lymphoma kinase (ALK) Anaplastic lymphoma kinase (ALK) is one of receptor-type tyrosine kinases, and its intracellular region has kinase activity. When a ligand binds to the extracellular region of ALK, the cell is activated and works for proliferation.
In addition, ALK is known to fuse with EML4 (Echinoderm microtubule-associated protein like protein 4) resulting from an inversion in the short arm of chromosome 2 (2p) (the fusion gene is called “EML4-ALK”) ). This fusion gene was obtained from analysis of Japanese lung cancer cases and was found to be found in about 10% of lung cancer cases.
  一方、ALKは脳の発生過程で重要な役目を果たし、神経系において特定の神経細胞に影響を与えることが知られている。小児固形腫瘍の一つである神経芽腫の発症にALK遺伝子の突然変異が関与していることが明らかにされ、その後、非遺伝的に発症する神経芽腫についてもALK遺伝子変異を有する症例のあることが確認された。
  従って、悪性リンパ腫、肺がん、さらに小児の神経芽腫において、ALK遺伝子変異ががん化の原因になると考えられている。
　このようなALKの基質としては、以下のペプチドが挙げられる（表３）。 On the other hand, ALK plays an important role in the developmental process of the brain and is known to affect specific neurons in the nervous system. It was revealed that ALK gene mutation was involved in the development of neuroblastoma, one of the children's solid tumors. It was confirmed that there was.
Therefore, ALK gene mutation is considered to cause canceration in malignant lymphoma, lung cancer, and neuroblastoma in children.
Examples of such ALK substrates include the following peptides (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　あるいは、上記配列番号５７～７２で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列において、1又は数個のアミノ酸が欠失、付加、挿入または置換されたアミノ酸配列を含み、ALKによりリン酸化されるペプチドも挙げられる。ここで「1又は数個」の意味は前記と同様であり、上記欠失、置換、付加等の変異の導入法も、前記と同様、部位特異的突然変異誘発法を利用した変異導入用キット等により行なうことができる。また、変異導入の確認法も、上記各種アミノ酸配列決定法、並びにX線及びNMR等による構造解析法などを用いて確認することができる。 Alternatively, in addition to the peptide consisting of the amino acid sequence shown in SEQ ID NOs: 57 to 72, the amino acid sequence includes an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted, and phosphorylated by ALK Also included is a peptide. Here, the meaning of “one or several” is the same as described above, and the mutation introduction kit using the site-directed mutagenesis method is the same as described above for the method of introducing mutation such as deletion, substitution, addition, etc. Etc. can be performed. In addition, the method for confirming the introduction of mutation can also be confirmed by using the above-mentioned various amino acid sequencing methods and the structural analysis methods such as X-ray and NMR.
　さらに、上記配列番号５７～７２で示されるアミノ酸配列からなるペプチドのほか、上記アミノ酸配列に対して、約80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上又は99％以上の相同性を有するアミノ酸配列を含み、ALKによりリン酸化されるペプチドも挙げられる。上記相同性の数値は一般的に大きい程好ましい。 Further, in addition to the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 57 to 72, about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more with respect to the amino acid sequence , Peptides containing an amino acid sequence having a homology of 98% or more or 99% or more and phosphorylated by ALK. In general, the larger the homology value, the better.
（４）ペプチドのスクリーニング
　上記基質ペプチドは、ペプチドマイクロアレイを用いたスクリーニング法、JPT peptide technologies社の「PepStar」と呼ばれるガラスペプチドアレイを用いたスクリーニング法（JPT法という）、あるいはLC Science社のマイクロ流路を利用したアレイを用いたスクリーニング法（LC法という）などの方法によってライブラリー等から選択されたものである。
　ペプチドマイクロアレイを用いたスクリーニング法では、例えば、チアゾリン環形成による方法が採用される。この方法では、高密度アミノ化ガラス基板（松浪ガラス）、あるいはAE基板又はGC基板（住友ベークライト開発）などの基板にグルタルアルデヒドを結合させ、これに、Ｎ末端をCysとした基質ペプチドを結合する操作を繰り返し、高密度にペプチドを配列させる。次に、抗リン酸化アミノ酸抗体を作製して蛍光標識し、標識された抗体とリン酸化ペプチドとを反応させて検出を行なう。そして、蛍光強度の高かったペプチドを基質ペプチドとして選択する。 (4) Peptide Screening The substrate peptide can be obtained by a screening method using a peptide microarray, a screening method using a glass peptide array called “PepStar” by JPT peptide technologies (referred to as JPT method), or a micro flow by LC Science. It is selected from a library or the like by a method such as a screening method using an array using a route (referred to as LC method).
In the screening method using a peptide microarray, for example, a method using thiazoline ring formation is employed. In this method, glutaraldehyde is bound to a substrate such as a high-density aminated glass substrate (Matsunami Glass), an AE substrate or a GC substrate (Sumitomo Bakelite Development), and a substrate peptide having an N-terminal Cys is bound thereto. Repeat the procedure to arrange peptides at high density. Next, an anti-phosphorylated amino acid antibody is prepared and fluorescently labeled, and detection is performed by reacting the labeled antibody with a phosphorylated peptide. Then, a peptide having high fluorescence intensity is selected as a substrate peptide.
　JPT法では、基質タンパク質のリン酸化部位を含む周辺のペプチド配列情報、あるいは公知文献等から得られた情報をもとにペプチド配列を設計し、これをガラスペプチドアレイにスポットする。これにキナーゼを作用させた後、Phos-tag biotin等を用いてリン酸化部位を検出する。そして、蛍光強度の高かったペプチドを基質ペプチドとして選択する。
　LC法では、基質タンパク質のリン酸化部位を含む周辺のペプチド配列情報、あるいは公知文献等から得られた情報をもとにペプチド配列を設計する。JPT法でのスクリーニング結果を基準に配列を再度設計することも可能である。次に、設計された配列を有するペプチドを合成し、これをマイクロ流路に添加してリン酸化の検出を行なう。そして、検出結果の高かったペプチドを本発明の基質ペプチドとして選択する。
　このようにしてスクリーニングされた基質ペプチドは、前記表１～３に示した。 In the JPT method, a peptide sequence is designed based on peripheral peptide sequence information including a phosphorylation site of a substrate protein or information obtained from known literature, and this is spotted on a glass peptide array. After making kinase act on this, a phosphorylation site | part is detected using Phos-tag biotin etc. Then, a peptide having high fluorescence intensity is selected as a substrate peptide.
In the LC method, a peptide sequence is designed based on peripheral peptide sequence information including a phosphorylation site of a substrate protein, or information obtained from known literature. It is also possible to redesign the sequence based on the screening result by the JPT method. Next, a peptide having the designed sequence is synthesized and added to the microchannel to detect phosphorylation. Then, the peptide having a high detection result is selected as the substrate peptide of the present invention.
The substrate peptides screened in this way are shown in Tables 1 to 3 above.
２．ペプチド合成
　前記スクリーニング方法により本発明の基質ペプチドが得られると、その後は、後述のペプチド合成法により、あるいは、ペプチドをコードする塩基配列を用いて遺伝子工学的手法によりペプチドを発現することにより、得ることができる。
  本発明のペプチドは、公知のペプチド合成方法、例えば全自動ペプチド合成装置を用いた方法により製造することができる。ペプチドは、天然物由来のペプチドであってもよいし、人工的に化学合成して得られたものであってもよく、限定はされない。化学合成ペプチドは、公知のペプチド合成方法を用いて得ることができる。合成方法としては、例えば、アジド法、酸クロライド法、酸無水物法、混合酸無水物法、DCC法、活性エステル法、カルボイミダゾール法及び酸化還元法等が挙げられる。また、その合成は、固相合成法及び液相合成法のいずれをも適用することができる。市販のペプチド合成装置を使用してもよい。合成反応後は、クロマトグラフィー等の公知の精製法を組み合わせてペプチドを精製することができる。 2. Peptide synthesis When the substrate peptide of the present invention is obtained by the screening method, it is obtained by expressing the peptide by a peptide synthesis method described later, or by genetic engineering using a nucleotide sequence encoding the peptide. be able to.
The peptide of the present invention can be produced by a known peptide synthesis method, for example, a method using a fully automatic peptide synthesizer. The peptide may be a peptide derived from a natural product, or may be obtained by artificial chemical synthesis, and is not limited. Chemically synthesized peptides can be obtained using known peptide synthesis methods. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer may be used. After the synthesis reaction, the peptide can be purified by combining known purification methods such as chromatography.
３．タンパク質キナーゼの活性測定方法
  本発明のペプチドは、タンパク質キナーゼの活性を測定するために用いることができる。
  本発明において、キナーゼ活性の測定方法は、具体的には、各キナーゼ基質ペプチドと、キナーゼ活性を有すると思われる被験試料とを適当なバッファー中などで接触させて反応させ、基質ペプチドがリン酸化され得る状況を経た後、リン酸化を検出する（すなわちリン酸基の存在を検出する）方法によりリン酸化ペプチドを検出及び定量する。ここで、リン酸化ペプチドの検出及び定量方法としては、限定はされないが、例えば、マトリックス支援レーザー脱離イオン化飛行時間型質量分析（MALDI-TOF/MS）を用いる方法、あるいは放射性同位体元素（ラジオアイソトープ）を用いる方法などが挙げられる。 3. Method for Measuring Activity of Protein Kinase The peptide of the present invention can be used for measuring the activity of protein kinase.
In the present invention, specifically, the kinase activity is measured by reacting each kinase substrate peptide with a test sample suspected of having kinase activity in an appropriate buffer or the like so that the substrate peptide is phosphorylated. After a situation that can be done, the phosphorylated peptide is detected and quantified by a method that detects phosphorylation (ie, detects the presence of phosphate groups). Here, the method for detecting and quantifying the phosphorylated peptide is not limited. For example, a method using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or a radioisotope element (radio) And a method using an isotope).
  このとき、各基質ペプチドを複数種類組み合わせて用いるとより精度の高い活性測定が可能となる。EGFR活性を測定する場合は、例えば配列番号１～２６によりそれぞれ表されるアミノ酸配列を含むペプチドのうち5種類以上、好ましくは10種類以上、最も好ましくは全種類を用いて活性測定を行うことが好ましい。また、MET活性を測定する場合は、例えば配列番号２７～５６によりそれぞれ表されるアミノ酸配列を含むペプチドのうち5種類以上、好ましくは10種類以上、最も好ましくは全種類を用いて活性測定を行うことが好ましい。さらに、ALK活性を測定する場合は、例えば配列番号５７～７２によりそれぞれ表されるアミノ酸配列を含むペプチドのうち5種類以上、好ましくは10種類以上、最も好ましくは全種類を用いて活性測定を行うことが好ましい。 At this time, the activity can be measured with higher accuracy if a plurality of substrate peptides are used in combination. When measuring the EGFR activity, for example, the activity measurement can be performed using 5 or more, preferably 10 or more, most preferably all of the peptides each containing the amino acid sequence represented by SEQ ID NOs: 1 to 26. preferable. When measuring MET activity, for example, activity measurement is performed using 5 or more, preferably 10 or more, and most preferably all types of peptides including the amino acid sequences represented by SEQ ID NOs: 27 to 56, respectively. It is preferable. Further, when measuring ALK activity, activity measurement is performed using, for example, 5 or more, preferably 10 or more, and most preferably all types of peptides including the amino acid sequences represented by SEQ ID NOs: 57 to 72, respectively. It is preferable.
  リン酸化されたペプチドの定量は常法により行うことができる。例えば、リン酸化されたペプチドに結合する抗体を用いて免疫学的方法により定量することが可能である。
  本発明のペプチドは、リン酸化されたペプチドの定量をより容易にする目的で標識物質により標識化されていてもよい。標識物質としては、通常の免疫学的測定方法に使用し得るものであれば特に限定されることなく使用できる。例えば、酵素、ビオチン、蛍光物質、放射性物質等を単独または組合せて使用することができる。酵素としてはペルオキシダーゼ、β-D-ガラクトシダーゼ、アルカリフォスファターゼ、マイクロペルオキダーゼが挙げられる。蛍光物質としてはフルオレッセインイソチオシアネート、フィコビリプロテイン、フィコエリスリン等が挙げられる。発光物質としてはイソルシノール、ルシゲニン等が挙げられる。放射性物質としては125I、131I、14C、3H 等が挙げられる。また、標識物質としてビオチンを用いた場合には、さらに酵素標識アビジンを用いることにより標識ビオチンの量を高い感度で測定できる点で好ましい。酵素標識アビジンの酵素としては、抗体に標識した場合の酵素と同様の酵素を用いることができる。 The quantification of the phosphorylated peptide can be performed by a conventional method. For example, it is possible to quantify by an immunological method using an antibody that binds to a phosphorylated peptide.
The peptide of the present invention may be labeled with a labeling substance for the purpose of facilitating the quantification of the phosphorylated peptide. Any labeling substance can be used without particular limitation as long as it can be used in a usual immunological measurement method. For example, an enzyme, biotin, a fluorescent substance, a radioactive substance, etc. can be used alone or in combination. Examples of the enzyme include peroxidase, β-D-galactosidase, alkaline phosphatase, and microperoxidase. Examples of the fluorescent substance include fluorescein isothiocyanate, phycobiliprotein, and phycoerythrin. Examples of the luminescent substance include isolucinol and lucigenin. Examples of radioactive substances include 125 I, 131 I, 14 C, and 3 H. Further, when biotin is used as a labeling substance, it is preferable in that the amount of labeled biotin can be measured with high sensitivity by using enzyme-labeled avidin. As the enzyme of the enzyme-labeled avidin, the same enzyme as that used when the antibody is labeled can be used.
  また、リン酸化部位に結合する試薬を利用して間接的にリン酸化を検出することも可能であり、この方法がより好ましい。リン酸化された部位に結合する試薬としては、抗リン酸化セリン/スレオニン抗体、抗リン酸化チロシン抗体、フォスタグ(Phos-tag）がある。フォスタグは、亜鉛の２核錯体であり、リン酸基そのものに結合する。これらの試薬を用いる場合において、抗体を用いるときは、抗体そのもの、あるいは２次抗体を上述と同様の検出基で標識したものを利用する。フォスタグでは、分子内にビオチン基を有しており、アビジンおよびその類縁体、あるいは抗ビオチン抗体を上述したものと同様の検出基を標識して、ビオチン部位に結合させることによって検出に供する。 It is also possible to detect phosphorylation indirectly using a reagent that binds to a phosphorylation site, and this method is more preferred. Examples of the reagent that binds to the phosphorylated site include an anti-phosphorylated serine / threonine antibody, an anti-phosphorylated tyrosine antibody, and a Phos-tag. The phos tag is a dinuclear complex of zinc and binds to the phosphate group itself. When using these reagents, when using an antibody, the antibody itself or a secondary antibody labeled with the same detection group as described above is used. Phostag has a biotin group in the molecule, and avidin and its analogs, or anti-biotin antibody is labeled with a detection group similar to that described above, and is used for detection by binding to the biotin site.
  また、本発明の基質ペプチド群は他のあらゆるキナーゼ活性評価法にも利用可能である。例えば、放射性の32Pを有するATPを利用したオートラジオグラフィーなどの放射化分析、あるいは、表面が負に帯電した金コロイドの凝集状態変化を利用したアッセイなどに利用できる。金コロイドを用いるアッセイでは、金コロイド表面の負荷電と、正荷電を有する基質ペプチドの静電相互作用による凝集が、ペプチドのリン酸化に伴うペプチド側の正荷電の減少による凝集能の低下により抑制されることに伴う色調の変化を利用する。
  本発明のペプチドは、測定用途で許容される担体又は賦形剤等の任意の成分と組み合わせて、EGFR、MET又はALK活性の測定用試薬として提供されてもよい。また、当該測定用試薬は、検出用抗体などと組み合わせて測定用キットとして提供することもできる。 In addition, the substrate peptide group of the present invention can be used in any other method for evaluating kinase activity. For example, it can be used for activation analysis such as autoradiography using ATP having radioactive 32 P, or an assay utilizing change in the aggregation state of gold colloid whose surface is negatively charged. In the assay using colloidal gold, negative charge on the colloidal gold surface and aggregation due to electrostatic interaction of the positively charged substrate peptide are suppressed by a decrease in aggregation ability due to a decrease in positive charge on the peptide side accompanying peptide phosphorylation. Use the color change that accompanies it.
The peptide of the present invention may be provided as a reagent for measuring EGFR, MET, or ALK activity in combination with any component such as a carrier or excipient that is acceptable for measurement applications. The measurement reagent can also be provided as a measurement kit in combination with a detection antibody or the like.
  タンパク質キナーゼの活性を測定するには、本発明のペプチドが基板上に固定化されたペプチドアレイを使用することができる。この場合には、ガラス、ポリジメチルシロキサン、金などの基板上に、化学修飾を介してペプチドを固定化する。基板の形状は、平板なもの、ピラーを並べたもの、ウェルを並べたものなど、これまでに報告されたいずれの基板にも適用可能である。これらの基板上にガラスではシランカップリング剤や、表面を多糖などでコートすることにより、ポリジメチルシロキサンでは、表面を多糖などでコートしたり、表面をプラズマなどで修飾することで、また、金ではチオール誘導体で修飾することにより、アミノ基、カルボキシル基などの官能基を導入する。これに架橋剤などを用いたり、末端カルボシキシル基を活性エステル化したり、ホルミル基やマレイミド基を導入後、ペプチド末端をシステイン残基として反応させることにより基質ペプチドを固定化することができる。このようにして多種類の基質を固定化したアレイを、酵素溶液や細胞破砕液で処理して、表面に固定化した基質をリン酸化して、抗リン酸化セリン・スレオニン抗体や抗リン酸化チロシン抗体、フォスタグなどをはじめとするリン酸基と結合する試薬を結合させてから、前述したものと同様の検出手段により、多くの基質のリン酸化の程度を同時に評価することが可能である。 In order to measure the activity of the protein kinase, a peptide array in which the peptide of the present invention is immobilized on a substrate can be used. In this case, the peptide is immobilized on a substrate such as glass, polydimethylsiloxane, or gold through chemical modification. The shape of the substrate can be applied to any of the substrates reported so far, such as a flat plate, a pillar array, and a well array. On these substrates, glass is coated with a silane coupling agent, or the surface is coated with a polysaccharide, and polydimethylsiloxane is coated with a surface such as a polysaccharide, or the surface is modified with plasma or the like. Then, functional groups such as amino groups and carboxyl groups are introduced by modification with thiol derivatives. The substrate peptide can be immobilized by using a cross-linking agent or the like for this, converting the terminal carboxy group into an active ester, or introducing a formyl group or a maleimide group and then reacting the peptide terminal as a cysteine residue. In this way, an array on which various types of substrates are immobilized is treated with an enzyme solution or a cell lysate to phosphorylate the substrate immobilized on the surface, thereby antiphosphorylated serine / threonine antibody or antiphosphorylated tyrosine. It is possible to simultaneously evaluate the degree of phosphorylation of many substrates by the same detection means as described above after binding a reagent that binds to a phosphate group such as an antibody or phostag.
４．タンパク質キナーゼ活性に関連する疾患の検出方法
  本発明のペプチドを用いて、哺乳動物（例えばヒト、ラット、ハムスター、ウサギ、ネコ、イヌ、ウシ、ウマ）から単離された成分（例えば血液、尿、哺乳動物の任意の組織(脳、肝臓、心臓、筋肉、皮膚など)の溶解産物（lysate）、哺乳動物の正常または癌培養細胞の溶解産物、および哺乳動物に発生した癌組織の溶解産物）を含む試料中のタンパク質キナーゼ活性を測定することにより、タンパク質キナーゼ活性に関連する疾患を診断することができる。
  「EGFR活性に関連する疾患」とは、罹患患者と健常者との間で上記試料中のEGFR活性が相違するような疾患を指す。このような疾患としては、例えば腎ガン、非小細胞肺癌、前立腺癌、頭頸部癌、卵巣癌、胃癌、大腸癌、乳癌、脳腫瘍（グリオーマ）、前立腺癌、口腔癌等が挙げられるがこれらには限定されるものではない。 4). Methods for detecting diseases associated with protein kinase activity Components (eg, blood, urine, isolated from mammals (eg, humans, rats, hamsters, rabbits, cats, dogs, cows, horses) using the peptides of the present invention. Lysate from any mammalian tissue (brain, liver, heart, muscle, skin, etc.), mammalian normal or cancer cell culture lysates, and cancer tissue lysates that arise in mammals By measuring the protein kinase activity in the containing sample, a disease associated with the protein kinase activity can be diagnosed.
“Disease related to EGFR activity” refers to a disease in which the EGFR activity in the sample differs between the affected patient and a healthy subject. Examples of such diseases include kidney cancer, non-small cell lung cancer, prostate cancer, head and neck cancer, ovarian cancer, stomach cancer, colon cancer, breast cancer, brain tumor (glioma), prostate cancer, oral cancer and the like. Is not limited.
  「MET活性に関連する疾患」とは、罹患患者と健常者との間で上記試料中のMET活性が相違するような疾患を指す。このような疾患としては、例えば結腸癌、腎ガン、非小細胞肺癌、前立腺癌、頭頸部癌、卵巣癌、胃癌、大腸癌、乳癌、脳腫瘍（グリオーマ）、前立腺癌、口腔癌等が挙げられるがこれらには限定されるものではない。
  「ALK活性に関連する疾患」とは、罹患患者と健常者との間で上記試料中のALK活性が相違するような疾患を指す。このような疾患としては、例えば腎ガン、非小細胞肺癌、前立腺癌、頭頸部癌、卵巣癌、胃癌、大腸癌、乳癌、脳腫瘍（グリオーマ）、神経芽腫、前立腺癌、口腔癌等が挙げられるがこれらには限定されるものではない。 “Disease related to MET activity” refers to a disease in which the MET activity in the sample differs between affected patients and healthy subjects. Examples of such diseases include colon cancer, renal cancer, non-small cell lung cancer, prostate cancer, head and neck cancer, ovarian cancer, gastric cancer, colon cancer, breast cancer, brain tumor (glioma), prostate cancer, oral cancer and the like. However, it is not limited to these.
The “disease related to ALK activity” refers to a disease in which ALK activity in the sample is different between affected patients and healthy subjects. Examples of such diseases include kidney cancer, non-small cell lung cancer, prostate cancer, head and neck cancer, ovarian cancer, stomach cancer, colon cancer, breast cancer, brain tumor (glioma), neuroblastoma, prostate cancer, oral cancer and the like. However, it is not limited to these.
  本発明の一つの態様として、EGFR活性が患者由来試料と健常者由来試料との間で相違したときは、当該患者の疾患は、EGFR活性に関連する疾患の可能性があることの指標となる。また、MET活性が患者由来試料と健常者由来試料との間で相違したときは、当該患者の疾患は、MET活性に関連する疾患の可能性があることの指標となる。そして、ALK活性が患者由来試料と健常者由来試料との間で相違したときは、当該患者の疾患は、ALK活性に関連する疾患の可能性があることの指標となる。
  本発明の診断方法は、投薬前診断、術中診断、臨床診断等の種々の診断に適用可能である。本発明のペプチドを用いたタンパク質キナーゼ活性測定方法については、前記と同様である。 As one aspect of the present invention, when the EGFR activity is different between the patient-derived sample and the healthy subject-derived sample, the disease of the patient is an indicator that there is a possibility of a disease related to EGFR activity. . Further, when the MET activity differs between the patient-derived sample and the healthy subject-derived sample, the disease of the patient is an indicator that there is a possibility of a disease related to the MET activity. When the ALK activity differs between the patient-derived sample and the healthy subject-derived sample, the disease of the patient is an indicator that there is a possibility of a disease related to the ALK activity.
The diagnostic method of the present invention can be applied to various diagnoses such as pre-medication diagnosis, intraoperative diagnosis, and clinical diagnosis. The method for measuring protein kinase activity using the peptide of the present invention is the same as described above.
  本発明のペプチドは、診断用途で許容される担体又は賦形剤等の任意の成分と組み合わせてタンパク質キナーゼ活性に関連する疾患の診断薬として提供することができる。また、当該診断薬は、検出用抗体などと組み合わせて診断用キットとして提供されてもよい。本発明の各種タンパク質キナーゼ活性検出用ペプチドアレイは、タンパク質キナーゼ活性に関連する上記疾患の診断の用途に使用することができる。 The peptide of the present invention can be provided as a diagnostic agent for diseases associated with protein kinase activity in combination with any component such as a carrier or excipient that is acceptable for diagnostic use. The diagnostic agent may be provided as a diagnostic kit in combination with a detection antibody or the like. The peptide arrays for detecting various protein kinase activities of the present invention can be used for diagnosis of the above-mentioned diseases related to protein kinase activity.
５．核酸
  本発明はまた、上記の基質ペプチドをコードする核酸に関する。核酸としてはDNAまたはRNAが挙げられ、DNAが好ましい。当該核酸をベクターに導入し、生体内において本発明の基質ペプチドを発現させることが可能である。また、当該核酸と、マーカー分子（例えば抗原抗体反応を使って検出可能なマーカー分子）をコードする核酸とを融合した核酸配列をベクターに組み込み、生体内において本発明の基質ペプチドとマーカー分子との融合ペプチドを発現させることにより、生体内のキナーゼ活性をインビボで解析することができるものと期待される。 5. Nucleic acid The present invention also relates to a nucleic acid encoding the above substrate peptide. Examples of the nucleic acid include DNA or RNA, and DNA is preferable. The nucleic acid can be introduced into a vector and the substrate peptide of the present invention can be expressed in vivo. In addition, a nucleic acid sequence fused with the nucleic acid and a nucleic acid encoding a marker molecule (for example, a marker molecule detectable using an antigen-antibody reaction) is incorporated into a vector, and the substrate peptide of the present invention and the marker molecule are incorporated in vivo. By expressing the fusion peptide, it is expected that in vivo kinase activity can be analyzed in vivo.
　以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれら実施例によって何ら限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  設計した基質をJPTpeptide technologies社にて委託合成し、これをN端でガラス基板へ化学結合修飾した。
　そして、各種のキナーゼ反応溶液[ATP(+)およびATP(-)]をそれぞれ300 μLずつ基板上に添加して、３７℃で2時間インキュベートしてリン酸化反応を行った。反応後、10×TBS溶液に浸漬して５分間洗浄するという操作を5回行った。そして、Blocking One-PをTBST溶液で5倍希釈した溶液を基板に室温で1時間作用し、ブロッキング操作を行った。その後、10μg/mL phos-tag/phos-tag buffer溶液を基板上に1000μL添加して、室温で1時間インキュベートした。インキュベート後、基板を超純水に浸漬した。続いて、2μg/mL Dylight647-streptavidin/Streptavidin buffer (Blocking One-Pを10 mM HEPES (pH 7.3)で20倍希釈した溶液)を基板上に1000μL添加して、室温で20分インキュベートした。インキュベート後、基板をTBS-T溶液に浸漬し２分間洗浄するという操作を5回行って、よく乾燥させた後、Scan Array Lite (PerkinElmer製)により蛍光イメージングを行った。 The designed substrate was commissioned and synthesized by JPTpeptide technologies, and this was chemically bonded to the glass substrate at the N-terminus.
Then, 300 μL of each kinase reaction solution [ATP (+) and ATP (−)] was added on the substrate and incubated at 37 ° C. for 2 hours for phosphorylation. After the reaction, the operation of immersing in 10 × TBS solution and washing for 5 minutes was performed 5 times. Then, a blocking operation was performed by applying a solution obtained by diluting Blocking One-P 5 times with TBST solution to the substrate at room temperature for 1 hour. Thereafter, 1000 μL of 10 μg / mL phos-tag / phos-tag buffer solution was added onto the substrate and incubated at room temperature for 1 hour. After incubation, the substrate was immersed in ultrapure water. Subsequently, 1000 μL of 2 μg / mL Dylight647-streptavidin / Streptavidin buffer (a solution obtained by diluting Blocking One-P with 10 mM HEPES (pH 7.3) 20-fold) was added to the substrate and incubated at room temperature for 20 minutes. After the incubation, the substrate was immersed in a TBS-T solution and washed for 2 minutes, and the substrate was thoroughly dried, followed by fluorescent imaging with Scan Array Lite (PerkinElmer).
　次に、それぞれの基質ペプチドについて、ATP(+)から得られた蛍光強度からATP(-)から得られた蛍光強度を差し引いた値を算出した。それぞれのキナーゼについて２回ずつ実験を行い、この値の平均値が一定の基準以上を示したものを選出し、そのキナーゼの良好な基質として選定した。
  以上の結果、EGFR、MET及びALKに対する基質ペプチド（配列番号1～48、57～72）を決定した（表１～３）。 Next, for each substrate peptide, a value obtained by subtracting the fluorescence intensity obtained from ATP (−) from the fluorescence intensity obtained from ATP (+) was calculated. Experiments were performed twice for each kinase, and those whose average value was above a certain standard were selected and selected as good substrates for the kinase.
As a result, substrate peptides (SEQ ID NOs: 1-48, 57-72) for EGFR, MET, and ALK were determined (Tables 1-3).
  培養したHCC827（ヒト肺がん細胞）を無血清培地で18時間インキュベートした。その後、D-PBS(-)で洗浄し、セルスクレーパーを用いて細胞を剥がし取り、細胞を回収した。遠心後、上清を除去した。そこへLysis bufferを加え、超音波処理を行った。超遠心後、上清（細胞質成分）を回収した。残ったペレットに界面活性剤を含むLysis bufferを加え、超音波処理を行った。超遠心後、上清として細胞膜成分を回収した。
　続いてペプチドアレイの作製を行った。次に、基質ペプチド（ペプチド番号109：配列番号25）とTCEPが濃度0.1 mMとなるように50 mM炭酸バッファー（pH9.5）中で混合した溶液をGenex Arrayer（カケンジェネックス製、ピン先直径150μm）にてマレイミド修飾GC基板にスポットして、室温で一晩インキュベートし、ペプチドを固定化した。インキュベート後、基板をTBS-T溶液に浸漬し、3分間洗浄するという操作を3回行い、未反応ペプチドを除去した。また、対照として既知のEGFR基質ペプチドであるEGFR pep（CGGEQEDEPEDYFEWLEP（配列番号73））を用いた。 Cultured HCC827 (human lung cancer cells) was incubated in serum-free medium for 18 hours. Thereafter, the cells were washed with D-PBS (−), and the cells were peeled off using a cell scraper, and the cells were collected. After centrifugation, the supernatant was removed. Lysis buffer was added thereto and sonication was performed. After ultracentrifugation, the supernatant (cytoplasmic component) was collected. Lysis buffer containing a surfactant was added to the remaining pellets, and sonication was performed. After ultracentrifugation, cell membrane components were collected as the supernatant.
Subsequently, a peptide array was prepared. Next, a solution prepared by mixing a substrate peptide (peptide No. 109: SEQ ID No. 25) and TCEP in 50 mM carbonate buffer (pH 9.5) so as to have a concentration of 0.1 mM is Genex Arrayer (manufactured by Kaken Genex, pin tip diameter 150 μm) ) Was spotted on a maleimide-modified GC substrate and incubated overnight at room temperature to immobilize the peptide. After incubation, the substrate was immersed in a TBS-T solution and washed for 3 minutes three times to remove unreacted peptides. As a control, EGFR pep (CGGEQEDEPEDYFEWLEP (SEQ ID NO: 73)), which is a known EGFR substrate peptide, was used.
　続いて、HCC827ライセートの細胞膜成分100μg/mL、5 mM MgCl2および0.1 mM ATPを含む50 mM HEPESを、無吸着ハイブリカバーを用いて基板上に添加し、37℃で2時間インキュベートした。その後、TBS-Tで8分間浸漬洗浄を3回行い、超純水で洗浄後、窒素ガスで乾燥させた。続いて、2μg/mLの蛍光修飾抗リン酸化チロシン抗体を作用させ、37℃で20分間インキュベートした。TBS-Tで2分間浸漬洗浄を2回行い、超純水で洗浄後、窒素ガスで乾燥させた。Scan Array Lite (PerkinElmer製)により蛍光イメージングを行った。 Subsequently, 50 mM HEPES containing 100 μg / mL of the cell membrane component of HCC827 lysate, 5 mM MgCl 2 and 0.1 mM ATP was added onto the substrate using a non-adsorbed hybrid cover, and incubated at 37 ° C. for 2 hours. Thereafter, the substrate was dipped and washed with TBS-T for 8 minutes three times, washed with ultrapure water, and then dried with nitrogen gas. Subsequently, 2 μg / mL of a fluorescence-modified antiphosphorylated tyrosine antibody was allowed to act and incubated at 37 ° C. for 20 minutes. Immersion cleaning was performed twice for 2 minutes with TBS-T, and after cleaning with ultrapure water, it was dried with nitrogen gas. Fluorescence imaging was performed using Scan Array Lite (PerkinElmer).
  その結果、HCC827ライセートでのキナーゼ活性の検出をすることができた（図１）。
  図１は、各ペプチドのHCC827ライセートによるリン酸化を示すものであり、本発明のペプチドにより、EGFRが高活性化していることで知られるHCC827細胞中の酵素活性を評価することができた。 As a result, it was possible to detect kinase activity in HCC827 lysate (FIG. 1).
FIG. 1 shows the phosphorylation of each peptide by HCC827 lysate, and the enzyme activity in HCC827 cells known to be highly activated by EGFR could be evaluated by the peptide of the present invention.
　培養したHCC827（ヒト肺がん細胞）を無血清培地で18時間インキュベートした。次に、培地を破棄し、EGFRの阻害剤であるIressa（ゲフィチニブ、Astra Zeneca)を加えた無血清培地に交換し、2時間インキュベートした。Iressaが含まれる無血清培地を破棄し、D-PBS(-)で洗浄した。その後、D-PBS(-)を加えてセルスクレーパーを用いて細胞を剥がし取り、細胞を回収した。遠心後、上清を除去した。そこへLysis bufferを加え、超音波処理を行った。超遠心後、上清（細胞質成分）を回収した。さらに界面活性剤を含むLysis bufferを加え、超音波処理を行った。超遠心後、上清として細胞膜成分を回収した。
　ペプチドアレイの作製およびアレイを用いた酵素活性の評価は実施例２と同じ手順で行った。 Cultured HCC827 (human lung cancer cells) was incubated in serum-free medium for 18 hours. Next, the medium was discarded and replaced with a serum-free medium supplemented with Iressa (Gefitinib, Astra Zeneca), an EGFR inhibitor, and incubated for 2 hours. The serum-free medium containing Iressa was discarded and washed with D-PBS (−). Thereafter, D-PBS (-) was added, and the cells were peeled off using a cell scraper, and the cells were collected. After centrifugation, the supernatant was removed. Lysis buffer was added thereto and sonication was performed. After ultracentrifugation, the supernatant (cytoplasmic component) was collected. Furthermore, a lysis buffer containing a surfactant was added and sonication was performed. After ultracentrifugation, cell membrane components were collected as the supernatant.
Preparation of a peptide array and evaluation of enzyme activity using the array were performed in the same procedure as in Example 2.
  その結果、IressaによるEGFR基質のリン酸化率低下を検出することができた（図２）。
  図２は、HCC827をEGFRの阻害剤であるIressaで処理した場合の各ペプチドのリン酸化率を示すものであり、本発明のペプチド（ペプチド番号109：配列番号25）により、Iressaの阻害活性の評価が可能であることを示された。 As a result, it was possible to detect a decrease in the phosphorylation rate of the EGFR substrate by Iressa (FIG. 2).
FIG. 2 shows the phosphorylation rate of each peptide when HCC827 is treated with Iressa, an inhibitor of EGFR, and the inhibitory activity of Iressa is increased by the peptide of the present invention (peptide number 109: SEQ ID NO: 25). It was shown that evaluation is possible.
  本発明により、EGFR、MET及びALKに対する基質ペプチドが提供される。本発明の基質ペプチドは、リン酸化酵素活性測定キット、疾病診断キット、投薬前診断キット、リン酸化酵素阻害剤のスクリーニング方法などに利用される。 According to the present invention, substrate peptides for EGFR, MET and ALK are provided. The substrate peptide of the present invention is used in a phosphorylase activity measurement kit, a disease diagnostic kit, a pre-medication diagnostic kit, a screening method for a phosphorylase inhibitor, and the like.
配列番号1～73：合成ペプチド
配列番号49：XaaはAsp又はGluを表す（存在位置：1～2）
配列番号49：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：3）
配列番号49：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：6）
配列番号50：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号50：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号50：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号50：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号51：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号51：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号51：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号51：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号52：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号52：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号52：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号52：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号53：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号53：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号53：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号53：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号54：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号54：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号54：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号54：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号55：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2）
配列番号55：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号55：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号55：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10）
配列番号56：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：1～2２）
配列番号56：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：5）
配列番号56：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：8）
配列番号56：Xaaは天然に存在する任意のアミノ酸を表す（存在位置：10） SEQ ID NO: 1-73: Synthetic peptide SEQ ID NO: 49: Xaa represents Asp or Glu (location: 1-2)
SEQ ID NO: 49: Xaa represents any naturally occurring amino acid (location: 3)
SEQ ID NO: 49: Xaa represents any naturally occurring amino acid (location: 6)
SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 50: Xaa represents a naturally occurring arbitrary amino acid (location: 5)
SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 50: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 51: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 52: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 52: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 52: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 52: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 53: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 53: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 53: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 53: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 54: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 54: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 54: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 54: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 55: Xaa represents any naturally occurring amino acid (location: 1-2)
SEQ ID NO: 55: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 55: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 55: Xaa represents any naturally occurring amino acid (location: 10)
SEQ ID NO: 56: Xaa represents any naturally occurring amino acid (location: 1-22)
SEQ ID NO: 56: Xaa represents any naturally occurring amino acid (location: 5)
SEQ ID NO: 56: Xaa represents any naturally occurring amino acid (location: 8)
SEQ ID NO: 56: Xaa represents any naturally occurring amino acid (location: 10)

Claims (24)

  1. 　以下の(a)又は(b)のペプチド。
      (a) 配列番号１～２６で表されるいずれかのアミノ酸配列からなるペプチド
      (b) 配列番号１～２６で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、上皮細胞成長因子受容体によりリン酸化されるペプチド     The following peptide (a) or (b).
    (a) a peptide consisting of any one of the amino acid sequences represented by SEQ ID NOs: 1 to 26 (b) one or several amino acids deleted or substituted in any amino acid sequence represented by SEQ ID NOs: 1 to 26 A peptide comprising an added or inserted amino acid sequence and phosphorylated by an epidermal growth factor receptor
  2. 　標識物質により標識化されている請求項1に記載のペプチド。 2. The peptide according to claim 1, which is labeled with a labeling substance.
  3. 　上皮細胞成長因子受容体活性の測定方法であって、
    (a) 請求項１又は２に記載のペプチドと被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for measuring epidermal growth factor receptor activity comprising:
    (a) reacting the peptide according to claim 1 or 2 with a test sample;
    (b) quantifying the peptide phosphorylated in step (a).
  4. 　上皮細胞成長因子受容体活性に関連する疾患の検出方法であって、
    (a) 請求項１又は２に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for detecting a disease associated with epidermal growth factor receptor activity comprising:
    (a) reacting the peptide of claim 1 or 2 with a test sample isolated from a mammal;
    (b) quantifying the peptide phosphorylated in step (a).
  5. 　請求項1又は2に記載のペプチドを含む、上皮細胞成長因子受容体活性の測定用試薬。 A reagent for measuring epidermal growth factor receptor activity, comprising the peptide according to claim 1 or 2.
  6. 　請求項1又は2に記載のペプチドを含む、上皮細胞成長因子受容体活性に関連する疾患の診断薬。 A diagnostic agent for a disease associated with epidermal growth factor receptor activity, comprising the peptide according to claim 1 or 2.
  7. 　基板上に請求項1又は2記載のペプチドが固定化された、上皮細胞成長因子受容体活性検出用ペプチドアレイ。 A peptide array for detecting epidermal growth factor receptor activity, wherein the peptide according to claim 1 or 2 is immobilized on a substrate.
  8. 　請求項1に記載のペプチドをコードする核酸。 A nucleic acid encoding the peptide according to claim 1.
  9. 　以下の(a)又は(b)のペプチド。
      (a) 配列番号２７～５６で表されるいずれかのアミノ酸配列からなるペプチド
      (b) 配列番号２７～５６で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、MET受容体チロシンキナーゼによりリン酸化されるペプチド     The following peptide (a) or (b).
    (a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 27 to 56 (b) one or several amino acids deleted or substituted in any one of the amino acid sequences represented by SEQ ID NOs: 27 to 56 A peptide consisting of an added or inserted amino acid sequence and phosphorylated by a MET receptor tyrosine kinase
  10. 　標識物質により標識化されている請求項９に記載のペプチド。 The peptide according to claim 9, which is labeled with a labeling substance.
  11. 　MET受容体チロシンキナーゼ活性の測定方法であって、
    (a) 請求項９又は１０に記載のペプチドと被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for measuring MET receptor tyrosine kinase activity, comprising:
    (a) reacting the peptide of claim 9 or 10 with a test sample;
    (b) quantifying the peptide phosphorylated in step (a).
  12. 　MET受容体チロシンキナーゼ活性に関連する疾患の検出方法であって、
    (a) 請求項９又は１０に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for detecting a disease associated with MET receptor tyrosine kinase activity, comprising:
    (a) reacting the peptide of claim 9 or 10 with a test sample isolated from a mammal;
    (b) quantifying the peptide phosphorylated in step (a).
  13. 　請求項９又は１０に記載のペプチドを含む、MET受容体チロシンキナーゼ活性の測定用試薬。 A reagent for measuring MET receptor tyrosine kinase activity, comprising the peptide according to claim 9 or 10.
  14. 　請求項９又は１０に記載のペプチドを含む、MET受容体チロシンキナーゼ活性に関連する疾患の診断薬。 A diagnostic agent for a disease associated with MET receptor tyrosine kinase activity, comprising the peptide according to claim 9 or 10.
  15. 　基板上に請求項９又は１０に記載のペプチドが固定化された、MET受容体チロシンキナーゼ活性検出用ペプチドアレイ。 A peptide array for detecting MET receptor tyrosine kinase activity, wherein the peptide according to claim 9 or 10 is immobilized on a substrate.
  16. 　請求項９に記載のペプチドをコードする核酸。 A nucleic acid encoding the peptide according to claim 9.
  17. 　以下の(a)又は(b)のペプチド。
      (a) 配列番号５７～７２で表されるいずれかのアミノ酸配列からなるペプチド
      (b) 配列番号５７～７２で表されるいずれかのアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換、付加若しくは挿入されたアミノ酸配列からなり、かつ、未分化リンパ腫キナーゼによりリン酸化されるペプチド     The following peptide (a) or (b).
    (a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 57 to 72 (b) one or several amino acids deleted or substituted in any amino acid sequence represented by SEQ ID NOs: 57 to 72 A peptide comprising an added or inserted amino acid sequence and phosphorylated by anaplastic lymphoma kinase
  18. 　標識物質により標識化されている請求項1７に記載のペプチド。 The peptide according to claim 17, which is labeled with a labeling substance.
  19. 　未分化リンパ腫キナーゼ活性の測定方法であって、
    (a) 請求項１０又は１１に記載のペプチドと被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for measuring anaplastic lymphoma kinase activity, comprising:
    (a) reacting the peptide according to claim 10 or 11 with a test sample;
    (b) quantifying the peptide phosphorylated in step (a).
  20. 　未分化リンパ腫キナーゼ活性に関連する疾患の検出方法であって、
    (a) 請求項１７又は１８に記載のペプチドと、哺乳動物から単離された被験試料とを反応させる工程と、
    (b) 工程(a)でリン酸化されたペプチドを定量する工程と
    を含む前記方法。     A method for detecting a disease associated with anaplastic lymphoma kinase activity comprising:
    (a) reacting the peptide of claim 17 or 18 with a test sample isolated from a mammal;
    (b) quantifying the peptide phosphorylated in step (a).
  21. 　請求項１７又は１８に記載のペプチドを含む、未分化リンパ腫キナーゼ活性の測定用試薬。 A reagent for measuring anaplastic lymphoma kinase activity, comprising the peptide according to claim 17 or 18.
  22. 　請求項１７又は１８に記載のペプチドを含む、未分化リンパ腫キナーゼ活性に関連する疾患の診断薬。 A diagnostic agent for a disease associated with anaplastic lymphoma kinase activity, comprising the peptide according to claim 17 or 18.
  23. 　基板上に請求項１７又は１８に記載のペプチドが固定化された、未分化リンパ腫キナーゼ活性検出用ペプチドアレイ。 A peptide array for detecting anaplastic lymphoma kinase activity, wherein the peptide according to claim 17 or 18 is immobilized on a substrate.
  24. 　請求項１７に記載のペプチドをコードする核酸。 A nucleic acid encoding the peptide according to claim 17.
PCT/JP2011/063699 2010-06-16 2011-06-15 Novel substrate peptide of protein kinase WO2011158863A1 (en)

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JP2008515783A (en) * 2004-09-15 2008-05-15 プロトメトリックス インコーポレイティッド Protein array and method of use thereof
JP2008289374A (en) * 2007-05-22 2008-12-04 Kyushu Univ New substrate polypeptide for protein kinase
WO2009012140A2 (en) * 2007-07-13 2009-01-22 Prometheus Laboratories, Inc. Drug selection for lung cancer therapy using antibody-based arrays

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JP2008515783A (en) * 2004-09-15 2008-05-15 プロトメトリックス インコーポレイティッド Protein array and method of use thereof
JP2008289374A (en) * 2007-05-22 2008-12-04 Kyushu Univ New substrate polypeptide for protein kinase
WO2009012140A2 (en) * 2007-07-13 2009-01-22 Prometheus Laboratories, Inc. Drug selection for lung cancer therapy using antibody-based arrays

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