WO2017061288A1 - チロシンホスファターゼ及びチロシンキナーゼ活性の測定方法 - Google Patents
チロシンホスファターゼ及びチロシンキナーゼ活性の測定方法 Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
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- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
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- 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
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- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Definitions
- the present invention relates to a method for measuring tyrosine phosphatase and tyrosine kinase activity.
- the present invention also provides a measurement kit used in this measurement method, a tyrosine phosphatase inhibitor or activator and a tyrosine kinase inhibitor or activator screening method using this measurement method, and a diagnosis using this measurement method.
- drugs Regarding drugs.
- Tyrosine kinase is an enzyme that phosphorylates tyrosine residues of proteins, while tyrosine phosphatase is an enzyme that catalyzes dephosphorylation using phosphorylated tyrosine residues as substrates.
- Tyrosine kinases and tyrosine phosphatases play an important role in vivo, and abnormal expression of these enzymes often causes diseases.
- EGFR epidermatitisine kinase
- PTP1B a tyrosine phosphatase
- Non-patent document 1 a method using 32 P radioisotope
- Non-patent document 2 a method using anti-phosphorylated antibody
- Non-patent document 3 A method using anti-phosphorylated antibody
- Non-Patent Document 3 A method using anti-phosphorylated antibody
- Non-Patent Document 3 A method using anti-phosphorylated antibody
- Non-Patent Document 3 A method using anti-phosphorylated antibody
- Non-Patent Document 3 is known.
- Non-Patent Document 3 there are not many known methods for measuring tyrosine phosphatase activity. Commonly used methods include quantifying inorganic phosphate using malachite green, and then estimating the activity (Non-patent Document 4) and phosphorylated tyrosine mimics and tyrosine phosphatases such as p-nitrophenyl phosphate (pNPP).
- pNPP p-nitrophenyl phosphate
- Non-patent Document 5 for determining the amount of dephosphoryl
- tyrosine phosphatase both of the above-mentioned two methods have great problems. Since the method using malachite green quantifies inorganic phosphoric acid, there is a problem that it cannot be used under conditions where inorganic phosphoric acid is present (for example, in a buffer containing phosphoric acid). Not satisfactory from the point of view.
- the method using phosphorylated tyrosine mimic has a problem that phosphorylated tyrosine mimic reacts with phosphatase in a non-selective manner, and cannot be used under conditions where phosphatase other than tyrosine phosphatase to be measured exists.
- a method for measuring tyrosine phosphatase activity comprising the following steps: (1) reacting a tyrosine phosphatase to be measured with a peptide containing a phosphorylated tyrosine residue and dephosphorylating the phosphorylated tyrosine residue; (2) The following general formula (I) is applied to the dephosphorylated tyrosine residue in the presence of an oxidizing agent and a metal catalyst.
- A represents a conjugated ring
- L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal
- R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- a step of binding a compound represented by: (3) A step of measuring the amount of the compound represented by the general formula (I) bound to the peptide and determining tyrosine phosphatase activity from the amount.
- Fluorescent substance is bound to a peptide containing a phosphorylated tyrosine residue and represented by the general formula (I) bound to the peptide using a carrier that specifically binds to the compound represented by the general formula (I).
- the tyrosine phosphatase activity according to [1] or [2], wherein the amount of the compound represented by the general formula (I) bound to the peptide by the fluorescent substance is measured Method.
- a fluorescent substance is bound to a peptide containing a phosphorylated tyrosine residue, and a fluorescent substance that forms a FRET pair with the fluorescent substance or a quencher for the fluorescent substance is bound to the compound represented by the general formula (I)
- the amount of the compound represented by the general formula (I) bound to the peptide by the change in fluorescence of the fluorescent material bound to the peptide and / or the fluorescent material bound to the compound represented by the general formula (I) The method for measuring tyrosine phosphatase activity according to [1] or [2], wherein the tyrosine phosphatase activity is measured.
- a kit for measuring tyrosine phosphatase activity comprising a compound represented by:
- a screening method for a tyrosine phosphatase inhibitor or activator comprising the following steps: (1) contacting a peptide containing tyrosine phosphatase and a phosphorylated tyrosine residue in the presence of a test substance; (2) A peptide containing a phosphorylated tyrosine residue brought into contact with tyrosine phosphatase in the presence of an oxidizing agent and a metal catalyst is represented by the following general formula (I) [Wherein, A represents a conjugated ring, L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- a fluorescent substance is bound to a peptide containing a phosphorylated tyrosine residue, and a carrier that specifically binds to a compound represented by the general formula (I) is used.
- a fluorescent substance is bound to a peptide containing a phosphorylated tyrosine residue, and a fluorescent substance that forms a FRET pair with the fluorescent substance or a quencher for the fluorescent substance is bound to the compound represented by the general formula (I)
- the amount of the compound represented by the general formula (I) bound to the peptide by the change in fluorescence of the fluorescent material bound to the peptide and / or the fluorescent material bound to the compound represented by the general formula (I) The method for screening a tyrosine phosphatase inhibitor or activator according to [7] or [8], wherein
- a peptide containing a phosphorylated tyrosine residue and the following general formula (I) [Wherein, A represents a conjugated ring, L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- a diagnostic agent for diabetes comprising a compound represented by:
- a method for measuring tyrosine kinase activity comprising the following steps: (1) reacting a tyrosine kinase to be measured with a peptide containing a non-phosphorylated tyrosine residue to phosphorylate the non-phosphorylated tyrosine residue; (2) In the presence of an oxidizing agent and a metal catalyst, the following general formula (I) [Wherein, A represents a conjugated ring, L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- a fluorescent substance is bound to a peptide containing a non-phosphorylated tyrosine residue, and a carrier that specifically binds to the compound represented by the general formula (I) is used to bind the peptide in the general formula (I)
- the compound represented by the general formula (I) bound to the peptide by the fluorescent substance is isolated and the amount of the tyrosine kinase activity described in [13] or [14] is measured Measuring method.
- a fluorescent substance is bound to a peptide containing a non-phosphorylated tyrosine residue, and a fluorescent substance that forms a FRET pair with the fluorescent substance or a quencher for the fluorescent substance is attached to the compound represented by the general formula (I)
- the fluorescent substance bound to the peptide and / or the fluorescence of the fluorescent substance bound to the compound represented by the general formula (I) is changed by the change in fluorescence of the compound represented by the general formula (I) bound to the peptide.
- the method for measuring tyrosine kinase activity according to [13] or [14], wherein the amount is measured.
- a kit for measuring tyrosine kinase activity comprising a compound represented by the formula:
- a screening method for a tyrosine kinase inhibitor or activator comprising the following steps: (1) contacting a tyrosine kinase with a peptide containing a non-phosphorylated tyrosine residue in the presence of a test substance; (2) A peptide containing a non-phosphorylated tyrosine residue brought into contact with a tyrosine kinase in the presence of an oxidizing agent and a metal catalyst is represented by the following general formula (I) [Wherein, A represents a conjugated ring, L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- R 2 and R 3 each have a hydrogen atom, an alkyl group, and an aromatic group that may have a substituent. Represents a group.
- a fluorescent substance is bound to a peptide containing a non-phosphorylated tyrosine residue, and a fluorescent substance that forms a FRET pair with the fluorescent substance or a quencher for the fluorescent substance is added to the compound represented by the general formula (I)
- the fluorescent substance bound to the peptide and / or the fluorescence of the fluorescent substance bound to the compound represented by the general formula (I) is changed by the change in fluorescence of the compound represented by the general formula (I) bound to the peptide.
- the method for screening a tyrosine kinase inhibitor or activator according to [19] or [20], wherein the amount is measured.
- the measurement method of the present invention can measure the activity of tyrosine phosphatase and tyrosine kinase with high accuracy.
- Tyrosine phosphatases and tyrosine kinases are thought to be involved in various diseases and are important targets for their therapeutic agents. For this reason, the measurement method of the present invention is useful for the development of a medicine targeting these enzymes.
- FIG. 3 is a diagram schematically illustrating a method for measuring the amount of a compound represented by the general formula (I) using FRET or a fluorescence quencher. Confirmation of tyrosine phosphatase and tyrosine kinase activity by mass spectrometry.
- (A) shows the dephosphorylation of pY-RR-Src by tyrosine phosphatase PTP1B
- (B) shows the phosphorylation of RR-Src by tyrosine kinase ErbB1.
- the upper row shows the MS chart of RR-Src only (same as control in Fig. 3), and the middle row shows the MS chart of the mixed solution of N 3 compound and RR-Src (same as N 3 comp. 10 eq in Fig. 3).
- the lower part shows an MS chart of a solution obtained by adding DBCO-biotin to a mixed solution of N 3 compound and RR-Src. Sensitive detection of chemically modified peptides by fluorescent labeling. Inorganic phosphate detection limit of Malachite Green method (Promega kit).
- the “alkyl group having 1 to 20 carbon atoms” is a linear or branched alkyl group having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an iso- Propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, neo-pentyl, hexyl, iso-hexyl, heptyl, iso-heptyl Group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group,
- the “alkyl group having 1 to 10 carbon atoms” is a linear or branched alkyl group having 1 to 10 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an iso- Propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, neo-pentyl, hexyl, iso-hexyl, heptyl, iso-heptyl Group, octyl group, nonyl group, decyl group and the like.
- the “alkyl group having 1 to 3 carbon atoms” is a linear or branched alkyl group having 1 to 3 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an iso- Such as a propyl group.
- the “alkoxy group having 1 to 20 carbon atoms” is a linear or branched alkoxy group having 1 to 20 carbon atoms, and includes, for example, a methoxy group, an ethoxy group, an n-propoxy group, an iso- Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl Oxy, iso-heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, heptadecyl An oxy group, an octa
- the “alkoxy group having 1 to 10 carbon atoms” is a linear or branched alkoxy group having 1 to 10 carbon atoms, and includes, for example, a methoxy group, an ethoxy group, an n-propoxy group, an iso- Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl An oxy group, an iso-heptyloxy group, an octyloxy group, a nonyloxy group, a decyloxy group, and the like.
- the “alkoxy group having 1 to 3 carbon atoms” is a linear or branched alkoxy group having 1 to 3 carbon atoms, and includes, for example, a methoxy group, an ethoxy group, an n-propoxy group, an iso- Such as a propoxy group.
- the “aromatic group optionally having a substituent” means an aromatic group having no substituent or an aromatic group having at least one substituent.
- the “aromatic group” refers to a group obtained by removing one hydrogen atom from an aromatic compound.
- the substituent can be selected from the group consisting of a methyl group, a fluorine atom, a chlorine atom, a bromine atom, a hydroxy group, a methoxy group, and the like.
- the aromatic group having at least one substituent include a phenyl group having at least one substituent, and specific examples thereof include a 2-methylphenyl group, 3-methylphenyl group, 4-methylphenyl group, 2,3-dimethylphenyl group, 2,4-dimethylphenyl group, 2,5-dimethylphenyl group, 2,6-dimethylphenyl group, 3,4-dimethylphenyl group Group, 3,5-dimethylphenyl group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2,3-difluorophenyl group, 2,4-difluorophenyl group, 2,5-difluorophenyl Group, 2,6-difluorophenyl group
- the “functional group used for the click reaction” is, for example, an azide group or an ethynyl group.
- conjugated ring refers to a ring having a conjugated double bond.
- the conjugated ring may be an aromatic ring or a non-aromatic ring. Further, the conjugated ring may be a ring composed of only carbon atoms or a heterocycle containing atoms other than carbon, for example, atoms such as nitrogen, oxygen, sulfur and the like.
- conjugated ring examples include a benzene ring, a 1,3-cyclohexadiene ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, a pyridazine ring, a triazine ring and the like, a cyclopentadiene ring, a furan ring, a thiophene ring, and a pyrrole.
- 5-membered rings such as a ring, a pyrazole ring, and an imidazole ring.
- the “radioisotope” means, for example, 11 C, 13 N, 15 O, 18 F, 62 Cu, 68 Ga, 76 Br, 99m Tc, 111 In, 67 Ga, 201 Tl, 123 I, 133 Xe etc. can be mentioned.
- preferred radioisotopes include 18 F.
- the “labeling substance” refers to a substance that enables the peptide to be detected by binding directly or indirectly to a peptide, for example, a fluorescent substance, a radioisotope, and a specific substance. Substances that act. Examples of fluorescent substances include fluorescein, fluorescein isothiocyanate (FITC), rhodamine and the like, and examples of substances that interact with specific substances include antigens of low molecular antibodies such as biotin and dinitrophenyl groups. And a substance having a molecular structure capable of forming a covalent bond such as Halo-Tag (registered trademark) and SNAP-tag (registered trademark).
- the “group containing a labeling substance” means, for example, a labeling substance: —X— [CH 2 CH 2 —Y] m — (CH 2 ) n —Z 1 [where X and Y are each CH 2 represents O, NH, S, NHCO or CO, Z 1 represents N 3 or CCH, and m and n each represents an integer of 0 to 12. ] Is a moiety other than —X— [CH 2 CH 2 —Y] m — (CH 2 ) n —.
- the method for measuring tyrosine phosphatase activity comprises (1) reacting a tyrosine phosphatase to be measured with a peptide containing a phosphorylated tyrosine residue, and dephosphorylating the phosphorylated tyrosine residue.
- A represents a conjugated ring
- L represents a hydrogen atom, or a linker having a functional group used for a click reaction at an arbitrary position on the conjugated ring or a linker having a labeling substance at the terminal
- R 1 represents a hydrogen atom, a single radioisotope present at any position on the conjugated ring, or a functional group used in a click reaction, or at any position on the conjugated ring.
- R 2 and R 3 are each a hydrogen atom, an alkyl group, an optionally aromatic may have a substituent Represents a group.
- tyrosine phosphatase Purifying tyrosine phosphatase from cell debris or cell lysate is a labor-intensive operation, and the method of the present invention is superior to conventional methods in that this can be omitted.
- the fact that tyrosine phosphatase is not purified but can be measured in the presence of cell debris or cell lysate means that the activity can be measured in a state closer to the living body, This is also superior to conventional methods.
- Step (1) a tyrosine phosphatase to be measured and a peptide containing a phosphorylated tyrosine residue are reacted to dephosphorylate the phosphorylated tyrosine residue.
- Any tyrosine phosphatase can be used as long as it can dephosphorylate phosphorylated tyrosine residues in the peptide.
- Some tyrosine phosphatases have been linked to specific diseases.
- PTP1B which is associated with diabetes
- SHP-2 which is associated with Noonan's syndrome associated with abnormal facial skeleton formation, short stature, hypertrophic cardiomyopathy, and colon cancer
- DEP1 has been pointed out. Such a tyrosine phosphatase may be measured.
- an unpurified tyrosine phosphatase can be used, but a purified one may be used.
- unpurified tyrosine phosphatase include biological samples containing tyrosine phosphatase (for example, cell debris, cell lysate, blood, plasma, urine, etc.).
- tyrosine phosphatase is associated with a specific disease
- the disease can be diagnosed by measuring the activity of this enzyme.
- diseases include cancer, in addition to diabetes described later.
- Specific examples of tyrosine phosphatases that can be used for disease diagnosis are shown below.
- PTP1B There is a report that activation of PTP1B is responsible for insulin resistance (Haftchenary et al., ACS Med. Chem. Lett. 2015, 6, 982-986). From this, by measuring the activity of PTP1B, It is thought that insulin resistance and diabetes can be diagnosed. Since PTP1B is mainly expressed in muscle, liver, and adipose tissue, PTP1B contained in crushed materials of these organs and tissues can be measured.
- LAR leukocyte common antigen-related
- PRL-1 Phosphatase of Regenerating Liver 1
- a method for diagnosing pancreatic cancer by collecting cells from a subject and evaluating the PRL-1 activity in the cells is known (Japanese Patent Publication No. 2006-519616).
- LMW-PTP Low molecular weight protein tyrosine phosphatase
- SHP1 SHP1 reduces the activity of CagA, an oncogenic protein of Helicobacter pylori, and the expression of this enzyme is suppressed by infection with EB virus (Saju et al., Nat Microbiol. 2016 Mar 14; 1: 16026. doi: 10.1038 / nmicrobiol.2016.26.). Therefore, the susceptibility to gastric cancer can be diagnosed by measuring the SHP1 activity in the epithelial cells of the stomach.
- the peptide may be any peptide as long as it contains a phosphorylated tyrosine residue.
- the chain length of the peptide is preferably about 10 to 20 from the viewpoint of ease of handling, etc., but it is very long and is generally recognized as “protein” instead of “peptide”.
- the peptide may be.
- the peptide does not need to have any function, but a peptide or protein having a specific function may be used. Examples of such a protein include a tyrosine phosphatase substrate protein in which there are no (or few) tyrosine residues modified in this reaction other than the phosphorylated tyrosine residue in the active center, or the phosphorylated tyrosine residue in the active center.
- a tyrosine phosphatase substrate protein in which a tyrosine residue other than the active center in a dephosphorylated state is capped in advance by a modification reaction can be used so that the modification reaction does not proceed other than the group
- the number of phosphorylated tyrosine residues contained in the peptide may be one, but there may be two or more.
- the peptide preferably contains only phosphorylated tyrosine residues and no non-phosphorylated tyrosine residues, but may contain non-phosphorylated tyrosine residues.
- the compound represented by the general formula (I) binds not only to the tyrosin residue dephosphorylated by the tyrosine phosphatase activity, but also to the originally non-phosphorylated tyrosine residue. It is necessary to subtract minutes to determine tyrosine phosphatase activity.
- the amount of tyrosine phosphatase used in this reaction is not particularly limited, but is usually 1 ⁇ 10 ⁇ 4 to 1 ⁇ 10 ⁇ 2 mol, preferably 1 ⁇ 10 ⁇ 3 to 1 ⁇ per 1 mol of peptide. 10 -2 mol.
- the buffer used for this reaction is not particularly limited, and a buffer containing sodium acetate, phosphoric acid, trishydroxymethylaminomethane, sulfonic acid buffer (HEPES, MES, etc.), etc. can be used.
- the pH during the reaction is not particularly limited, but is usually 3 to 9, and preferably 5 to 7.
- the reaction temperature is not particularly limited, but is usually 4 to 40 ° C., and preferably 20 to 40 ° C.
- the reaction time is not particularly limited, but is usually 5 minutes to 24 hours, preferably 30 minutes to 2 hours.
- Step (2) the compound represented by the general formula (I) is bound to the dephosphorylated tyrosine residue in the presence of an oxidizing agent and a metal catalyst.
- hydrogen peroxide is usually used, but is not limited to this as long as the reaction can proceed.
- APS ammonium persulfate
- tertiary butyl peroxide tertiary butyl peroxide
- cumene peroxide and the like are used. Also good.
- any metal catalyst can be used as long as the reaction can proceed, but it is preferable to use a porphyrin metal complex.
- a porphyrin metal complex a porphyrin copper complex or a porphyrin cobalt complex may be used, but a porphyrin iron complex is preferably used.
- a porphyrin iron complex a protein containing a porphyrin iron complex may be used in addition to the porphyrin iron complex itself.
- Specific examples of the porphyrin iron complex include hemin, hemoglobin, horseradish peroxidase (HRP), myoglobin, cytochrome, etc. Among them, it is preferable to use HRP.
- A represents a conjugated ring.
- A may be a conjugated ring, but is preferably a benzene ring.
- R 1 represents a hydrogen atom, one radioisotope present at any position on the conjugated ring, or a functional group used for a click reaction, or any one on the conjugated ring. 1 or 2 amino groups, acetamide groups, hydroxy groups, alkyl groups, or alkoxy groups present at the position of.
- the number of carbon atoms of the alkyl group and the alkoxy group is not particularly limited, but is preferably 1-20, more preferably 1-10, and 1-3. It is particularly preferred.
- R 1 may be any group as described above, but is preferably a hydrogen atom, or one amino group, an acetamido group, or a methoxy group, and more preferably a hydrogen atom or one methoxy group. .
- each substituent may be the same or different.
- the substituent may be present at any position on the conjugated ring, but is preferably present at a position away from the adjacent heterocyclic ring for ease of synthesis.
- R 2 represents a hydrogen atom, an alkyl group, or an aromatic group which may have a substituent.
- the number of carbon atoms of the alkyl group is not particularly limited, but it is preferably 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable.
- R 2 may be any group as described above, but is preferably a methyl group or a phenyl group, and more preferably a methyl group.
- R 3 represents a hydrogen atom, an alkyl group, or an aromatic group which may have a substituent.
- the number of carbon atoms of the alkyl group is not particularly limited, but it is preferably 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable.
- R 3 may be any group as described above, but is preferably a hydrogen atom.
- L represents a hydrogen atom, a linker having a functional group used for a click reaction at the terminal, or a linker having a labeling substance at the terminal.
- the linker having a functional group used for the click reaction at the terminal may have any structure as long as such a functional group does not lose the binding property, but -X- [CH 2 CH 2 -Y] m- (CH 2 ) n -Z 1 (where X and Y represent CH 2 , O, NH, S, NHCO or CO, respectively, Z 1 represents N 3 or CCH, and m and n represent Each represents an integer of 0-12.
- the linker having a labeling substance at the terminal may have any structure as long as the labeling substance does not lose its function, but -X- [CH 2 CH 2 -Y] m- (CH 2 ) n -Z 2 [where X and Y each represent CH 2 , O, NH, S, NHCO, or CO, Z 2 represents a group including a group including a labeling substance, and m and n are each an integer of 0 to 12] Represents. ] Is preferable.
- Preferred examples of the compound represented by the general formula (I) include the following general formula (Ia)
- L may be present at any position on the benzene ring, but is preferably present at the 6-position or 7-position on the dihydrophthalazine ring.
- the compound represented by the general formula (Ia) only one kind of compound may be used, but a mixture of two or more kinds of compounds may be used. For example, a mixture of a compound having L at the 6-position and a compound having L at the 7-position may be used.
- the compound represented by the general formula (I) binds to a dephosphorylated tyrosine residue in the peptide, and may bind one molecule or two molecules to this tyrosine residue.
- the amount of the compound represented by the general formula (I) is relatively smaller than the amount of the peptide, the oxidizing agent and the metal catalyst, the compound represented by the general formula (I) binds two molecules to the tyrosine residue. In the case of relatively large amounts, the compound represented by the general formula (I) binds to one molecule of tyrosine residue.
- the number of the compounds represented by the general formula (I) bound to the tyrosine residue can be determined. Can be controlled.
- the amount of the peptide to be used is not particularly limited, but is usually 0.001 to 1 mol per 1 mol of the compound represented by the general formula (I), and is represented by the general formula (I) of two molecules at the tyrosine residue. When the compound is bound, it is 0.0005 to 0.5 mol.
- the amount of the oxidizing agent to be used is not particularly limited, but is usually 1 to 100 mol per 1 mol of the compound represented by the general formula (I), and represented by the general formula (I) of two molecules per tyrosine residue. Even when a compound to be bonded is bound, it is 1 to 100 mol.
- the amount of the metal catalyst to be used is not particularly limited, but is usually 1 ⁇ 10 ⁇ 5 to 1 ⁇ 10 ⁇ 3 mol per 1 mol of the compound represented by the general formula (I), and 2 molecules per tyrosine residue. Even when the compound represented by the general formula (I) is bonded, the amount is 1 ⁇ 10 ⁇ 5 to 1 ⁇ 10 ⁇ 3 mol.
- the buffer used for this reaction is not particularly limited, and a buffer containing phosphate buffer, trishydroxymethylaminomethane, sulfonic acid buffer (HEPES, HMES, etc.), etc. can be used.
- a buffer containing phosphate buffer, trishydroxymethylaminomethane, sulfonic acid buffer (HEPES, HMES, etc.), etc. can be used.
- a phosphate-containing buffer cannot be used to quantify the phosphate produced by the reaction, but it should be used without any problem in the method of the present invention. Can do.
- the pH during the reaction is not particularly limited, but is usually 5 to 9, and preferably 7 to 8.
- the reaction temperature is not particularly limited, but is usually 4 to 70 ° C, and preferably 20 to 30 ° C.
- the reaction time is not particularly limited, but is usually 15 minutes to 24 hours, and preferably 30 minutes to 2 hours.
- Step (2) may be performed after the end of step (1) or may be performed simultaneously with step (1). That is, the reaction between tyrosine phosphatase and the peptide was terminated by inactivating tyrosine phosphatase, and then the compound represented by the general formula (I), an oxidizing agent and a metal catalyst were added, and dephosphorylated.
- the tyrosine residue may be bound to the compound represented by the general formula (I), or tyrosine phosphatase, peptide, the compound represented by the general formula (I), an oxidizing agent and a metal catalyst are mixed to obtain a tyrosine residue.
- the dephosphorylation of the group and the binding of the compound represented by the general formula (I) to the dephosphorylated tyrosine residue may occur simultaneously.
- step (3) the amount of the compound represented by the general formula (I) bound to the peptide is measured, and the tyrosine phosphatase activity is determined from the amount.
- the method for measuring the amount of the compound represented by the general formula (I) bound to the peptide is not particularly limited.
- the label when the compound represented by the general formula (I) contains a labeling substance, the label
- a labeling substance is bound to the functional group, and the measurement is performed with the labeling substance. can do.
- the amount of the compound represented by the general formula (I) bound to the peptide can also be measured by the following method.
- a method using a carrier that specifically binds to a compound represented by the general formula (I) In this method, a fluorescent substance is bound to a peptide containing a phosphorylated tyrosine residue, which is represented by the general formula (I).
- the compound represented by the general formula (I) bound to the peptide is isolated using a carrier that specifically binds to the compound, and the amount of the compound represented by the general formula (I) by the fluorescent substance bound to the peptide Measure.
- Fluorescent substances such as fluorescein, FITC, rhodamine can be used.
- the compound represented by the general formula (I) contains biotin, beads such as avidin or streptavidin bound thereto may be used. Can be used. Also, a plate on which avidin or streptavidin is fixed can be used.
- FIG. 1 is a diagram schematically illustrating a method of using this FRET.
- Fluorescent substances that form FRET pairs include FITC and rhodamine, FITC and Cy3, and Cy3 and Cy5.
- the fluorescence intensity of the fluorescent substance serving as a donor is weakened while the fluorescence intensity of the fluorescent substance serving as an acceptor is increased. Accordingly, the amount of the compound represented by the general formula (I) bound to the peptide is determined by measuring the fluorescence intensity of the fluorescent substance serving as a donor or the fluorescence intensity of the fluorescent substance serving as an acceptor, or a change in both fluorescence intensities. Can be measured.
- the phosphor containing a phosphorylated tyrosine residue is used as a donor and the phosphor bound to the compound represented by the general formula (I) is used as an acceptor.
- a fluorescent substance bonded to a peptide may be used as an acceptor, and a fluorescent substance bonded to a compound represented by the general formula (I) may be used as a donor.
- This method is suitable for high-throughput measurement of tyrosine phosphatase activity because no washing operation is required.
- FIG. 1 is a diagram schematically illustrating a method of using this fluorescence quencher.
- Quenchers include DABCYL, BHQ-1, BHQ-2, and QSY-7.
- This method is also suitable for high-throughput measurement of tyrosine phosphatase activity because no washing operation is required.
- Kit for measuring tyrosine phosphatase activity of the present invention comprises a peptide containing a phosphorylated tyrosine residue and a compound represented by the general formula (I).
- This kit may contain a peptide containing a phosphorylated tyrosine residue and a compound other than the compound represented by the general formula (I).
- a compound other than the compound represented by the general formula (I) For example, in order for the compound represented by the general formula (I) to bind to the dephosphorylated tyrosine residue, an oxidizing agent or a metal catalyst is necessary, and therefore, these may be included.
- it contains reagents and equipment for measuring the amount of the compound represented by the general formula (I) bound to the peptide, or a substance for stabilizing the peptide and the compound represented by the general formula (I). You may go out.
- Screening method for tyrosine phosphatase inhibitor or activator comprises (1) a tyrosine phosphatase and a phosphorylated tyrosine residue in the presence of a test substance.
- Step (1) a tyrosine phosphatase and a peptide containing a phosphorylated tyrosine residue are contacted in the presence of a test substance.
- the test substance may be any substance, either a natural product or a synthetic compound. Specific examples include proteins, peptides, vitamins, hormones, polysaccharides, oligosaccharides, monosaccharides, low molecular compounds, nucleic acids (DNA, RNA, oligonucleotides, mononucleotides, etc.), lipids, and the like.
- the test substance may be purified, but may be an unpurified cell extract, cell disruption solution, cell lysate or the like.
- the amount of the test substance to be used is not particularly limited, but is usually 10 to 10,000 mol, preferably 100 to 10,000 mol, per 1 mol of tyrosine phosphatase.
- tyrosine phosphatase as described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- tyrosine phosphatases described in “[1] Measuring method of tyrosine phosphatase activity those that cause various diseases by being activated are included. For example, activation of PTP1B and LAR PTP may cause insulin resistance and diabetes, and activation of PRL-1 and LMW-PTP may cause cancer. Therefore, these inhibitors of tyrosine phosphatase can be candidates for therapeutic and prophylactic drugs for insulin resistance, diabetes, and cancer.
- peptide containing a phosphorylated tyrosine residue the same peptide as described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- Step (2) a peptide containing a phosphorylated tyrosine residue that has been contacted with tyrosine phosphatase is contacted with a compound represented by general formula (I) in the presence of an oxidizing agent and a metal catalyst.
- the metal catalyst As the oxidizing agent, the metal catalyst, and the compound represented by the general formula (I), the same compounds as those described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- the contact between the peptide and the compound represented by the general formula (I) is carried out by changing the compound represented by the general formula (I) to the dephosphorylated tyrosine residue described in “[1] Method for measuring tyrosine phosphatase activity”.
- the reaction can be performed under the same conditions as those for the reaction to be combined.
- Step (2) may be performed after the end of step (1) or may be performed simultaneously with step (1).
- step (3) the amount of the compound represented by the general formula (I) bound to the peptide is measured, and the tyrosine phosphatase activity is determined from the amount.
- the amount of the compound represented by the general formula (I) bound to the peptide can be measured in the same manner as described in “[1] Method for measuring tyrosine phosphatase activity”.
- the test substance can be determined to be a tyrosine phosphatase inhibitor and is high. In some cases, the test substance can be determined as a tyrosine phosphatase activator.
- the diabetes diagnostic agent of the present invention comprises a peptide containing a phosphorylated tyrosine residue and a compound represented by the general formula (I).
- PTP1B a type of tyrosine phosphatase
- a sample blood, cell lysate, etc.
- the method for measuring tyrosine kinase activity of the present invention comprises (1) reacting a tyrosine kinase to be measured with a peptide containing a non-phosphorylated tyrosine residue, A step of phosphorylation, (2) a step of binding a compound represented by the general formula (I) to a tyrosine residue that has not been phosphorylated in the presence of an oxidizing agent and a metal catalyst, (3) step (2) The step of measuring the amount of the compound represented by the general formula (I) bound to the peptide in (4), the non-phosphorylated tyrosine residue of the peptide containing the non-phosphorylated tyrosine residue used in step (1), A step of binding the compound represented by the general formula (I) in the presence of an oxidizing agent and a metal catalyst, and (5) the amount of the compound represented by the general formula (I) bound to the peptide in the step
- Measuring step and (6) step Tyrosine kinase activity from the amount of the compound represented by general formula (I) bound to the peptide measured in (3) and the amount of the compound represented by general formula (I) bound to the peptide measured in step (5) It includes the process of calculating
- step (1) the tyrosine kinase to be measured is reacted with a peptide containing a non-phosphorylated tyrosine residue to phosphorylate the non-phosphorylated tyrosine residue.
- the tyrosine kinase to be measured may be any tyrosine kinase as long as it can phosphorylate a non-phosphorylated tyrosine residue in the peptide, and examples thereof include ErbB1, ErbB2, VEGFR, and ALK. Some tyrosine kinases have been linked to specific diseases. For example, ErbB1, ErbB2, ALK, and VEGFR that have been pointed out to be associated with pulmonary fibrosis. Such a tyrosine kinase may be measured. A purified tyrosine kinase may be used, but an unpurified one, for example, a biological sample containing kilocin kinase may be used.
- the peptide may be any peptide as long as it contains a non-phosphate tyrosine residue.
- the chain length of the peptide is preferably about 10 to 20 from the viewpoint of ease of handling, etc., but it is very long and is generally recognized as “protein” instead of “peptide”.
- the peptide may be.
- the peptide does not need to have any function, but a peptide or protein having a specific function may be used. Examples of such a protein include a tyrosine kinase substrate protein having no (or few) tyrosine residues modified by this reaction other than the tyrosine residue of the phosphorylation target of the active center, or the tyrosine residue of the active center.
- a tyrosine kinase substrate protein or the like in which a tyrosine residue other than the active center is capped in advance by a tyrosine modification reaction can be used so that the modification reaction does not proceed other than the group.
- the number of non-phosphorylated tyrosine residues contained in the peptide may be one, but there may be two or more.
- the peptide may contain only non-phosphorylated tyrosine residues or may contain both non-phosphorylated tyrosine residues and phosphorylated tyrosine residues.
- the amount of tyrosine kinase used in this reaction is not particularly limited, but is usually 1 ⁇ 10 ⁇ 4 to 1 ⁇ 10 ⁇ 2 mol per 1 mol of peptide, preferably 1 ⁇ 10 ⁇ 3 to 1 ⁇ 10 -2 mol.
- the buffer used for this reaction is not particularly limited, and sulfonic acid buffers (HEPES, MES, etc.), phosphoric acid, trishydroxymethylaminomethane, and the like can be used.
- the pH during the reaction is not particularly limited, but is usually 5 to 9, and preferably 6 to 8.
- the reaction temperature is not particularly limited, but is usually 4 to 40 ° C., and preferably 20 to 40 ° C.
- the reaction time is not particularly limited, but is usually 5 minutes to 24 hours, preferably 30 minutes to 2 hours.
- Step (2) a compound represented by the general formula (I) is bound to a tyrosine residue that has not been phosphorylated in the presence of an oxidizing agent and a metal catalyst.
- the metal catalyst As the oxidizing agent, the metal catalyst, and the compound represented by the general formula (I), the same compounds as those described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- the reaction for binding the compound represented by the general formula (I) to a tyrosine residue that has not been phosphorylated is a general formula of the dephosphorylated tyrosine residue described in “[1] Method for Measuring Tyrosine Phosphatase Activity”.
- the reaction can be carried out under the same conditions as in the reaction for bonding the compound represented by (I).
- step (3) the amount of the compound represented by general formula (I) bound to the peptide in step (2) is measured.
- the amount of the compound represented by the general formula (I) bound to the peptide can be measured in the same manner as described in “[1] Method for measuring tyrosine phosphatase activity”.
- Step (4) the non-phosphorylated tyrosine residue of the peptide used in the step (1) is represented by the general formula (I) in the presence of an oxidizing agent and a metal catalyst. The compound is allowed to bind.
- the metal catalyst As the oxidizing agent, the metal catalyst, and the compound represented by the general formula (I), the same compounds as those described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- the reaction for binding the compound represented by the general formula (I) to the non-phosphorylated tyrosine residue is carried out by subjecting the dephosphorylated tyrosine residue described in “[1] Method for measuring tyrosine phosphatase activity” to the general formula (I ) Can be carried out under the same conditions as those for the reaction for bonding the compound represented by.
- the coupling reaction in this step is for comparison with the coupling reaction in step (2), it is performed under the same conditions as the coupling reaction in step (2).
- step (5) the amount of the compound represented by general formula (I) bound to the peptide in step (4) is measured.
- the amount of the compound represented by the general formula (I) bound to the peptide can be measured in the same manner as described in “[1] Method for measuring tyrosine phosphatase activity”.
- Step (6) the amount of the compound represented by the general formula (I) bound to the peptide measured in the step (3) and the general formula (I) bound to the peptide measured in the step (5) are represented.
- the tyrosine kinase activity is determined from the amount of the compound.
- the non-phosphorylated tyrosine residue in the peptide is phosphorylated by the action of tyrosine kinase. Since the compound represented by the general formula (I) specifically binds to a non-phosphorylated tyrosine residue, the amount of the compound represented by the general formula (I) binding to the peptide was reacted with tyrosine kinase.
- the case (measured amount in step (3)) is smaller than the case where tyrosine kinase is not reacted (measured amount in step (5)). Therefore, the activity of tyrosine kinase can be determined from the difference between the measured amount in step (5) and the measured amount in step (3).
- Kit for measuring tyrosine kinase activity of the present invention comprises a peptide containing a non-phosphorylated tyrosine residue and a compound represented by the general formula (I). .
- a peptide containing a non-phosphorylated tyrosine residue and a compound represented by the general formula (I) contained in this kit are added to a sample containing tyrosine kinase, the tyrosine kinase causes the non-phosphorylated tyrosine residue in the peptide to be phosphorylated. Oxidized. Since the compound represented by the general formula (I) binds to the tyrosine residue not phosphorylated by the tyrosine kinase, the activity of the tyrosine kinase is based on the amount of the compound represented by the general formula (I) bound to this peptide. Can be measured.
- the same peptide as described in “[5] Measuring method of tyrosine kinase activity” can be used, and the compound represented by the general formula (I) is “[1] The same as described in “Method for Measuring Tyrosine Kinase Activity” can be used.
- This kit may contain a peptide containing a non-phosphorylated tyrosine residue and a compound other than the compound represented by the general formula (I).
- a compound other than the compound represented by the general formula (I) For example, an oxidant and a metal catalyst are necessary for the compound represented by the general formula (I) to bind to a tyrosine residue that has not been phosphorylated.
- it contains reagents and equipment for measuring the amount of the compound represented by the general formula (I) bound to the peptide, or a substance for stabilizing the peptide and the compound represented by the general formula (I). You may go out.
- Screening method for tyrosine kinase inhibitor or activator comprises (1) tyrosine kinase and non-phosphorylated tyrosine residue in the presence of a test substance. (2) contacting a peptide containing a non-phosphorylated tyrosine residue contacted with a tyrosine kinase with a compound represented by the general formula (I) in the presence of an oxidizing agent and a metal catalyst.
- a step, (3) a step of measuring the amount of the compound represented by the general formula (I) bound to the peptide in step (2), and (4) a peptide comprising a non-phosphorylated tyrosine residue used in step (1)
- (5) the compound represented by the general formula (I) bound to the peptide in step (4)
- Amount The step of measuring, (6) the amount of the compound represented by general formula (I) bound to the peptide measured in step (3) and the general formula (I) bound to the peptide measured in step (5)
- Step (1) a tyrosine kinase and a peptide containing a non-phosphorylated tyrosine residue are contacted in the presence of a test substance.
- test substance can be the same as described in “[3] Screening method of tyrosine phosphatase inhibitor or activator”, and the peptide containing tyrosine kinase and non-phosphorylated tyrosine residue is “[5] The same as described in “Method for Measuring Tyrosine Kinase Activity” can be used.
- Step (2) a peptide containing a non-phosphorylated tyrosine residue that has been contacted with tyrosine kinase is contacted with a compound represented by general formula (I) in the presence of an oxidizing agent and a metal catalyst.
- the metal catalyst As the oxidizing agent, the metal catalyst, and the compound represented by the general formula (I), the same compounds as those described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- the contact between the peptide and the compound represented by the general formula (I) binds the compound represented by the general formula (I) to the tyrosine residue not phosphorylated as described in “[5] Method for measuring kinase activity”. It can carry out on the same conditions as reaction to make.
- step (3) the amount of the compound represented by general formula (I) bound to the peptide in step (2) is measured.
- the amount of the compound represented by the general formula (I) bound to the peptide can be measured in the same manner as described in “[1] Method for measuring tyrosine phosphatase activity”.
- step (4) the peptide containing the non-phosphorylated tyrosine residue used in step (1) is contacted with the compound represented by general formula (I) in the presence of an oxidizing agent and a metal catalyst.
- an oxidizing agent the metal catalyst, and the compound represented by the general formula (I)
- the same compounds as those described in “[1] Method for measuring tyrosine phosphatase activity” can be used.
- the contact between the peptide and the compound represented by the general formula (I) binds the compound represented by the general formula (I) to the tyrosine residue not phosphorylated as described in “[5] Method for measuring kinase activity”. It can carry out on the same conditions as reaction to make.
- the contact reaction in this step is for comparison with the contact reaction in step (2), it is performed under the same conditions as the contact reaction in step (2).
- step (5) the amount of the compound represented by general formula (I) bound to the peptide in step (4) is measured.
- the amount of the compound represented by the general formula (I) bound to the peptide can be measured in the same manner as described in “[1] Method for measuring tyrosine phosphatase activity”.
- Step (6) the amount of the compound represented by the general formula (I) bound to the peptide measured in the step (3) and the general formula (I) bound to the peptide measured in the step (5) are represented.
- the tyrosine kinase activity is determined from the amount of the compound.
- the activity of tyrosine kinase in the presence of the test substance can be determined. This activity is determined in the absence of the test substance. If the test substance is low, the test substance can be determined as a tyrosine kinase inhibitor. If the test substance is high, the test substance can be determined as a tyrosine kinase activator.
- Example 1 Confirmation of phosphatase kinase activity (mass spectrometry of substrate peptide) (1) Confirmation of dephosphorylation of pY-RR-Src by tyrosine phosphatase PTP1B 100 ⁇ M solution of pY-RR-Src (amino acid sequence: RRLIEDAE (pY) AARG, Millipore 12-217) (60 mM NaOAc buffer pH5.5 1 ⁇ g of human recombinant PTP1B (Funakoshi 6301-100, strictly prohibited from repeated freezing and thawing) was added to 50 ⁇ L and incubated at room temperature for 1 hour.
- FIG. 2A shows the MS chart after 1 hour.
- Example 2 Chemical modification reaction of RR-Src in a dephosphorylated state The binding of N 3 compound having the following structure to tyrosine residues was examined.
- N 3 compound was used as a mixture of isomers in a ratio of 1: 0.65 at the 6th and 7th positions.
- RR-Src was dissolved in 100 mM Phosphate buffer (pH 7.4) to prepare a 1 mM peptide stock solution. 0.6 ⁇ l so that the final concentration is 100 ⁇ M peptide, 100 ⁇ M HRP (horse radish peroxidase, Aldrich) 50 nM, N 3 compound 0.1-1 mM in 100 mM Phosphate buffer (pH 7.4) on a 50 ⁇ L scale for each reaction. Prepared in ml Eppendorf tubes.
- the mixture was mixed on an MS plate, dried at room temperature, and a covalent bond formation reaction was confirmed by MALDI-TOF-MS analysis (Bruker, UltrafleXtreme) (FIG. 4).
- the covalent modification reaction does not proceed with the phosphorylated substrate peptide (pY-RR-Src), and it reacts specifically only with the dephosphorylated substrate peptide (RR-Src). Became clear. In addition, it was suggested that the reaction can be carried out almost quantitatively even when the reaction conditions are 10 ⁇ M peptide concentration.
- Example 5 Detection of chemically modified non-phosphorylated substrate peptide
- N 3 compound modified RR-Src and biotin modified RR-Src (100 ⁇ M peptide) solution prepared by the method described in Examples 2 and 4 FITC (5-Isothiocyanatofluorescein, TCI) in 1 M DMSO was added to a final concentration of 4 mM and incubated at room temperature for 1 hour.
- the solution labeled with N 3 compound-modified RR-Src is labeled sample 1
- the solution labeled with biotin-modified RR-Src is labeled sample 2.
- the assay kit determines the amount of free inorganic phosphate produced in 1 equivalent of the peptide as a result of dephosphorylation by phosphatase. Is. Therefore, there is a limitation that a buffer containing phosphate cannot be used in the assay.
- the effective detection range of inorganic phosphate is described as 200-4000 pmol.
- the detection limit is about 200 mmpmol.
- the present invention is considered to be a method that exceeds the conventional method.
- the method for measuring tyrosine phosphatase and tyrosine kinase activity of the present invention is useful for the development of new drugs, and can be used in industrial fields such as the pharmaceutical industry.
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Abstract
Description
〔1〕以下の工程を含むことを特徴とするチロシンホスファターゼ活性の測定方法、
(1)測定対象とするチロシンホスファターゼとリン酸化チロシン残基を含むペプチドを反応させ、リン酸化チロシン残基を脱リン酸化する工程、
(2)脱リン酸化させたチロシン残基に、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物を結合させる工程、
(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程。
で表される化合物を含むことを特徴とするチロシンホスファターゼ活性測定用キット。
(1)被験物質の存在下で、チロシンホスファターゼとリン酸化チロシン残基を含むペプチドを接触させる工程、
(2)チロシンホスファターゼと接触させたリン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物と接触させる工程、
(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程。
で表される化合物であることを特徴とする〔7〕に記載のチロシンホスファターゼ阻害剤又は活性化剤のスクリーニング方法。
で表される化合物を含むことを特徴とする糖尿病の診断薬。
(1)測定対象とするチロシンキナーゼと非リン酸化チロシン残基を含むペプチドを反応させ、非リン酸化チロシン残基をリン酸化する工程、
(2)リン酸化しなかったチロシン残基に、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物を結合させる工程、
(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドの非リン酸化チロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる工程、
(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程。
で表される化合物を含むことを特徴とするチロシンキナーゼ活性測定用キット。
(1)被験物質の存在下で、チロシンキナーゼと非リン酸化チロシン残基を含むペプチドを接触させる工程、
(2)チロシンキナーゼと接触させた非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物と接触させる工程、
(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる工程、
(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程。
で表される化合物であることを特徴とする〔19〕に記載のチロシンキナーゼ阻害剤又は活性化剤のスクリーニング方法。
本発明のチロシンホスファターゼ活性の測定方法は、(1)測定対象とするチロシンホスファターゼとリン酸化チロシン残基を含むペプチドを反応させ、リン酸化チロシン残基を脱リン酸化する工程、(2)脱リン酸化させたチロシン残基に、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物を結合させる工程、及び(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程を含むことを特徴とするものである。
工程(1)では、測定対象とするチロシンホスファターゼとリン酸化チロシン残基を含むペプチドを反応させ、リン酸化チロシン残基を脱リン酸化する。
PTP1Bの活性化がインスリン抵抗性の原因となっているという報告があり(Haftchenary et al., ACS Med. Chem. Lett. 2015, 6, 982-986)、このことから、PTP1Bの活性測定により、インスリン抵抗性や糖尿病を診断できると考えられる。PTP1Bは、主に筋肉、肝臓、脂肪組織で発現しているので、これらの器官や組織の破砕物中に含まれるPTP1Bを測定対象とすることができる。
筋肉におけるLAR PTPの高活性化がインスリン抵抗性の発症に関与しているという報告がある(Zabolotny et al., Proc Natl Acad Sci U S A, 2001, 98(9):5187-92)。このことから、LAR PTPの活性測定により、インスリン抵抗性や糖尿病を診断できると考えられる。
被検者から細胞を採取し、その細胞中のPRL-1活性を評価し、膵臓癌の診断をする方法が知られている(特表2006-519616)。
哺乳類から得られる細胞可溶化物中で、LMW-PTPが過剰発現しているかどうかにより、癌を診断する方法が知られている(特表2006-501153)。
SHP1は、ピロリ菌の発癌タンパク質であるCagAの活性を低下させ、また、この酵素は、EBウイルスの感染により発現が抑制される(Saju et al., Nat Microbiol. 2016 Mar 14;1:16026. doi: 10.1038/nmicrobiol.2016.26.)。従って、胃の上皮細胞におけるSHP1活性を測定することにより、胃がんの罹り易さを診断できる。
工程(2)では、脱リン酸化させたチロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる。
工程(3)では、ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める。
この方法では、リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物と特異的に結合する担体を用いて、ペプチドに結合した一般式(I)で表される化合物を単離し、ペプチドに結合した蛍光物質によって一般式(I)で表される化合物の量を測定する。
この方法では、リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質とFRETペアを形成する蛍光物質を結合させ、ペプチドに結合させた蛍光物質及び/又は一般式(I)で表される化合物に結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定する。
この方法では、リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質に対するクエンチャーを結合させ、ペプチドに結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定する。
本発明のチロシンホスファターゼ活性測定用キットは、リン酸化チロシン残基を含むペプチドと一般式(I)で表される化合物を含むことを特徴とする。
本発明のチロシンホスファターゼ阻害剤又は活性化剤のスクリーニング方法は、(1)被験物質の存在下で、チロシンホスファターゼとリン酸化チロシン残基を含むペプチドを接触させる工程、(2)チロシンホスファターゼと接触させたリン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる工程、及び(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程を含むことを特徴とするものである。
工程(1)では、被験物質の存在下で、チロシンホスファターゼとリン酸化チロシン残基を含むペプチドを接触させる。
工程(2)では、チロシンホスファターゼと接触させたリン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる。
工程(3)では、ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める。
本発明の糖尿病の診断薬は、リン酸化チロシン残基を含むペプチドと一般式(I)で表される化合物を含むことを特徴とするものである。
本発明のチロシンキナーゼ活性の測定方法は、(1)測定対象とするチロシンキナーゼと非リン酸化チロシン残基を含むペプチドを反応させ、非リン酸化チロシン残基をリン酸化する工程、(2)リン酸化しなかったチロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる工程、(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドの非リン酸化チロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる工程、(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、及び(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程を含むことを特徴とするものである。
工程(1)では、測定対象とするチロシンキナーゼと非リン酸化チロシン残基を含むペプチドを反応させ、非リン酸化チロシン残基をリン酸化する。
工程(2)では、リン酸化しなかったチロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる。
工程(3)では、工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する。
工程(4)では、工程(1)で使用した非リン酸化チロシン残基を含むペプチドの非リン酸化チロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる。
工程(5)では、工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する。
工程(6)では、工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める。
本発明のチロシンキナーゼ活性測定用キットは、非リン酸化チロシン残基を含むペプチドと一般式(I)で表される化合物を含むことを特徴とするものである。
本発明のチロシンキナーゼ阻害剤又は活性化剤のスクリーニング方法は、(1)被験物質の存在下で、チロシンキナーゼと非リン酸化チロシン残基を含むペプチドを接触させる工程、(2)チロシンキナーゼと接触させた非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる工程、(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる工程、(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程を含むことを特徴とするものである。
工程(1)では、被験物質の存在下で、チロシンキナーゼと非リン酸化チロシン残基を含むペプチドを接触させる。
工程(2)では、チロシンキナーゼと接触させた非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる。
工程(3)では、工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する。
工程(4)では、工程(1)で使用した非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる。
酸化剤、金属触媒、及び一般式(I)で表される化合物は、「〔1〕チロシンホスファターゼ活性の測定方法」に記載したものと同様のものを使用できる。
工程(5)では、工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する。
工程(6)では、工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める。
(1)チロシンホスファターゼPTP1BによるpY-RR-Srcの脱リン酸化の確認
pY-RR-Src(アミノ酸配列:RRLIEDAE(pY)AARG、ミリポア12-217)の100 μM溶液(60 mM NaOAc buffer pH5.5に溶解)50 μLに対して、human recombinant PTP1B(フナコシ 6301-100、凍結融解繰り返し厳禁)を1 μg分加え、室温で1時間インキュベートした。PTP1Bを加える前(control)とインキュベート後の100 μMペプチド溶液のサンプルを10倍量の0.1% TFA水溶液で希釈し、その溶液0.5-1 μMとCHCA溶液 (0.5 mg/mL in acetonitrile : 0.1% TFA = 1 : 1) 1 μMをMALDI-TOF-MSプレート上で混合し、室温で乾燥、MALDI-TOF-MS解析(Bruker, UltrafleXtreme)により、基質ペプチドpY-RR-Srcの脱リン酸化反応を確認した。図2(A)に1時間後のMSチャートを示した。
RR-Src(アミノ酸配列:RRLIEDAEYAARG、Anaspec AS22581)の100 μM溶液(12.5 mM HEPES buffer pH7.4, 10 mM MnCl2, ATP 1 mMに溶解)50 μLに対して、ErbB1(life PR7295B、凍結融解繰り返し厳禁)を1 μg分加え、37 ℃でインキュベートし、上記と同様のMALDI-TOF-MS測定手法により、基質ペプチドのリン酸化の経時変化を観測した。図2(B)に24時間後のMSチャートを示した。
下記の構造のN3 compoundのチロシン残基への結合性を調べた。
各反応50 μLのスケールで、100 mM Phosphate buffer (pH 7.4)中、最終の濃度がペプチドRR-Src/pY-RR-Src = 100/100 μM、HRP (horse radish peroxidase, Aldrich) 50 nM, N3 compound 1 mMになるように0.6 mlのエッペンドルフチューブ中に調整した。
図3に示したN3 comp. 10 eqと同じ組成のペプチド溶液(ペプチド 100 μM、加えたN3 compound 1 mM)に対して2 mMのDBCO-biotin(Aldrich)を加え、37 ℃で1時間インキュベートした。その溶液1 μMとCHCA 溶液(0.5 mg/mL in acetonitrile : 0.1% TFA = 1 : 1) 1 μMをMALDI-TOF-MSプレート上で混合し、室温で乾燥、MALDI-TOF-MS解析(Bruker, UltrafleXtreme)により、共有結合形成反応を確認した(図5下段)。
実施例2及び4に記載の方法により調製したN3 compound修飾RR-Srcおよび、ビオチン修飾RR-Src (100 μM peptide)溶液に対してFITC (5-Isothiocyanatofluorescein, TCI)の1 M DMSO溶液を最終濃度が4 mMになるように加え、室温で1時間インキュベートした。N3 compound修飾RR-Srcを蛍光標識した溶液をサンプル1、ビオチン修飾RR-Srcを蛍光標識した溶液をサンプル2とし、両サンプルの溶液からペプチド100 pmol相当分を取り、FG-beads Streptavidin beads(多摩川精機)200 μgと100 mM Phosphate buffer (pH7.4) 中で30分撹拌し、ビオチン修飾ペプチドを磁気ビーズに結合させた。その後、1 mLの100 mM Phosphate buffer (pH7.4)で3回ビーズを洗浄し、100 μLにビーズを懸濁させてプレートリーダー(TECAN、Ex/Em = 485/510 nm)で蛍光測定した。
数少ない従来のチロシンホスファターゼ測定法であるマラカイトグリーン法との比較のため、Progmega社Tyrosine Phosphatase Assay Systemの検出限界を実験により算出した。
Claims (22)
- 以下の工程を含むことを特徴とするチロシンホスファターゼ活性の測定方法、
(1)測定対象とするチロシンホスファターゼとリン酸化チロシン残基を含むペプチドを反応させ、リン酸化チロシン残基を脱リン酸化する工程、
(2)脱リン酸化させたチロシン残基に、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物を結合させる工程、
(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程。 - リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物と特異的に結合する担体を用いて、ペプチドに結合した一般式(I)で表される化合物を単離し、前記蛍光物質によってペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項1又は2に記載のチロシンホスファターゼ活性の測定方法。
- リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質とFRETペアを形成する蛍光物質又は前記蛍光物質に対するクエンチャーを結合させ、ペプチドに結合させた蛍光物質及び/又は一般式(I)で表される化合物に結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項1又は2に記載のチロシンホスファターゼ活性の測定方法。
- リン酸化チロシン残基を含むペプチドと下記の一般式(I)
で表される化合物を含むことを特徴とするチロシンホスファターゼ活性測定用キット。 - 以下の工程を含むことを特徴とするチロシンホスファターゼ阻害剤又は活性化剤のスクリーニング方法、
(1)被験物質の存在下で、チロシンホスファターゼとリン酸化チロシン残基を含むペプチドを接触させる工程、
(2)チロシンホスファターゼと接触させたリン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物と接触させる工程、
(3)ペプチドに結合した一般式(I)で表される化合物の量を測定し、その量からチロシンホスファターゼ活性を求める工程。 - リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物と特異的に結合する担体を用いて、ペプチドに結合した一般式(I)で表される化合物を単離し、ペプチドに結合した蛍光物質によって一般式(I)で表される化合物の量を測定することを特徴とする請求項7又は8に記載のチロシンホスファターゼ阻害剤又は活性化剤のスクリーニング方法。
- リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質とFRETペアを形成する蛍光物質又は前記蛍光物質に対するクエンチャーを結合させ、ペプチドに結合させた蛍光物質及び/又は一般式(I)で表される化合物に結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項7又は8に記載のチロシンホスファターゼ阻害剤又は活性化剤のスクリーニング方法。
- リン酸化チロシン残基を含むペプチドと下記の一般式(I)
で表される化合物を含むことを特徴とする糖尿病の診断薬。 - 以下の工程を含むことを特徴とするチロシンキナーゼ活性の測定方法、
(1)測定対象とするチロシンキナーゼと非リン酸化チロシン残基を含むペプチドを反応させ、非リン酸化チロシン残基をリン酸化する工程、
(2)リン酸化しなかったチロシン残基に、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物を結合させる工程、
(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドの非リン酸化チロシン残基に、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物を結合させる工程、
(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程。 - 非リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物と特異的に結合する担体を用いて、ペプチドに結合した一般式(I)で表される化合物を単離し、前記蛍光物質によってペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項13又は14に記載のチロシンキナーゼ活性の測定方法。
- 非リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質とFRETペアを形成する蛍光物質又は前記蛍光物質に対するクエンチャーを結合させ、ペプチドに結合させた蛍光物質及び/又は一般式(I)で表される化合物に結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項13又は14に記載のチロシンキナーゼ活性の測定方法。
- 非リン酸化チロシン残基を含むペプチドと下記の一般式(I)
で表される化合物を含むことを特徴とするチロシンキナーゼ活性測定用キット。 - 以下の工程を含むことを特徴とするチロシンキナーゼ阻害剤又は活性化剤のスクリーニング方法、
(1)被験物質の存在下で、チロシンキナーゼと非リン酸化チロシン残基を含むペプチドを接触させる工程、
(2)チロシンキナーゼと接触させた非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、下記の一般式(I)
で表される化合物と接触させる工程、
(3)工程(2)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(4)工程(1)で使用した非リン酸化チロシン残基を含むペプチドを、酸化剤及び金属触媒の存在下で、一般式(I)で表される化合物と接触させる工程、
(5)工程(4)においてペプチドに結合した一般式(I)で表される化合物の量を測定する工程、
(6)工程(3)で測定したペプチドに結合した一般式(I)で表される化合物の量と工程(5)で測定したペプチドに結合した一般式(I)で表される化合物の量からチロシンキナーゼ活性を求める工程。 - 非リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物と特異的に結合する担体を用いて、ペプチドに結合した一般式(I)で表される化合物を単離し、ペプチドに結合した蛍光物質によって一般式(I)で表される化合物の量を測定することを特徴とする請求項19又は20に記載のチロシンキナーゼ阻害剤又は活性化剤のスクリーニング方法。
- 非リン酸化チロシン残基を含むペプチドに蛍光物質を結合させ、一般式(I)で表される化合物に、前記蛍光物質とFRETペアを形成する蛍光物質又は前記蛍光物質に対するクエンチャーを結合させ、ペプチドに結合させた蛍光物質及び/又は一般式(I)で表される化合物に結合させた蛍光物質の蛍光の変化によって、ペプチドに結合した一般式(I)で表される化合物の量を測定することを特徴とする請求項19又は20に記載のチロシンキナーゼ阻害剤又は活性化剤のスクリーニング方法。
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