WO2008140230A1 - Processus d'identification de la spécificité d'un substrat kinase au moyen d'une bibliothèque de peptides - Google Patents

Processus d'identification de la spécificité d'un substrat kinase au moyen d'une bibliothèque de peptides Download PDF

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WO2008140230A1
WO2008140230A1 PCT/KR2008/002632 KR2008002632W WO2008140230A1 WO 2008140230 A1 WO2008140230 A1 WO 2008140230A1 KR 2008002632 W KR2008002632 W KR 2008002632W WO 2008140230 A1 WO2008140230 A1 WO 2008140230A1
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peptide
amino acid
kinase
solid support
acid sequence
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PCT/KR2008/002632
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English (en)
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Yoon-Sik Lee
Byung-Gee Kim
Eun-Mi Kim
Dong-Sik Shin
Yun Gon Kim
Mira Kim
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Seoul National University Industry Foundation
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Publication of WO2008140230A1 publication Critical patent/WO2008140230A1/fr

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • 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

Definitions

  • the present invention relates to a process for the identification of a kinase substrate specificity by using a peptide library. More particulary, the present invention is directed to a process for indent ifying a kinase substrate specificity, comprising: i ) linking a plurality of peptides, having a same amino acid sequence, to the surface of a solid support resin to obtain a peptide library which has peptides, with the same amino acid sequence, on the surface of the solid support resin; ii) repeating the step i ) with changing the amino acid sequence to give a random mixture of peptide libraries, wherein each solid suppot resin has peptides with a same amino acid sequence, however the amino acid sequence liked to each solid support is different from one another; iii) phosphorylating selectively a specific amino acid of a peptide which has a certain amino acid sequence by reacting the random mixture of the peptide libraries with a kinase, to give a
  • kinase refers to an enzyme that transfers phosphate groups from ATP to serine, threonine or tyrosine residues of a protein
  • tyrosine kinase refers to an enzyme that phosphorylates specifically a tyrosine residue of a protein
  • antibody refers to an immunoglobulin that is found in blood or other bodily fluids and is a protein which binds to an antigen. Antigenic specificity of the antibody is diverse.
  • substrate specificity refers to a characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. The substrate specificity is defined by a protein portion of an enzyme.
  • phosphorylation refers to a reaction that introduces a phosphate group to a protein molecule or a small molecule such as tyrosine, serine or threonine.
  • KIPP-MS kinase phosphorylation profiling by mass spectrometry
  • MALDI-TOF matrix assisted laser desorption/ionization time-of flight
  • phosphatase refers to an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxy1 group.
  • peptide library refers to a collection of cloned free peptides, frequently consisting of all possible combinations of amino acids making up an n-amino acid peptide.
  • ladder peptide sequence refers to a peptide that is synthesized in a shape of a ladder in which a series of polypeptides are anchored to one bead.
  • split and pool synthesis refers to a method for synthesizing a peptide library that involves splitting a sample of solid supports into a given number of fractions, charging each subset to its own reaction vessel for reaction, collecting and thoroughly mixing the solid supports back together, with successive splitting, reacting, and remixing.
  • OBOC one-bead one-component
  • photolabile linker refers to a compound that is designed to facilitate the functional connection of a peptide to a bead and is liable to be decomposed by an ultra violet ray of a certain wavelength.
  • a solid support resin made of polystyrene is used as a bead of the present invention.
  • the term “docking” refers to a method which, using a computer program, predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex, the reaction between proteins and ligands, and the interaction between reactive residues and ligands.
  • the term “docking energy” refers to an energy that is calculated from the interaction between proteins and ligands, such as a hydrogen bond, electron affinity, electron repulsion, etc., by using a quantum chemical program package.
  • the term “flexible docking” refers to a docking method that allows the protein and/or ligand to change their moleculare conformation throughout the course of the simulation.
  • FlexX refers to a commercial docking software that predicts protein-ligand interactions and the geometry of the complex as well as an estimate for the strength of binding for a given protein and a ligand so as to give a optimal docking mode.
  • P-O distance refers to a distance between a phosphorous atom of the phosphate group of adenosine triphosphate (ATP) and an oxygen atom of the hydroxyl group of the substrate such as tyrosine.
  • RMS gradient refers to an energe change rate accompanied by the energy change according to the structure change when calculating the structure of protein. The most stable structure can be found in which the energy change is minimized.
  • the present invention is directed to a method for fast identification of a kinase substrate specificity by using a peptide library and a mass spectrometer. Therefore, the method can be applied to find out the phosphorylation of a kinase of which a substrate specificity is not known thereby investigating the signal transduction activated by the phosphorylation. Also, the method can be applied to the research into pretease or phosphatase which participates in the signal transduction in a cell as well as the study of a protein kinase.
  • An enzyme has a substrate specificity that it is extremely selective for its substrate since it binds specifically the substrate by recognizing the specific structure of the substrate. Therefore, it is very important to verify the substrate specificity of an enzyme in order to understand the function of the enzyme within a living organism.
  • a kinase is a kind of a phosphotransferase that transfers phosphate groups from nucleotide triphosphate, such as ATP, to specific target molecules (substrates), and activates a substrate.
  • nucleotide triphosphate such as ATP
  • target molecules such as ATP
  • kinases such as a adenylate kinase, a pyruvate kinase, a phosphokinase, etc.
  • a protein kinase plays an important role in the metabolic ragulation in a human body by the activation or inactivation of an enzyme.
  • a tyrosine kinase is likely to be associated with the regulation of cell proliferation.
  • Most cell growth factor receptors such as an epithelial growth factor receptor has a tyrosine kinase.
  • a protein produced by the representative oncogene, src, is alos a kind of a tyrosine kinase.
  • Lam et al. disclose first a method for synthesizing an OBOC peptide library in which the library is synthesized on a TentaGel bead, and this method has been recently used for identifying bioactive materials (Lam; K. S., Salmon; S. E., Hersh; E. M., Hruby; V., Kazmierski; W. M., Knapp; R. J.. Nature 354, 82 (1991)).
  • the OBOC peptide library developed by Lam et al. cannot be used with a photolabile linker due to the intrinsically uniform distribution of functional groups of the TentaGel, and has a disadvantage that peptides are extricated by using chemicals and therefore an additional peptide purification procedure is required (Franz; A. H., Liu; R., Song; A., Lam; K. S., Lebrilla; C. B., J. Comb. Chem. 5, 125 (2003)).
  • St. Hilaire et al used a photochemical method for extricating peptides from a peptide library in order to identify the substrate specificity of protease, in 2002.
  • the method developed by St. Hilaire et al . utilizes large PEGA resins which have sizes up to from 300 nm to 80 nm, and therefore it is difficult to synthesize millions of peptide libraries (St. Hilaire; Alves! L. C, Herrera; F., Renil; M., Sanderson; S. J., Mottram; J. C; Coombs; G. H., Juliano; M. A., Juliano; L., Arevalo; J., Meldal; M. J. Med. Chem. 45, 1971 (2002)).
  • a kinase is associated with the mechanism of disease transfer and it is very important to understand exactly the function of the kinase based on the clear understanding of the signal transduction by the phosphorylation. It is also very important to sequencing the substrate to which the kinase acts in order to analyze a novel kinase and map the gene of the novel kinase according to the Human Kinome Map thereby using as a marker for drug discovery and development.
  • fluoresein-labeled antiphosphotyrosine antibody was used to analyze the phosphorylation pattern of a substrate, and the study for the analysis of the kinase substrate specificity was performed with analyzing the active profile of the fluoresein-labeled antiphosphotyrosine antibody (Mahesh, U. (2003), US Patent No. 6,806,056).
  • Franz et al discloses a method for preparing a small-sized compound library on a solid support resin and extricating the library using chemicals J. Comb. Chem. , 2003, Vol. 5, 125).
  • Franz et al prepared the bioactive compound library with TentaGel and a methionine linker, and finally extricated the analyte using cyanogen bromide (CNBr).
  • CBr cyanogen bromide
  • the present inventors have recognized that, after synthesizing a peptide on a core-shell bead as a ladder sequence, phosphorylat ing the synthesized peptide with a kinase, reacting the phosphorylated peptide library with an antibody which is conjugated with one of a phosphatase and a peroxidase and binds selectively to a phosphorylated amino acid, the phosphorylated peptide sequence can be more easily and exactly identified than the prior arts by paying attention to the selection of a solid support resin by detecting the change of property, e.g. color, due to the reaction of the sbustrate with one of the phosphatase and the peroxidase. Also, the present inventors have recognized that the mass analysis of the peptide can be made easy by the introduction of a photolabile linker to the core-shell bead. The present invention has been completed. [Disclosure] [Technical Problem]
  • the primary object of the present invention is to provide a process for indentifying a kinase substrate specificity, comprising: i ) linking a plurality of peptides, having a same amino acid sequence, to the surface of a solid support resin to obtain a peptide library which has peptides, with the same amino acid sequence, on the surface of the solid support resin; ii) repeating the step i) with changing the amino acid sequence to give a random mixture of peptide libraries, wherein each solid suppot resin has peptides with a same amino acid sequence, however the amino acid sequence liked to each solid support is different from one another; iii) phosphorylating selectively a specific amino acid of a peptide which has a certain amino acid sequence by reacting the random mixture of the peptide libraries with a kinase, to give a phosphorylated peptide library; iv) reacting the phosphorylated peptide library with an antibody which is conjugated with
  • Another object of the present invention is to provide a peptide library represented by the following formula: ⁇ 29> Z-X-X-Y-X-X-Z-Spacer-Linker-sol id support resin
  • Z represents one of the essential amino acids excluding cysteine!
  • X represents an amino acid which is one of the essential amino acids excluding cysteine and not identical to Y;
  • Y represents tyrosine, threonine or serine!
  • Spacer represents a linear or branched peptide which consists of 1 to 6 amino acids and contains alkyl and/or ether! and
  • Linker represents a 2- nitrobenzyl derivative, a 2-(2-nitrophenyl)propyl derivative, a benzoin derivative or a phenacyl derivative.
  • the abovement ioned primary object of the present invention can be achieved by providing a process for indentifying a kinase substrate specificity, comprising: i) linking a plurality of peptides, having a same amino acid sequence, to the surface of a solid support resin to obtain a peptide library which has peptides, with the same amino acid sequence, on the surface of the solid support resin! ii) repeating the step i) with changing the amino acid sequence to give a random mixture of peptide libraries, wherein each solid suppot resin has peptides with a same amino acid sequence, however the amino acid sequence liked to each solid support is different from one another!
  • the present invention employs a mass spectrometer to identify a kinase substrate specificity.
  • the present invention employs a OBOC synthetic technique including a ladder peptide to investigate fast and effectively the phosphorylation of the kinase.
  • the process according to the present invention can be applicable to not only a tyrosine kinase but also a serine and threonine kinase.
  • the substrate specificity of p60 and ZAP-70 was identified by the process of the present invention.
  • an OBOC ladder peptide is synthesized.
  • the peptide library is synthesized by the split and pool synthesis using a resin in the form of a core-shell.
  • the photolysis efficacy of the core-shell resin is at least twice higher than that of the TentaGel since amino groups are present on the surface of the core-shell resin (Kim, H., Cho, J. K., Chung, W. J., Lee, Y. S., Org. Lett., 6, 3273 (2004); Korean patent application No. 10- 2004-0078860).
  • the peptide library is randomly synthesized with 18 amino acids excluding cysteine and tyrosine at two sites respectively on either side of tyrosine, total four sites.
  • Alanine is introduced at N-terminal and C- terminal of the peptide such that the kinase approaches easily the peptide synthesized on the bead.
  • the amino acid at N- and C-terminal can be any of all the amino acids excluding alanine.
  • a photolabile linker is introduced onto the surface of the bead in order to dissociate the peptide from the bead.
  • the spacer composed of ( ⁇ -Ala)-( ⁇ - ACA)-C ⁇ -AIa)-C ⁇ -ACA) (BEBE) is also introduced next to the linker in order to avoid coinciding the mass of the peptide with that of matrix, an auxiliary agent, used for mass analysis.
  • the photolabile linker may be selected from a 2-nitrobenzyl derivative, a 2-(2-nitrophenyl)propyl derivative, a benzoin derivative or a phenacyl derivative and the spacer may be a linear or branched peptide which consists of 1 to 6 amino acids and contains alkyl and/or ether. Consequently, the peptide library sequence is Ala-X-X-Tyr-X-X-AIa-BEBE-NH 2 (Fig. 1). About 30 mg of the synthesized beads has more than about 100,000 random sequences, and therefore the beads can fully contain four amino acid combinations.
  • peptide libraries are prepared.
  • the beads are washed with a phosphate-buffered solution (PBS), and are treated with a blocking solution in order to prevent a non-specific protein adsorption.
  • PBS phosphate-buffered solution
  • phosphorylation takes place.
  • the phosphorylation is proceeded by mixing a Tris-HCL buffer, ATP and a kinase. After the phosphorylation is completed, the reacting solution is removed and the beads are washed with Type EI water.
  • an antibody (Ab) is conjugated with the bead and the antibody used is an anti-phosphotyrosine Ab to which an alkaline phosphatase is bound.
  • the Ab is reacted with the bead, the Ab is conjugated with only the phosphorylated bead. After reaction, the resultants are washed.
  • the selection of the peptide library is performed. That is, when the substrates are reacted with the alkaline phosphatase conjugated with the Ab used in the step iv), phosphorylated beads can be detected by their color change.
  • the alkaline phosphatase is reacted with the beads in the PBS solution in which BCIP (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt) and NBT (nitro-blue tetraazolium chloride) are dissolved.
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
  • NBT nitro-blue tetraazolium chloride
  • UV rays with a wavelength of 365 nm is irradiated to the color-changed beads which has been selected one by one with forceps and a microscope and then the peptides linked to the beads are dissociated.
  • the dissociated peptides are analyzed by MALDI-TOF.
  • the organic auxiliary agent used during the analysis is DHB (2,5-dihydroxybenzoic acid in 70% MeOH).
  • a molecular modeling study was performed to analyze the difference of reactivity between the tyrosine kinase and the optimal substrates. The molecular modeling was carried out by using the molecular modeling software c-src package, SYBYL 7.1.
  • the structure of p60 and ZAP-70 kinases were obtained from the Protein Data Bank. Flexible docking was performed by using FlexX in order to predict X-ray crystal structure of the kinase-substrate complex.
  • the optimal structure of the kinase-substrate complex can be obtained by minimizing the RMS gradient of amino acid residues within the range of 15 A from the active site so as to be below 0.05 kcal/mol/A.
  • Another object of the present invention can be achieved by providing a peptide library represented by the following formula:
  • Z represents one of the essential amino acids excluding cysteine
  • X represents an amino acid which is one of the essential amino acids excluding cysteine and not identical to Y
  • Y represents tyrosine, threonine or serine
  • Spacer represents a linear or branched peptide which consists of 1 to 6 amino acids and contains alkyl and/or ether
  • Linker represents a 2- nitrobenzyl derivative, a 2-(2-nitrophenyl)propyl derivative, a benzoin derivative or a phenacyl derivative.
  • the solid support resin has a core-shell structure.
  • the kinase substrate specificity can be fast determined by the combination of MALDI-TOF mass spectrometry and the synthetic technique of the OBOC peptide library comprising the ladder peptide according to the present invention.
  • the thus obtained substrate sequence is well in accord with the motif on which a kinase actually acts in vivo, which is very important to understand exactly the function of the protein kinase and is able to give significant information to develop a protein kinase inhibitor.
  • the process of the present invention can be applicable to the serine and threonine kinases as well as to the tyrosine kinase.
  • Fig. 1 is a schematic diagram of the library bead according to the present invention.
  • Fig. 2 is a flow diagram of the identification of the kinase substrate specificity by using KIPP-MS according to the present invention.
  • Fig. 3 shows the results of the mass analysis of Example 5 of the present invention.
  • Fig. 4 shows the sequence of the p60 phosphorylated by the tyrosine kinase.
  • Fig. 5 is a graph showing the number of appearance of amino acid c ⁇ src according to the position of the tyrosine kinase, p60 , which is obtained by the statistical calculation.
  • Fig. 6 shows the results of the analysis of the sequences phosphorylated by the tyrosine kinase, ZAP-70, by using MALDI-TOF.
  • Fig. 7 is a graph showing the number of appearance of amino acid according to the position of the tyrosine kinase, ZAP-70, which is obtained by the statistical calculation.
  • Fig. 8 shows proteins of which sequence is in accord with the substrate specificity sequence of the tyrosine kinase, ZAP-70, which is identified by NCBI human protein database and KIPP-MS.
  • Fig. 6 shows the results of the analysis of the sequences phosphorylated by the tyrosine kinase, ZAP-70, by using MALDI-TOF.
  • Fig. 7 is a graph showing the number of appearance of amino acid according to the position of the tyrosine kinase, ZAP-70, which is obtained by the statistical calculation.
  • Fig. 8 shows proteins of which sequence is
  • FIG. 9 (a) shows the results of the three dimensional docking of the optimal substrate peptide obtained by KIPP-MS on the active site of the tyrosine kinase, p60
  • Fig. 9 (b) shows the results of the three dimensional docking of the optimal substrate peptide obtained by KIPP-MS on the active site of the tyrosine kinase, ZAP-70.
  • HiCore support resin (0.3 mmol/g) was swelled within 30 mL of NMP (N-methyl-2-pyrrolidone), and then 6 mmol of Fmoc-photolabile linker (Fmoc-PLL, Fmoc-4-[4-(l-aminoethyl)-2-methoxy-5-nitrophenoxy]butyric acid) , 0.6 mmol of benzotriazol-l-yloxytris(dimethylamino) ⁇ hosphonium hexafluoro- phosphate, BOP), 0.6 mmol of 1-hydroxybenzotriazole (HOBt), and 1.2 mmol of diisopropylethylene (DIEA) were dissolved in 10 mL of NMP.
  • NMP N-methyl-2-pyrrolidone
  • the peptide library was prepared using split and pool synthesis. Alanine was introduced at N-terminal and C-terminal of the peptide and tyrosine was introduced in the middle of the peptide to synthesize the peptide library having a structure of Ala-X-X-Tyr-X-X-AIa-BEBE-PLL-HiCore, in order that the kinase approaches easily the peptide synthesized on the bead. As shown in Fig. 1, the peptide sequence of one-bead one-sequence, was synthesized as a form of a ladder.
  • PLL is a photolabile linker.
  • the PLL is decomposed by the exposure to 365 nm UV rays.
  • the BEBE which is a spacer is introduced to distingush from matrix used for mass analysis.
  • the X represents 18 amino acids excluding cysteine and tyrosine.
  • Example 3 Preparation of a phosphorylated peptide library ⁇ 75>
  • the peptide library support resin was washed with 50 mM PBS.
  • blocking solution 3% BSA, Tween20 in PBS
  • beads were washed with the blocking solution.
  • Kinase was reacted with 25 mM Tris- HCl buffer solution (pH 7.4, 7 mM MgCl 2 , 0.5 mM EGTA), 100 M ATP and 2 units c-src of p60 or ZAP-70 for 1 to 2 hr at 20 to 30 °C .
  • the reaction solution was removed and the beads were washed with Type HI water.
  • the alkaline phosphatase was dissolved in the 50 mM PBS solution in which 0.1 mg/mL of BCIP (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt) and 0.1 mg/mL of NBT (nitro-blue tetrazolium chloride) had been dissolved, and then the solution was reacted with the beads for 30 min to 1 hr at 37 ° C followed by the washing with Type IH water.
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
  • NBT nitro-blue tetrazolium chloride
  • Fig. 2 (A) the OBOC library beads were phosphorylated by the reaction with the kinase. Then, the phosphorylated beads were reacted with the anti-phosphoamino acid Ab bound to a phosphatase. The phosphatase bound to the antibody was used for detecting the phosphorylated bead. That is, when the phosphatase was bound to the antibody and then the phosphatase substrate was added to the library bead, the color of the bead phosphorylated with the phosphatase was changed to clear red. The color- changed beads were picked out. In the step of Fig. 2 (B), the peptides were cleaved from the selected beads by the irradiation of UV rays, and analyzed by using MALDI-TOF.
  • the kinase substrate specificity of ⁇ 60 is c-src characterized in that the phosphorylation reaction of p60 most effectively takes place when acidic amino acids (GIu and Asp) are located at +1 and +2 positions and He is located at -1 position.
  • acidic amino acids GIu and Asp
  • Tyr-Glu-Glu was selected as the optimum substrate through such experiments. He at -1 position interacted electrostatically with Gly406 of p60c-src, and the P-O distance between the hydroxyl group of the substrate, Tyr, and the phosphate group of ATP was as close as about 4.16 A, and consequently the hydrogen bond between Arg388 and As ⁇ 286 which were located within the active site of the kinase was formed.
  • the docking energy ( ⁇ G) of the optimum substrate was -15.76 kcal/mol.
  • the docking energy ( ⁇ G) became -11.22 kcal/mol and the P-O distance 4.34 A (Fig. 9(a)).
  • Glus at +1 and +2 positions were changed with other amino acid, substrates were not able to be inserted into c-src the active site of p60 .

Abstract

La présente invention concerne un processus d'identification de la spécificité d'un substrat kinase au moyen d'une bibliothèque de peptides. Plus particulièrement, ce processus d'identification de la spécificité d'un substrat kinase consiste: (1) à relier une pluralité de peptides présentant la même séquence d'acides aminés à la surface d'une résine support solide pour obtenir une bibliothèque dont les peptides ayant la même séquence d'acides aminés se trouvent sur la surface de la résine support solide; (ii); à répéter l'opération (i) en changeant la séquence des acides aminés de manière à obtenir un mélange aléatoire de bibliothèques de peptides, chaque résine support solide portant des peptides qui ont la même séquence d'acides aminés, mais les séquences d'acides aminés reliées à chaque résine support solide étant différentes les unes des autres; (iii) à phosphoryler sélectivement un acide aminé spécifique d'un peptide présentant une certaine séquence d'acides aminés en faisant réagir le mélange aléatoire des bibliothèques de peptides avec une kinase, ce qui donne une bibliothèque de peptides phosphorylés; (iv) à faire réagir la bibliothèque de peptides phosphorylés avec un anticorps qui est conjugué soit avec une phosphatase, soit une peroxydase et qui se lie sélectivement à un acide aminé phosphorylé, puis à ajouter un substrat qui agit sélectivement sur le mélange de réaction ainsi obtenu; (v) a sélectionner une résine support solide comportant une bibliothèque de peptides dont la propriété a été modifiée en détectant le changement de propriété due à la réaction du substrat avec une phosphatase ou une peroxydase; et (vi) à séparer un peptide de la résine support solide, puis à analyser une séquence dudit peptide.
PCT/KR2008/002632 2007-05-09 2008-05-09 Processus d'identification de la spécificité d'un substrat kinase au moyen d'une bibliothèque de peptides WO2008140230A1 (fr)

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