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WO2009069980A2 - Puce à protéines pour détermination de l'activité kinase ou phosphatase - Google Patents

Puce à protéines pour détermination de l'activité kinase ou phosphatase Download PDF

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Publication number
WO2009069980A2
WO2009069980A2 PCT/KR2008/007069 KR2008007069W WO2009069980A2 WO 2009069980 A2 WO2009069980 A2 WO 2009069980A2 KR 2008007069 W KR2008007069 W KR 2008007069W WO 2009069980 A2 WO2009069980 A2 WO 2009069980A2
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Prior art keywords
protein
kinase
substrate
chip
phosphatase
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WO2009069980A3 (fr
Inventor
Moon-Hi Han
Hye-Jin Lim
Yong-Wan Cho
Yoon-Suk Lee
In-Cheol Kang
Eun-Kyoung Lee
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Proteogen Inc
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Proteogen Inc
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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Definitions

  • the present invention relates to a protein chip for determining kinase or phosphatase activity, and a method of determining the kinase or phosphatase activity.
  • a biochip such as a protein chip or a DNA chip, is obtained by micro-arraying a gene, a piece of DNA, a certain protein, or the like on a substrate.
  • an analytical experiment on a biochemical substance by using the chips there is an advantage in that a large amount of substances can be analyzed in a short period of time by using a small amount of test samples.
  • protein chip research has been actively conducted in diagnostics and new drug discovery, because although biochemical activities occurring in all living things including humans are basically based on DNA information, disease expression is actually caused by a protein having a specific function in a cell, rather than in DNA.
  • kinase is an enzyme which covalently bonds a phosphate group to a residue of tyrosine, serine, or threonine with a specific sequence in a substrate protein, in order to determine the activity of kinase, the measurement of the phosphorylation of various different protein substrates is required. Also, since kinase includes 500 or more various kinds of kinases, there has been a problem in determining the activity on a chip.
  • the inventors of the present invention found that when a substrate protein is immobilized on a well-on-a-chip type substrate by using a calixcrown derivative, it is possible to directly immobilize a substrate protein of an enzyme on a substrate without an additional chemical treatment. In addition, it is possible to measure the extent of the substrate phosphorylation by the reaction of the substrate protein with kinase or phosphatase on a substrate, and to simply analyze an activity of a enzyme and a large amount of enzyme inhibitors on a solid substrate by using a micro- amount (IuI) of expensive test samples, such as an enzyme and a detecting antibody.
  • IuI micro- amount
  • a Well-on-a-Chip type protein chip for determining kinase or phosphatase activity in which a Calixcrown derivative capable of recognizing a cationic functional group of a protein is immobilized on a chip base plate, and a substrate protein of kinase or phosphatase is immobilized by multiple ion recognition of the calixcrown derivative included in a well.
  • a method of determining kinase or phosphatase activity including the steps of: a) treating the protein chip with kinase or phosphatase capable of reacting with a substrate protein; and b) measuring phosphorylation extent of the substrate protein by the treatment.
  • a substrate immobilized on a protein chip it is possible to successfully subject a substrate immobilized on a protein chip to phosphorylation or dephosphorylation by an enzyme, and to detect phosphorylation of the substrate on the protein chip (refer to Examples 2 to 8) .
  • ⁇ a calixcrown derivative' can recognize a cationic functional group of an amino acid on a protein surface, preferably an ammonium group.
  • calixcrown derivative may be a compound represented by Formula 1 or Formula 2.
  • n 1, each of Ri, R 2 , R 3 and R 4 independently represents -CHO, -SH, or -COOH, and each of R 5 and Re independently represents -H, -methyl, -ethyl, propyl, -isopropyl, or -isobutyl; n represents 1, each of Ri, R 2 , R 3 and R 4 independently represents -CH 2 SH, or each of Ri and R 3 represents -CH 2 SH and each of R 2 and R 4 independently represents -H, and each of R 5 and R ⁇ independently represents -H, -methyl, -ethyl, propyl, -isopropyl, or -isobutyl; or n represents 2, each of Ri, R 2 , R 3 and R 4 independently represents -CH 2 SH, or each of Ri and R 3 independently represents -CH 2 SH, and each of R 2 and R 4 represents -H, and each of R 5 and R 6 independently represents -H, -methyl, - e
  • each of Ri, R 2 , R 3 and R 4 independently represents -CH 2 SH, or two of Ri to R 4 are coupled together to form a group (-CH 2 -S-S-CH 2 -) .
  • a substrate protein of kinase or phosphatase is immobilized. Therefore, some problems in concentration, activity, and orientation may be solved.
  • a calixcrown derivative in immobilizing a substrate protein of kinase or phosphatase on a chip base plate by using a calixcrown derivative, it is possible to simply immobilize a protein on a solid substrate surface by multiple ion recognition (which is a molecular recognition method) without any additional processes which have been used in the conventional protein immobilization reaction, for example, chemical treatment of a protein molecule, or genetic conversion (such as a fusion protein) .
  • ion recognition which is a molecular recognition method
  • additional processes which have been used in the conventional protein immobilization reaction for example, chemical treatment of a protein molecule, or genetic conversion (such as a fusion protein) .
  • a substrate protein is immobilized by multiple ion recognition having a weaker strength than a chemical binding strength occurring in the conventional immobilization reaction, surface attraction reduces the function of the substrate protein to a relatively small extent, compared to the conventional method.
  • the protein chip according to the present invention is useful in that the substrate protein' s structure and activity can be maintained during immobilization.
  • a chip base plate may include glass, fused quartz, silicon wafer, plastic, or the like, but glass is preferred.
  • a chip base plate on which a calixcrown derivative is immobilized may be prepared by the following method.
  • the substrate In order to aminate a substrate, such as a glass slide, the substrate is immersed in a piranha solution
  • a calixcrown derivative may be densely immobilized on a chip base plate with uniform distribution, and functions as a molecular linker which makes it possible to immobilize a substrate protein having reactivity with kinase or phosphatase on a protein chip.
  • a Well-on-a-Chip type protein chip in which a substrate protein of kinase or phosphatase is immobilized by the calixcrown derivative in a well, may be prepared by the following method.
  • an adhesive tape in which well-forming holes with an average diameter of 0.1 to 5mm are arranged is attached.
  • the adhesive tape is attached as described above, it is possible to make a well size uniform, to prevent samples between wells from mixing with each other, to reduce an accompanying fluorescence noise, and to minimize an experimental error caused by a micro-amount of sample. This improves accuracy in quantitative analysis. Then, after the substrate protein is diluted with a substrate dilution buffer down to a certain concentration
  • the diluted substrate protein aqueous solution is spotted into each protein chip well by
  • a Well-on-a-Chip type protein chip is prepared. Also, substrate protein concentrations in respective wells may be uniformly or non-uniformly adjusted.
  • a phosphorylated substrate protein may be used as the substrate protein. Also, in using such a phosphorylated protein, before addition of kinase to the protein chip according to the present invention, the substrate protein immobilized on the protein chip may be dephosphorylated.
  • a 'Well-on-a-Chip' is also referred to as a ⁇ well-chip' .
  • ⁇ a substrate protein' includes a peptide as well as a protein.
  • a substrate peptide has a sequence to be phosphorylated by a specific kinase.
  • a purificated substrate protein is used.
  • 'kinase' may be selected from the group including MAPKl (Mitogen-activated protein kinase 1), Aurora kinase A, Aurora kinase B, Akt kinase, Cdkl/cyclin B kinase (Cyclin dependent kinasel) , Cdk2/cyclin A kinase (Cyclin dependent kinase2), IKKa, IKK ⁇ (IkBa kinase- ⁇ / ⁇ ) , MEKl (Mitogen-activated or extracellular signal- regulated protein kinasel) , ZAP-70(Zeta chain-associated protein-70), FGFRl (Fibroblast growth factor receptor 1), GSK3 ⁇ (Glycogen synthase kinase-3 ⁇ / ⁇ ) , JAK3 (Janus kinase 3), AbI kinase (Abelson tyrosine kinase), JNKl
  • phosphatase' may be selected from the group including ⁇ PP (Bacteriophage ⁇ protein phosphatase), PP2A (Protein Phosphatase 2A), PPl
  • Phosphatase IB Phosphatase IB
  • present invention is not limited thereto, and other phosphatases may be used.
  • 'a substrate protein of kinase' may be selected from the group including MBP (Myelin basic protein), Histone H3, FKHR(FOXOIa, Forkhead box 01a), Histone Hl, I ⁇ -B ⁇ (Inhibitor-kappa-B alpha, NF ⁇ BI ⁇ (Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha) ) , MAPK2 (Mitogen-activated protein kinase 2), LATl (Linker for activation of T cell-1) , PLC ⁇ - 1 (Phospho-lipase C gamma-1), GS (Glycogen Synthase), STAT3 (signal transducers and activators of transcription 3), Crk(V-crk sarcoma virus CTlO oncogene homolog (avian) - like protein), Bcl-2 (B-cell lymphoma 2), c-Jun (cellular Jun) ,
  • MBP
  • 'a substrate protein of phosphatase' may be selected from the group including GS (Glycogen Synthase), IR (Insulin Receptor) .
  • the present invention is not limited thereto, and there is no limitation in the substrate protein, as long as it is capable of reacting with phosphatase.
  • a phosphorylated substrate that is, a substrate in an activated state, may be used. In using a substrate which has been expressed by E. coli or other expression systems and then purified, the substrate may already be in a phosphorylated state through physiological activation of a host or a purification process.
  • a substrate of kinase corresponds to another kinase of downstream signal transduction, and is in a phosphorylated state.
  • the phosphorylated substrate needs to be dephosphorylated.
  • treatment with a certain phosphatase may remove a phosphate group attached to the substrate prior to the reaction of a kinase and the substrate. This may increase signal strength against a kinase activity.
  • the protein chip according to the present invention it is possible to carry out both dephosphorylation by phosphatase, and re-phosphorylation by kinase (refer to Example 6) .
  • MAP Kinase Mitogen- activated protein kinase
  • An MAP Kinase signal transduction pathway includes three or more kinases, and transfers external stimulus into a cell by phosphorylating and activating a kinase in the following step. Also, in many cases, the MAPK is phosphorylated itself, thereby amplifying the stimulus.
  • residue of certain amino acids Threonine/tyrosine
  • MEKl various proteins having the specific amino acid sequence of P-X-S/T-P (a cytoskeletal protein, a translation factor, a transcription factor, an Rsk protein, etc.
  • MAP kinase signal transduction system has been revolutionarily conserved in all tissues of all eukaryotes and prokaryotes. Accordingly, inhibitors that specifically inhibit an MAP kinase pathway are very excellent in their applications and very valuable, because they can be developed as pharmaceuticals for inhibiting various diseases (such as cancer, abnormal cell differentiation, rheumatoid arthritis, etc. ) caused by an abnormal MAP kinase pathway as well as reagents for signal transduction research.
  • various diseases such as cancer, abnormal cell differentiation, rheumatoid arthritis, etc.
  • MBP Myelin basic protein
  • an 'Aurora kinase' refers to a kinase which is for centrosome replication during mitosis of an eucaryotic cell, bipolar spindle formation by mitosis, chromosome arrangement on the equatorial plate by a spindle, and accuracy monitoring of a spindle check point. It was found by genetic mutation showing centrosome/chromosome division abnormalities in drosophilia and yeast. Three mammalian isomers of aurora kinases have been identified (aurora-A, aurora-B and aurora-C) . They all share a kinase domain located in the carboxyl terminus, and have similar protein structures.
  • Aurora A has been reported to be over-expressed in tissues of various kinds of cancers, such as breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, etc. and thus there is a possibility that it is an oncogene.
  • Aurora A has been reported that when Aurora A is artificially over-expressed in cells, the cells are transformed into cancer by the increase in centrosome numbers, aneuploidy (abnormal numbers of chromosomes) , and chromosome instability. Accordingly, the Aurora A has been proven to be an oncogene.
  • small molecule inhibitors such as ZM447439, VX- 680, Hesperadin, have been recently developed, the inhibitors being capable of selectively inhibiting phosphorylation by using a competitive inhibitor in an ATP- binding site in a kinase domain. Also, according to clinical tests, such inhibitors have recently been reported to be effective in inhibiting the growth of cancer cells.
  • the inhibitors inhibit phosphorylation of SerlO of Histone H3 by inserting into the ATP-binding site of Aurora kinase. However, they are not anti- mitosis compounds, because they cannot inhibit cell cycle progression.
  • Aurora kinase inhibitors have effects on chromosome arrangement/segregation, but do not cause the delay or retention of mitosis. Nevertheless, after mitosis, the inhibitors induce apoptosis of cells. Accordingly, since a material inhibiting the activity of Aurora kinase as an oncogene is expected to be a novel anti-cancer therapeutic, a representative target substrate for Aurora Kinase, Histone H3, was used in the test in an embodiment of the present invention.
  • a kinase activity determining protein chip may be used to examine specific reactivity in MBP phosphorylation by MAPKl, H3 phosphorylation by Aurora kinase A, Histone H3 phosphorylation by Aurora kinase B, FKHR phosphorylation by Akt kinase, Histone Hl phosphorylation by Cdkl/cyclin B kinase, Histone Hl phosphorylation by Cdk2/cyclin A kinase, I ⁇ -B ⁇ phosphorylation by IKK ⁇ , I ⁇ -B ⁇ phosphorylation by IKKa, MAPK2 phosphorylation by MEKl, LATl phosphorylation by ZAP-70, PLC ⁇ -1 phosphorylation by FGFRl, GS phosphorylation by G
  • a Well-on-a-Chip type protein chip in which a calixcrown derivative is immobilized on a chip base plate, and a substrate protein of kinase or phosphatase is immobilized by the calixcrown derivative included in a well, is treated with kinase or phosphatase, and then, the extent of phosphorylation/ dephosphorylation of a substrate protein by the treatment is measured to determine the activity of the kinase or phosphatase.
  • a kinase reaction solution obtained by mixing a kinase dilution buffer with an ATP/Mg solution is spotted into each well to make the kinase react with a substrate protein.
  • Kinase phosphorylates a substrate protein through a reaction with the substrate protein.
  • a phosphorylated substrate in an activated state, or an auto-phosphorylated substrate by mixing with an ATP/Mg solution may be spotted into each well and immobilized, and the addition of phosphatase may induce dephosphorylation of the substrate.
  • a phospho-specific antibody specifically capable of binding to a phosphorylated substrate may be used.
  • a labeled secondary antibody specifically capable of binding to the Fc fragment of the bounded phospho-specific antibody is used for a reaction.
  • the reactions by these various enzymes and antibodies are finally analyzed through fluorescence distribution/intensity on a protein chip surface by using a microarray fluorescence scanner, and thereby kinase or phosphatase activity on a substrate may be determined.
  • the phospho-specific antibody is diluted to a predetermined concentration and spotted into each well, and then a secondary antibody labeled with a marker is diluted to an appropriate concentration, and spotted into each well.
  • the marker for example, fluorescence distribution
  • the marker may include a fluorescent substance, an enzyme, a radioactive material, a fine article, a colorant, or the like, but the present invention is not limited thereto.
  • another step of adding a candidate for an inhibitor of the kinase or phosphatase may be further included so as to screen for potential inhibitors of the kinase or phosphatase by comparing the inhibition extent of the reaction of the substrate protein and the kinase or phosphatase with a control group.
  • Staurosporine which have been widely used as a kinase inhibitor may be used.
  • a specific inhibitor of each enzyme may be easily determined (refer to Examples 2-9, and 2-10) .
  • a kinase or phosphatase inhibitor screening method is safe and easy to use, compared to a conventional method which uses radioactivity, and thus may be widely used for research. Moreover, the method makes it possible to determine the enzyme activity on a protein chip, and thus may be widely applied to high throughput screening system for economically and efficiently screening a specific inhibitor for a specific enzyme.
  • the kinase or phosphatase activity determination protein chip according to the present invention may be used to screen activators of the enzymes in the similar manner as described for inhibitors, the method of screening activators is within the spirit and scope of the present invention.
  • the present invention relates to a protein chip for determining kinase or phosphatase activity closely related to diseases. Unlike a conventional method in which enzyme activity on a chip cannot be determined, the protein chip according to the present invention has an advantage in that the enzyme activity can be determined on a chip. Also, since it is possible to screen specific inhibitors of various kinases or phosphatases by using a small amount of test samples, the method is expected to act a leading role in novel drug development through new paradigm. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. Ia illustrates the structure of a well chip and overall test processes in the well chip.
  • FIG. Ib schematically illustrates the overall test processes from the standpoint of a protein substrate molecule.
  • FIG. 2 illustrates specific reactivity of an MAPKl enzyme to an MBP substrate.
  • FIG. 3 is a graph illustrating the results from a test for obtaining an appropriate MAPKl concentration in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 4 shows a graph illustrating the results from a test for obtaining an appropriate MBP concentration in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 5 shows a graph illustrating the results from a test for obtaining an appropriate phospho-specific antibody concentration in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 6 shows a graph illustrating the results from a test for obtaining an appropriate MAPKl reaction time against an MBP substrate concentration.
  • FIG. 7 shows a graph illustrating reactivity of a phospho-specific antibody over time in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 8 shows a graph illustrating the results from a test for obtaining an appropriate ATP concentration in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 9 shows a graph illustrating reactivity against a secondary antibody concentration in the enzyme reaction of an MAPKl with an MBP substrate.
  • FIG. 10 shows a graph illustrating an inhibitory effect of phosphorylation against a concentration of ERK inhibitor II specifically recognizing MAPKl (Mitogen- activated protein kinase 1), and an inhibitory effect against an ATP concentration, as numerical values converted from fluorescent images.
  • MAPKl Mitogen- activated protein kinase 1
  • FIG. 11a and lib illustrate specific reactivity of Aurora kinase A to Histone H3 (substrate), and specific reactivity of a phospho-specific antibody, as scanned fluorescent images.
  • FIG. 12 shows a graph illustrating the results from a test for obtaining an appropriate Aurora kinase A concentration in the enzyme reaction of Aurora kinase A with a Histone H3 substrate.
  • FIG. 13 shows a graph illustrating the results from a test for obtaining an appropriate Histone H3 concentration in the enzyme reaction of Aurora kinase A with a Histone H3 substrate.
  • FIG. 14 shows a graph illustrating the results from a test for obtaining an appropriate phospho-specific antibody concentration in the enzyme reaction of Aurora kinase A with a Histone H3 substrate.
  • FIG. 15 shows a graph illustrating the results from a test for obtaining an appropriate ATP concentration in the enzyme reaction of Aurora kinase A with a Histone H3 substrate.
  • FIG. 16a and 16b illustrate specific reactivity of Aurora kinase B to Histone H3 (substrate) , and specific reactivity of a phospho-specific antibody.
  • FIG. 17 shows a graph illustrating the results from a test for obtaining an appropriate Aurora kinase B concentration in the enzyme reaction of Aurora kinase B with a Histone H3 substrate.
  • FIG. 18 shows a graph illustrating the results from a test for obtaining an appropriate Histone H3 concentration in the enzyme reaction of Aurora kinase B with a Histone H3 substrate.
  • FIG. 19 shows a graph illustrating the results from a test for obtaining an appropriate phospho-specific antibody concentration in the enzyme reaction of Aurora kinase B with a Histone H3 substrate.
  • FIG. 20 shows a graph illustrating the results from a test for obtaining an appropriate Aurora kinase B reaction time in the enzyme reaction of Aurora kinase B with a Histone H3 substrate, which is based on an enzyme reaction time to a substrate concentration.
  • FIG. 21 shows a graph illustrating the results from a test for obtaining an appropriate ATP concentration in the enzyme reaction of Aurora kinase B with a Histone H3 substrate.
  • FIG. 22 shows a graph illustrating the results from a test for obtaining an appropriate concentration of GSK3 ⁇ and GSK3 ⁇ ⁇ to a dephosphorylated GS substrate.
  • FIG. 23 shows a graph illustrating the results from three times repetition of dephosphorylation and phosphorylation of a GS substrate.
  • FIG. 24 shows a graph illustrating phosphorylation against an APT treatment of an insulin receptor substrate.
  • FIG. 25 shows a graph illustrating dephosphorylation of a phosphorylated substrate against a PTPlB phosphatase concentration .
  • FIG. 26 shows a graph illustrating the results from a test for obtaining an appropriate substrate concentration in dephosphorylation of an insulin receptor substrate by PTPlB phosphatase.
  • FIG. 27 shows scanned fluorescent images obtained by screening inhibitors for Aurora kinase A.
  • Example 1 Preparation of a protein chip As shown in FIG. 1-b, the following method is commonly applied to a kinase or phosphatase activity determination using a protein chip used in the present invention.
  • a glass substrate on which a calixcrown derivative represented by Formula 1 (n represents 1, each of Ri, R2, R3 and R 4 independently represents -CHO, and each of R 5 and Re represents -methyl) is immobilized was prepared.
  • An adhesive tape in which 1.5mm sized holes are uniformly arranged was attached to the glass substrate to provide a well chip.
  • a substrate protein aqueous solution diluted with a substrate dilution buffer to a predetermined concentration was spotted to each well by IuI by using a pipette, and then the well was incubated for about 1 day in an incubator where a humidity of 70% or more is sufficiently maintained.
  • the incubated well chip was immersed in a washing buffer and washed by a shaker for 20 minutes, and then was blocked for 1 hour in a blocking buffer. Then, the well chip was washed with a washing solution for 20 minutes to remove the blocking buffer, and was rinsed with distilled water. Next, compressed nitrogen gas was used to dry the well chip.
  • a kinase or phosphatase dilution buffer containing a predetermined amount of enzyme was optionally mixed with an ATP/Mg solution to provide a kinase or phosphatase reaction solution.
  • a kinase or phosphatase reaction solution was spotted into each well by IuI, and then the well chip was incubated for a predetermined time in a 30 ° C incubator where humidity of 70% or more is maintained.
  • the well chip was taken, washed with a washing solution for 20 minutes, and then was dried by nitrogen gas. After a phospho-specific antibody was diluted to a predetermined concentration and spotted into each well by IuI, the well chip was incubated in a incubator for 30 minutes.
  • the well chip was washed with a washing solution for 20 minutes and was dried by nitrogen gas.
  • a fluorescence-labeled secondary antibody which specifically recognizes an Fc fragment of a phospho-specific antibody derived from antibody producing animals (rats, rabbits, sheep, goats, or the like) was diluted to an appropriate concentration, and was spotted into each well by IuI. Then, the well chip was incubated in a incubator for 30minutes. The incubated well chip was washed with a washing solution for 20 minutes and dried, and then the wells were removed.
  • Example 1 a microarray scanner (GenePix 4000B, Axon Instruments, USA) was used to scan fluorescence distribution on the chip surface, and then the results were analyzed (see FIG. Ia) .
  • the following Examples were carried out in the same manner as described in Example 1, but other conditions which were not specifically described, such as concentrations, times, etc. were varied to find optimum solutions. Meanwhile, all substances used in Examples of the present invention, such as enzymes, substrates, phospho-specific antibodies, secondary antibodies and buffers, are commercially available. Table 1 shows substrates, enzymes, and corresponding phospho-specific antibodies which were used in Examples.
  • a buffer Korean assay buffer in Table 2
  • an ATP/Mg solution (5x ATP/Mg stock solution in Table 2) were purchased from Upstate (USA) .
  • MBP a substrate
  • a 5x kinase assay buffer and a 5x ATP/Mg solution were mixed with distilled water in such a manner that they are diluted to Ix concentration, respectively, and the mixed solution was diluted to an enzyme concentration of 20ng/ul and was spotted into each well by IuI.
  • a phospho- specific antibody was diluted with an antibody dilution buffer to a concentration of lOug/ml, and also a secondary antibody was diluted with an antibody dilution buffer to a concentration of 2ug/ml and was spotted into each well by IuI.
  • the fluorescence scan images as shown in FIG. 2 were obtained.
  • a fluorescence scan image scanned by a microarray scanner is shown by rainbow color displays according to fluorescence intensity so that the image can be instinctively recognized.
  • Example 2-2 reactivity against an enzyme concentration
  • MAPKl an enzyme
  • a substrate diluted with a substrate dilution buffer (refer to Table 5) to a concentration of lOOug/ml was spotted into each well by IuI, and then was immobilized on a chip and was subjected to BSA blocking. Then, an enzyme was gradually diluted with an enzyme dilution solution and an ATP solution to stepwise decreasing concentrations and was spotted by IuI, thereby inducing phosphorylation of the substrate immobilized on a well chip. After incubation, a phospho-specific antibody was diluted with an antibody dilution solution to lOug/ml, and was spotted into respective wells by IuI. Finally, a secondary antibody diluted with an antibody dilution solution to 2ug/ml was spotted by IuI. After incubation and washing, the results were analyzed through the fluorescent images scanned by a microarray scanner.
  • a substrate dilution buffer (refer to Table 5) to a concentration of lOOug/
  • FIG. 3 shows reactivity against a concentration of an MAPKl enzyme, and it can be seen that reactivity gradually increased as concentration increased, and was saturated at a concentration of 10ng/ul. Accordingly, in the following
  • MBP a substrate
  • a substrate was gradually diluted with a substrate dilution buffer to stepwise decreasing concentrations, and was spotted into each well by IuI, and then was immobilized on a chip and was subjected to BSA blocking. Then, an enzyme was diluted with an enzyme dilution reaction solution and an ATP solution to 10ng/ul and was spotted by IuI, thereby inducing phosphorylation of the substrate immobilized on the well chip. After incubation, a phospho-specific antibody diluted to 10ug/ml was spotted into respective wells by IuI. Finally, a secondary antibody diluted with an antibody dilution solution to 2ug/ml was spotted by IuI.
  • FIG. 4 shows reactivity against a concentration of MBP (a substrate) , and it can be seen that the reactivity gradually increased as concentration increased, and was saturated at about 500ng/ul. However, at kinase (-), although fluorescence intensity was 1000 or less, there existed some non-specific reactions. Accordingly, it was determined that a substrate concentration has to be adjusted to 100 ⁇ 250ug/ml.
  • Example 2-5 reactivity against an enzyme reaction time
  • a substrate concentration that is, the values of Vmax, Km
  • 9 well chips were prepared.
  • MBP with different concentrations was spotted and immobilized.
  • IOng a predetermined amount of enzyme was added to the well chips and reacted for different reaction times thereby inducing phosphorylation.
  • one column as a negative control group was not subjected to an enzyme reaction so as to determine whether the phosphorylation was caused by an enzyme reaction or not, and was used for treatment of result values in test result analysis.
  • each chip was subjected to washing, drying, and spotting of a phospho-specific antibody, and then was subjected to washing, drying, and spotting of a secondary antibody.
  • a fluorescence scanner was used to scan fluorescent images, and the images were converted to numerical values. Then, graphs against a concentrations were drawn. As shown in FIG. 6, it can be seen that the saturation time depended on the substrate concentration. Also, it was found that as the substrate concentration increased, the reaction time decreased. For example, at a substrate concentration of 100ug/ml (as shown in Example 2-3), a saturation time was about 30 minutes, and at a concentration higher than lOOug/ml, a saturation time was less than 20 minutes.
  • the preferable reaction time ranges from 30 to 60 minutes.
  • reaction time of a phospho-specific antibody varied from 0 minute to 2 hours. Since an enzyme was added to alternate columns under the same conditions, the columns were divided into phosphorylated columns by substrate immobilization and enzyme reaction, and non- phosphorylated columns. With different reaction times of 0, 15, 30, 45, 60, 90, and 120 minutes, phospho-specific antibodies were spotted into respective wells. A secondary antibody directed against a phospho-specific antibody was spotted with a concentration of 2ug/ml by IuI, and fluorescence patterns were analyzed by a microarray scanner.
  • fluorescence intensity rapidly increased within 15 minutes, and then after 15 minutes, the intensity gently increased over time.
  • fluorescence intensity increased as phospho- specific antibody reaction time increased, the increase was not significant after 15 minutes.
  • the phospho- specific antibody reaction time was adjusted to 30 minutes.
  • Example 2-7 Reactivity against an ATP concentration
  • a kinase reaction in order to introduce a phosphate group to a substrate, the enzyme activity change against a concentrations of ATP (another substrate) was measured.
  • MBP substrate
  • MAPKl enzyme activity change against a concentrations of ATP (another substrate) was measured.
  • the amount of ATP included in an enzyme reaction solution was adjusted in such a manner that the final concentrations are 0, 10, 50, 100, and 30OuM, and then 1 hour incubation was carried out. After washing, lOug/ml of phospho-specific antibody solution corresponding to each substrate was spotted by IuI, and was reacted.
  • fluorescent images resulting from all Examples for the present invention indicate enzyme reactions by kinase.
  • the most preferable dilution concentration is 1:100.
  • test conditions were optimized, and in order to screen an inhibitor by using such optimum enzyme reaction conditions obtained as above, Staurosporine which has been widely used as a kinase inhibitor was used as a positive control group.
  • DMSO dimethyl methacrylate
  • distilled water instead of DMSO
  • the well chip was taken, and washed for 20 minutes and dried.
  • a phospho-specific antibody (lOug/ml) was spotted by IuI, and after 30-minute incubation, washing, and drying, a secondary antibody was spotted by IuI.
  • a microarray scanner was used to obtain the results of fluorescent images.
  • Table 3 shows a phosphorylation extent against a
  • Staurosporine concentration as numerical values by calculating values resulting from fluorescent images. As the Staurosporine concentration increased, the phosphorylation reaction of an enzyme slightly reduced. Quantitatively, IC50 was obtained at a relatively high concentration of about 3IuM. In other words, it was found that an enzyme inhibitory effect by Staurosporine hardly occurred in MAPKl.
  • ERK inhibitor II Cat. No. 328007, negative control: 328008
  • the material for a negative control group (negative control, 328008) of the inhibitor has a structure where in a small molecule inhibitor structure, one branched amine group (-NH 2 ) is substituted by a hydroxyl group (-0H) , and has an IC50 value of lOOuM or more.
  • FIG. 10 illustrates graphs showing the inhibitory effect of a phosphorylation reaction against an inhibitor concentration through numerical value conversion of fluorescent images.
  • IC50 was about 4uM.
  • a negative control group as described in product information, even at 10OuM, an inhibitory effect hardly occurred.
  • Histone H3 (a substrate) was diluted with a substrate dilution buffer to lOOug/ml, and then a 5x kinase assay buffer and a 5x ATP/Mg solution were mixed in distilled water in such a manner that they are diluted to Ix concentration, respectively. Then, the mixed solution was diluted to an enzyme concentration of 10ng/ul and was spotted into each well by IuI, thereby inducing an enzyme reaction.
  • Phospho-specific antibodies CDAnti-phospho Ser- 10 Histone H3 Ab and (2)Anti-phospho Ser-28 Histone H3 Ab, were diluted with an antibody dilution buffer to lOug/ml. Also, a secondary antibody was diluted with an antibody dilution solution to a 1:100 concentration and was spotted into each well by IuI. In other words, under the same buffer condition, against an existence/non existence of a substrate and an enzyme, the fluorescence scan images as shown in FIG. 11a were obtained. Herein, only the first well satisfying all conditions showed fluorescence intensity.
  • a phosphorylation antibody is bound to a phosphorylated site, which is detected by a secondary antibody, thereby providing fluorescent images shown in a state where all conditions are satisfied.
  • Example 2 In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine an enzyme concentration appropriate for a reaction, Aurora kinase A (an enzyme) was diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing an enzyme reaction. This Example was carried out in the same manner as Example 2-2.
  • FIG. 12 shows the reactivity against a concentrations of Aurora kinase A, and it can be seen that the reactivity gradually increased as concentration increased, and was saturated at a concentration of about 0.23uM( ⁇ 10 ng/ul) . Accordingly, in the following Examples, an enzyme concentration was adjusted to 10ng/ul. [Example 3-3] reactivity against a substrate concentration
  • FIG. 13 shows the reactivity against a concentration of Histone H3, and the reactivity gradually increased as concentration increased. However, since even at a concentration of 5.8uM ( ⁇ 100 ug/ml) , a significant result can be shown, it was determined that the preferable concentration is about 100ug/ml.
  • Example 2 In determining an appropriate concentration of a phospho-specific antibody used for detecting Histone H3 phosphorylation by Aurora kinase A, the detection extent of a phosphate group induced in a substrate was examined by varying only the concentration of the phospho-specific antibody.
  • This Example was carried out in the same manner as Example 2-4. As a result, as shown in FIG. 14, it can be seen that the fluorescence intensity gradually increased as antibody concentration increased, and was saturated at a concentration of about 50ug/ml. However, since even at a concentration of lOug/ml, fluorescence intensity was significantly higher than a negative control group, in the following Examples, phospho-specific antibody was adjusted to about 10ug/ml.
  • Example 3-5 reactivity against an ATP concentration
  • a phosphate group in introducing a phosphate group to a substrate, the enzyme activity change against a concentration of ATP (another substrate) was measured.
  • This Example was carried out in the same manner as Example 2-7.
  • the reactivity was examined by varying only the concentration of ATP while maintaining other conditions. As a result, it can be seen that the reactivity was saturated at 5OuM or more (see FIG. 16) .
  • Table 4 shows a phosphorylation extent against a
  • Staurosporine concentration as numerical values by calculating values resulting from fluorescent images. As the Staurosporine concentration increased, enzyme phosphorylation reduced. Quantitatively, IC50 for MBP-MAPKl
  • Example 2-9 was 3IuM, and IC50 for Histone H3-Aurora A was about 1.3uM. In other words, it was found that an inhibitory effect by Staurosporine on enzymes was higher in
  • Example 3-1 In order to examine specific reactivity of Histone H3 phosphorylated by Aurora kinase B, different conditions were applied to wells. This Example was carried out in the same manner as Example 3-1, except that some conditions, such as the existence/non existence of a substrate, an enzyme, and a phosphorylation antibody, etc. varied.
  • Histone H3 (a substrate) was diluted with a substrate dilution buffer to 100ug/ml, and then a 5x kinase assay buffer and a 5x ATP/Mg solution were mixed in distilled water in such a manner that they are diluted to Ix concentration, respectively. Then, the mixed solution was diluted to an enzyme concentration of 10ng/ul and was spotted into each well by IuI.
  • phospho-specific antibodies Anti-phospho Ser-10 Histone H3 Ab and Anti- phospho Ser-28 Histone H3 Ab were diluted with an antibody dilution buffer to lOug/ml.
  • a secondary antibody was diluted with an antibody dilution solution to a 1:100 concentration and was spotted into each well by IuI.
  • Example 2 In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine the enzyme concentration appropriate for a reaction, Aurora kinase B (an enzyme) was diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing an enzyme reaction. This Example was carried out in the same manner as Example 2-2.
  • FIG. 17 shows the reactivity against a concentration of Aurora kinase B, and it can be seen that the reactivity gradually increased as concentration increased, and was saturated at a concentration of about 5ng/ul. Accordingly, in the following Examples, an enzyme concentration was adjusted to 5ng/ul.
  • Histone H3 a substrate was diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing an enzyme reaction. This Example was carried out in the same manner as Example 2-3.
  • FIG. 18 shows the reactivity against a concentration of Histone H3, and it can be seen that the reactivity gradually increased as concentration increased, and was saturated at a concentration of 5ug/ml or more. Accordingly, in the following Examples, a substrate concentration was adjusted to 5ug/ml.
  • Example 2 In determining the appropriate concentration of a phospho-specific antibody used for detecting Histone H3 phosphorylated by Aurora kinase B, the detection extent of a phosphate group was examined by varying only the concentration of the phospho-specific antibody while maintaining other conditions. This Example was carried out in the same manner as Example 2-4.
  • the fluorescence intensity increased as antibody concentration increased.
  • the fluorescence intensity was saturated at a concentration of 50ug/ml or more, the increase of fluorescence intensity was not significant at a concentration higher than lOug/ml.
  • the concentration was adjusted to about 10ug/ml.
  • Example 2-5 On immobilized substrates at different concentrations, a test for obtaining the optimum reaction time of an enzyme was carried out. This Example was carried out in the same manner as Example 2-5.
  • the reactivity was examined by varying only the ATP concentration while maintaining other conditions. As a result, as shown in FIG. 21, it can be seen that the reactivity gradually increased as APT concentration increased, showed a maximum value at 10OuM, and slightly decreased at a high concentration of 30OuM.
  • Example 2-9 test conditions were optimized, and in order to screen an inhibitor inhibiting such optimum enzyme reaction conditions obtained as above, Staurosporine which has been widely used as a kinase inhibitor was used as a positive control group.
  • This Example was carried out in the same manner as Example 2-9.
  • Table 5 shows a phosphorylation extent against a a Staurosporine concentration as numerical values by calculating values resulting from fluorescent images.
  • Staurosporine concentration increased, the phosphorylation reaction of an enzyme was reduced.
  • IC50 was obtained at a low concentration of about 78uM, compared to the above described Examples (IC50 for MBP-MAPKl at 3IuM, and IC50 for Histone H3-Aurora A at about 1.3uM) . It is expected that it is possible to screen an enzyme reaction inhibitor from several libraries by using the results from this Example.
  • Example 4-1 In order to examine specific reactivity of a specific substrate phosphorylated by a specific kinase, different conditions were applied to wells. This Example was carried out in the same manner as Example 4-1. Under the same conditions, the examination was carried out by varying some conditions, such as the existence/non existence of a substrate, an enzyme, and a phosphorylation antibody, etc. The results, as noted in Table 6, were obtained, and only the first well satisfying all conditions showed high fluorescence intensity.
  • fluorescence intensity of the first well satisfying all conditions was 300 or more times higher than those of negative control groups.
  • the fluorescence intensity value of the positive control group was 10 times higher than those of the negative control groups.
  • a substrate protein immobilized on the protein chip prepared according to Example 1 was treated with a specific phosphatase before a phosphorylation reaction by kinase, so that a preliminarily attached phosphate group can be removed from the substrate thereby increasing signal intensity of kinase activity.
  • Example 6-1 specific reactivity of GSK3 ⁇ / ⁇
  • different test conditions were applied to respective wells. Detailed test processes were the same as described in Example 2-1.
  • the extent of substrate phosphorylation caused by the addition of GSK3 (kinase) was slightly (about 1.5 times) higher than that of the wells excluding kinase. It is assumed that since the GS (substrate) was already phosphorylated, the phospho- specific antibody bound to the substrate.
  • Example 6-1 in order to dephosphorylate a preliminarily phosphorylated substrate GS, a Protein Phosphatase 2A (PP2A) was treated. Detailed test processes were the same as described in Example 6-1, except that phosphatase, instead of kinase, was treated. As noted in Table 2, as a reaction buffer, buffers appropriate for respective phosphatases were used. Also, after dephosphorylation, under the same conditions, kinase was treated, thereby inducing phosphorylation again. Under the same conditions, against an existence/non existence of phosphatase and kinase, fluorescence intensity values were obtained as noted in Table 9 (GSK3 ⁇ ) and Table 10 (GSK3 ⁇ ) .
  • the phosphorylation extent of the substrate was shown (Table 9:11745, Table 10:13730), and in the third well which was treated with only phosphatase, the phosphorylation extent was about 2.5 times smaller than that of the first well (Table 9:4670, Table 10:5365) . Also, in the fourth well which was dephosphorylated and then was phosphorylated by kinase, the phosphorylation extent (Table 9:27548, Table 10:34169) was almost similar to that (Table 9:26526, Table 10:34257) of the second well where phosphorylation was treated on a non- dephosphorylated substrate.
  • a site dephosphorylated by a phosphatase corresponds to a site of an amino acid residue phosphorylated by kinase.
  • Example 6-3 phosphorylation of dephosphorylated substrate against a kinase concentration
  • GS substrate
  • ⁇ PP ⁇ -phosphate
  • GSK3 ⁇ and ⁇ enzyme reaction were diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing an enzyme reaction.
  • This Example was carried out in the same manner as Examples 2-2 and 6-2.
  • FIG. 22 shows the reactivity against a concentrations of GSK3 ⁇ and ⁇ on a dephosphorylated substrate GS.
  • fluorescence intensity corresponds to the phosphorylation extent of a substrate dephosphorylated by ⁇ PP treatment.
  • Example 6-2 In order to confirm the results from Example 6-2, dephosphorylation by using ⁇ PP with dephosphorylation activity higher than PP2A, phosphorylation by treatment of GSK3 ⁇ / ⁇ , and dephosphorylation by using ⁇ PP were repeatedly performed three times.
  • the results as shown in FIG. 23 were caused by GSK3 ⁇ , and showed that within an error deviation range, dephosphorylation-phosphorylation were repeated with the same fluorescence intensity.
  • kinase assay buffer-1 and ATP/Mg buffer as noted in Table 2 were diluted with a Ix aqueous solution and were added to make MnCl 2 with a concentration of 20 Mm, thereby inducing phosphorylation.
  • anti- phospho-IR (Tyrll58/Tyrll62/Tyrll63) antibody capable of recognizing a tyrosine residue (Tyrll58/Tyrll62/Tyrll ⁇ 3) of the substrate was used for 1-hour reaction, and then anti- rabbit IgG-Cy5 fluorescent antibody capable of recognizing the above antibody was used to examine the phosphorylation extent of the tyrosine residue.
  • FIG. 24 shows phosphorylation extents of a substrate against a whether ATP was treated or not, under the same substrate concentration condition. It can be seen that ATP treatment induced a high extent of phosphorylation, and a treatment with a concentration of 20ng/ul induced the highest extent of phosphorylation.
  • tyrosine phosphorylation of a substrate was induced through APT treatment in an aqueous solution, and the substrate was immobilized on a protein chip. Then, in order to examine the dephosphorylation extent against a
  • a phosphatase was treated with a concentration of 10 to 0.2ng/ul for 1-hour reaction.
  • a phospho antibody capable of recognizing a tyrosine residue, and anti-rabbit IgG-Cy5 fluorescent antibody were treated to examine dephosphorylation of a tyrosine residue against a PTPlB phosphatase concentration.
  • Example 7-2 a substrate was phosphorylated against concentrations, and was immobilized on a protein chip. After treatment of PTPlB phosphatase with a concentration of 5ng/ul, in the same manner as Example 7-1, phospho antibody capable of recognizing a tyrosine residue of the substrate and anti-rabbit IgG-Cy5 fluorescent antibody were treated to detect dephosphorylation .
  • Example 3-6 Staurosporine which has been widely used as a kinase inhibitor was used as the positive control group.
  • the library material was selected from about 800 crude drug samples extracted from ethanol and water, which were provided from the plant extract bank of Korea bio venture center. Each library was diluted to a concentration of 50ug/ml. Detailed test processes were the same as described in Example 2-9.

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Abstract

Puce à protéines de type Well-on-a-Chip (puits sur puce) permettant de déterminer une activité kinase ou phosphatase, dans laquelle un dérivé Calixcrown capable de reconnaître un groupe fonctionnel cationique d'une protéine kinase ou phosphatase est immobilisé sur une plaque de base pour puce, un substrat protéique de kinase ou de phosphatase étant immobilisé par reconnaissance à ions multiples d'un dérivé de Calixcrown inclus dans le puits. Est également décrite une méthode faisant intervenir la puce à protéine de l'invention, qui consiste: (a) traiter la puce à protéine avec une kinase ou une phosphatage pouvant réagir avec un substrat protéique; et (b) à mesurer l'ampleur de la phosphorylation de la protéine substrat provoquée par le traitement. La puce à protéine et la méthode de l'invention permettent d'analyser de façon l'activité d'une kinase ou d'une phosphatase sur une plaque de base pour puce au moyen d'une faible quantité de réactif.
PCT/KR2008/007069 2007-11-29 2008-11-28 Puce à protéines pour détermination de l'activité kinase ou phosphatase Ceased WO2009069980A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013152967A1 (fr) 2012-04-13 2013-10-17 Universite Libre De Bruxelles (Ulb) Matériaux enduits de calixarènes
WO2013136334A3 (fr) * 2012-03-14 2013-11-07 Marx Stephen Moyens et procédés pour le diagnostic et la thérapeutique de maladies
CN108823180A (zh) * 2018-06-29 2018-11-16 云南大学 使蛋白激酶b特定位点去磷酸化的蛋白及其核酸
WO2020188470A1 (fr) * 2019-03-15 2020-09-24 Innopharmascreen Inc. Calixcrowns et utilisations associées

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US20060057026A1 (en) * 2004-09-14 2006-03-16 Boiadjiev Vassil I Gold thiolate and photochemically functionalized microcantilevers using molecular recognition agents

Cited By (9)

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Publication number Priority date Publication date Assignee Title
WO2013136334A3 (fr) * 2012-03-14 2013-11-07 Marx Stephen Moyens et procédés pour le diagnostic et la thérapeutique de maladies
EP2825886A4 (fr) * 2012-03-14 2015-11-18 Stephen Marx Moyens et procédés pour le diagnostic et la thérapeutique de maladies
EP3173788A3 (fr) * 2012-03-14 2017-07-12 Marx, Stephen Moyens et procédés pour le diagnostic et la thérapeutique de maladies
WO2013152967A1 (fr) 2012-04-13 2013-10-17 Universite Libre De Bruxelles (Ulb) Matériaux enduits de calixarènes
US10329232B2 (en) 2012-04-13 2019-06-25 Universite Libre De Bruxelles (Ulb) Materials coated with calixarenes
US11124617B2 (en) 2012-04-13 2021-09-21 Universite Libre De Bruxelles (Ulb) Materials coated with calixarenes
CN108823180A (zh) * 2018-06-29 2018-11-16 云南大学 使蛋白激酶b特定位点去磷酸化的蛋白及其核酸
WO2020188470A1 (fr) * 2019-03-15 2020-09-24 Innopharmascreen Inc. Calixcrowns et utilisations associées
CN113614072A (zh) * 2019-03-15 2021-11-05 创新药物筛选有限公司 杯冠化合物及其用途

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