WO2009069980A2 - Protein chip for determining kinase or phosphatase activity - Google Patents

Abstract

Disclosed is 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. Also, a method using the disclosed protein chip includes 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. Through the disclosed protein chip and method, it is possible to simply analyze the activity of kinase or phosphatase on a chip base plate by using a small amount of reagent.

Description

PROTEIN CHIP FOR DETERMINING KINASE OR PHOSPHATASE ACTIVITY

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention relates to a protein chip for determining kinase or phosphatase activity, and a method of determining the kinase or phosphatase activity.

2. Description of the Prior Art

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. In 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. Especially, 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.

However, on various enzymes existing in a living body, such as kinase, phosphatase, hydrolase, oxidation- reductase, or the like, a method of determining the activity on a chip has not been commercialized yet.

Moreover, since 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.

Thus, various methods of determining activity of an enzyme such as kinase have been proposed, but most of them require expensive analyzing/testing equipment. A method of determining activity of an enzyme on a plate has also been proposed. However, in this method, immobilization process of a substrate protein is complicated. In addition, there is a problem in that during a formation of a chemical bond, the structure of the substrate protein may be changed, by which the activity is lost. Thus, this method is not appropriate for a screening system of novel drug candidates. Also, one reason a chip-based enzyme activity determining system has not been developed is that enzyme activity determination has been generally carried out in an aqueous solution, which makes it difficult to find a technical breakthrough in a protein chip using a surface- immobilized substrate or a phospho-specific antibody.

SUMMARY OF THE INVENTION

Although various methods for activity determination of an enzyme such as kinase have been proposed, a method of determining the activity of an enzyme on a chip by using a protein chip without an additional chemical treatment has not been known, the protein chip including a substrate protein immobilized thereon. Until now, a method of immobilizing a substrate protein on a substrate without a structural/chemical change of the substrate protein has not been known. Also, in the case of an immobilized substrate protein, it has not been known that it is possible to directly measure a phosphorylation extent by the reaction between an enzyme and a protein 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.

The present invention is based on this finding. In accordance with an aspect of the present invention, there is provided 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. In accordance with another aspect of the present invention, there is provided a method of determining kinase or phosphatase activity, the method 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. Hereinafter, the present invention will be described in detail.

According to the present invention, 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) .

In the present invention, ^Λa calixcrown derivative' can recognize a cationic functional group of an amino acid on a protein surface, preferably an ammonium group.

One cation is bound per one calixcrown derivative molecule, and one substrate protein molecule is immobilized on two or more calixcrown derivatives by multiple ion recognition. One example of the calixcrown derivative may be a compound represented by Formula 1 or Formula 2.

[Formula 1]

In Formula 1, n represents 1, each of Ri, R₂, R₃ and R₄ independently represents -CHO, -SH, or -COOH, and each of R₅ and Re independently represents -H, -methyl, -ethyl, propyl, -isopropyl, or -isobutyl; n represents 1, each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or each of Ri and R₃ represents -CH₂SH and each of R₂ and R₄ independently represents -H, and each of R₅ and Rξ independently represents -H, -methyl, -ethyl, propyl, -isopropyl, or -isobutyl; or n represents 2, each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or each of Ri and R₃ independently represents -CH₂SH, and each of R₂ and R₄ represents -H, and each of R₅ and R₆ independently represents -H, -methyl, - ethyl, propyl, -isopropyl, or -isobutyl.

In Formula 2, each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or two of Ri to R₄ are coupled together to form a group (-CH₂-S-S-CH₂-) .

In the present invention, through multiple ion recognition for recognizing a great amount of cations (of an ammonium group, or the like) distributed on the opposite side of an active site of a protein by using a crown ring of a calixcrown derivative, a substrate protein of kinase or phosphatase is immobilized. Therefore, some problems in concentration, activity, and orientation may be solved.

In the present invention, 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) . Moreover, it is possible to provide a protein monolayer on a chip base plate, in which substrate protein molecules are densely packed so that other protein molecules cannot be immobilized in an empty space on the chip base plate by a non-specific immobilization reaction.

In the above described substrate protein monolayer, there is little empty space capable of having an influence on the following measurements, and thus, it is possible to simultaneously solve problems which have occurred in a conventional immobilization method, such as a concentration problem, a non-specific immobilization reaction problem, or the like.

In the present invention, since 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.

Also, in the substrate protein of kinase or phosphatase, since a site where a great amount of cations of an ammonium group, or the like are distributed is opposite to a site where an enzyme is bonded or a phosphorylation/ dephosphorylation site, an orientation problem may be solved to a proper extent.

According to the present invention, when a substrate protein of kinase or phosphatase is immobilized on a self- assembled monolayer of a calixcrown derivative, the additional extra chemical treatment may be omitted. Thus, the protein chip according to the present invention is useful in that the substrate protein' s structure and activity can be maintained during immobilization.

Meanwhile, 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.

In order to aminate a substrate, such as a glass slide, the substrate is immersed in a piranha solution

(obtained by mixing hydrogen peroxide and concentrated sulfuric acid in a ratio of 1:2~3) for 1 hour, and then washed with water and acetone and dried. In a solution prepared by dissolving a calixcrown derivative compound in an organic solvent, such as CHCI₃, at a concentration of l~3mM, the glass slide is immersed for 4 to 6 hours, and then taken and washed with an acetone solution and water, respectively, followed by drying. Herein, 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.

Meanwhile, 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.

On a chip base plate where a calixcrown derivative is immobilized, an adhesive tape in which well-forming holes with an average diameter of 0.1 to 5mm are arranged is attached. When 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

(several nmoles to ]i moles) , the diluted substrate protein aqueous solution is spotted into each protein chip well by

0.1 to 5ul, and is incubated and immobilized with a humidity of 70 to 90%, so that protein denaturation by the drying of the substrate protein solution does not occur. Then, a Well-on-a-Chip type protein chip is prepared. Also, substrate protein concentrations in respective wells may be uniformly or non-uniformly adjusted. Herein, as the substrate protein, a phosphorylated substrate protein may be used. 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.

In the present invention, a 'Well-on-a-Chip' is also referred to as a ^λwell-chip' .

In the present invention, ^λa substrate protein' includes a peptide as well as a protein. There is no limitation in the molecular weight of a substrate peptide as long as the substrate peptide has a sequence to be phosphorylated by a specific kinase. Preferably, a purificated substrate protein is used.

In the present invention, '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, IKKβ(IkBa kinase- α/β) , MEKl (Mitogen-activated or extracellular signal- regulated protein kinasel) , ZAP-70(Zeta chain-associated protein-70), FGFRl (Fibroblast growth factor receptor 1), GSK3α (Glycogen synthase kinase-3α/β) , JAK3 (Janus kinase 3), AbI kinase (Abelson tyrosine kinase), JNKl (c-Jun N-terminal Kinase 1), JNK2(c-Jun N-terminal kinase 2) . However, the present invention is not limited thereto, and other kinases may be used. In the present invention, phosphatase' may be selected from the group including λPP (Bacteriophage λ protein phosphatase), PP2A (Protein Phosphatase 2A), PPl

(Protein Phosphatase 1), PTPlB (Protein Tyrosine

Phosphatase IB) . However, the present invention is not limited thereto, and other phosphatases may be used.

In the present invention, '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, Iκ-Bα(Inhibitor-kappa-B alpha, NFκBIα (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) , PLCγ- 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) , or the like. However, 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 kinase.

In the present invention, 'a substrate protein of phosphatase' may be selected from the group including GS (Glycogen Synthase), IR (Insulin Receptor) . However, 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. Also, as the substrate protein of kinase or 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. Also, in a commercially available substrate, there is a case where a substrate of kinase corresponds to another kinase of downstream signal transduction, and is in a phosphorylated state. Thus, when such a substrate is used, there is a problem in that the kinase-dependent signal is weak. Therefore, the phosphorylated substrate needs to be dephosphorylated. In the present invention, in determining the enzyme activity of kinase, after a substrate protein is immobilized on a protein chip base plate, 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. Meanwhile, on 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) .

In the present invention, 'MAP Kinase (Mitogen- activated protein kinase) ' is in a down-stream of signal transduction pathways. 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. When the residue of certain amino acids (Threonine/tyrosine) is doubly phosphorylated and activated by MEKl /2, 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. ) are phosphorylated. Thus, finally, a signal is transferred into a nucleus, thereby activating a transcription factor, etc. and phosphorylating other intracellular target proteins. This induces the growth, differentiation and physiological activity of a cell. Such an 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.

Therefore, in an embodiment of the present invention, in order to prepare a protein chip for determining the enzyme activity of MAP Kinasel, MBP (Myelin basic protein) which is generally used for MAPKl activity test, was used as a substrate.

In the present invention, 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. They have different intracellular positions despite structural similarity, and show different functions. They are positioned in specific positions of chromosomes which have been reported to have relations with the pathogenesis/progress of cancer. Especially, 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. Actually, it 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.

Based on the fact that Aurora kinase functions as an oncogene, in order to inhibit the activity of Aurora kinase, 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. In other words, 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.

In addition to the above mentioned two targets, it is possible to use various couples of enzymes and substrates, as substrate proteins and kinases: activity of Akt (an oncogene) using FKHR (a transcription factor) as a substrate, activity of Cdkl, 2 (a cell cycle control enzyme) using Histone Hl (for forming nucleosomes, together with Histone H3) as a substrate, activity of IKKa, β using Iκ-Ba (playing an important role in NF-κB signal transduction) as a substrate, activity of MEKl using MAPK2 (an upstream signal transduction factor of the above mentioned MAPK signal transduction) as a substrate, activity of ZAP-70 using LAT (playing an important role in signal transduction of T-lymphocyte) as a substrate, activity of FGFRl using PLCγ-1 (playing an important role in receptor signal transduction) as a substrate. Also, since commercially available substrates and kinases are used, it is possible to test the activity of GSK3 using Glycogen synthase as a substrate in substrate-dependent embodiments. Also, a kinase activity determining protein chip according to the present invention 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, Iκ-Bα phosphorylation by IKKβ, Iκ-Bα phosphorylation by IKKa, MAPK2 phosphorylation by MEKl, LATl phosphorylation by ZAP-70, PLCγ-1 phosphorylation by FGFRl, GS phosphorylation by GSK3α, STAT3 phosphorylation by JAK3, Crk phosphorylation by AbI kinase, Bcl-2 phosphorylation by JNKl, c-Jun phosphorylation by JNK2, IR dephosphorylation by PTPlB (refer to Examples 2 to 7) .

According to the present invention, 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.

Herein, in kinase treatment, 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.

Meanwhile, in phosphatase treatment, 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.

Next, in an example method of determining the extent of phosphorylation of a substrate protein by the above mentioned treatment, a phospho-specific antibody specifically capable of binding to a phosphorylated substrate may be used. Then, a labeled secondary antibody specifically capable of binding to the Fc fragment of the bounded phospho-specific antibody is used for a reaction. Next, 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. Herein, 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. After incubation, the marker (for example, fluorescence distribution) on the protein chip surface is analyzed to determine the enzyme activity of the kinase or phosphatase. Such a 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.

Meanwhile, before the treatment of kinase or phosphatase on the Well-on-a-Chip type protein chip according to the present invention, 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.

In order to screen a reaction activity inhibitor of kinase, as a positive control group, Staurosporine which have been widely used as a kinase inhibitor may be used. By comparing the inhibitory effect in a reaction between kinase and a substrate protein with an inhibitory effect in the positive control group, a specific inhibitor of each enzyme may be easily determined (refer to Examples 2-9, and 2-10) .

As described above, a kinase or phosphatase inhibitor screening method according to the present invention 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.

Meanwhile, since 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.

As described above, 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.

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 GSK3α and GSK3α β 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.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Hereinafter, an embodiment of the present invention will be described in detail, but the present invention is not limited thereto.

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. First, a glass substrate on which a calixcrown derivative represented by Formula 1 (n represents 1, each of Ri, R2, R3 and R₄ independently represents -CHO, and each of R₅ 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. In order to remove unimmobilized remaining substrates, 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. Such 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. When the enzyme reaction was completed, 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. After the incubation was completed, 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. Next, 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.

Table 1

(Mo: Mouse, Rb: Rabbit)

Besides Table 1, the following phospho-specific antibodies (D^, and secondary antibodies (►) to bind to Fc fragments of the phospho-specific antibodies were used. D> Rabbit Anti-phospho-Ser /Ab (Zymed, 61-8100) ϊ> Rabbit Anti-phospho-Thr Ab (Zymed, 71-8200) t> Rabbit Anti-phosphorylated protein Ab (Zymed, 61- 8300)

> Anti-phospho Ser Ab #1~#4 (CalBiochem, 525282) > Mouse Anti-phospho Ser Ab-TAMRA (Anaspec, 29535)

D> Rabbit Anti-phospho Ser28 Histone H3 Ab (Upstate, 07-145) D> Rabbit Anti-Histone H3, CT, pan clone A3S (Upstate,

05-928 )

►Cy5-Goat Anti-Mouse IgG Conjugate (Zymed, 81-6516) ►Cy5-Goat Anti-Rabbit IgG Conjugate (Zymed, 81-6116) ► Alexa 633-Goat Anti-Mouse IgM Conjugate (Molecular

Probes, A21046)

Also, for an enzyme reaction, a buffer (Kinase assay buffer in Table 2) and an ATP/Mg solution (5x ATP/Mg stock solution in Table 2) were purchased from Upstate (USA) .

Other buffers (in Table 2, a substrate dilution solution, a blocking solution, a washing solution, an antibody dilution solution) , except for the above mentioned commercially available buffers, are known to those skilled in the art, and were prepared in a research laboratory. Meanwhile, Staurosporine used in Example 2-9, Example 3-6, and Example 4-7 was purchased from Sigma (USA) .

Table 2

Example 2 Examination of the reaction of MBP-MAPKl

[Example 2-1] Examination of specific reactivity

In order to examine the specific reactivity of MBP phosphorylated by MAPKl on the protein chip prepared according to Example 1, different test conditions were applied to respective wells. MBP (a substrate) was diluted with a substrate dilution buffer to 100ug/ml. In an enzyme reaction, 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. In other words, under the same buffer condition, against an existence/non existence of an enzyme, a phospho- specific antibody, and a secondary antibody, 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. In other words, from high fluorescence intensity to low fluorescence intensity, white-red-orange-yellow-light green-green-blue-dark blue- black were shown. Then, when the fluorescence intensity was converted into numerical values by using analysis software included in GenePix program, only the first well satisfying all conditions showed fluorescence intensity. Herein, the fluorescence intensity of the first well was about 50 times higher than that of the other negative control groups.

Based on the above results, it was found that only a well where all reactions were carried out can show specific reactivity. Thus, according to the present invention, it can be seen that it is possible to successfully phosphorylate even a substrate immobilized on a protein chip by an enzyme, and to detect substrate phosphorylation by the enzyme on the protein chip.

The following examples were carried out to find more accurate concentration/time conditions, and it was proved that the present invention is not limited to MBP-MAPKl and is applicable to other substrates-enzymes.

[Example 2-2] reactivity against an enzyme concentration In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine the enzyme concentration appropriate for a reaction, MAPKl (an enzyme) was diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing enzyme reactions.

First, 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.

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

Examples, an enzyme concentration was adjusted to 10ng/ul.

[Example 2-3] reactivity against a substrate concentration

In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine a substrate concentration appropriate for a reaction, MBP (a substrate) was diluted to stepwise decreasing concentrations according to different well chip columns, thereby inducing enzyme reactions.

First, 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. After incubation and washing, the results were analyzed through the fluorescent images scanned by a microarray scanner. 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 2-4] reactivity against a phospho-specific antibody concentration

In determining an appropriate concentration of a phospho-specific antibody used for detecting MBP phosphorylated by MAPKl, the detection extent of a phosphate group of a substrate was examined by varying only the concentration of the phospho-specific antibody while maintaining other conditions. MBP (a substrate) was immobilized on a well chip, and then MAPKl diluted with an ATP containing enzyme reaction solution to a concentration of 10ng/ul was spotted by IuI, thereby inducing phosphorylation of the immobilized substrate. Then, the phospho-specific antibody was gradually diluted to stepwise decreasing concentrations from 100ug/ml, thereby carrying out reactions. After washing and drying, a secondary antibody was spotted, and the results were scanned by a microarray scanner. As a result, as shown in FIG. 5, it can be seen that fluorescence intensity gradually increased as antibody concentration increased, and was saturated at a concentration of about 10ng/ul or more. Also, there was no non-specific reaction as shown in FIG. 4.

In conclusion, since the optimum result can be obtained at a phospho-specific antibody concentration of lOug/ml, in the following Examples, the concentration was adjusted to 10ug/ml.

[Example 2-5] reactivity against an enzyme reaction time In order to determine the appropriate reaction time of an enzyme against a substrate concentration (that is, the values of Vmax, Km) , 9 well chips were prepared. In respective rows of each well chip, MBP with different concentrations was spotted and immobilized. After blocking, washing and drying, a predetermined amount (IOng) of enzyme was added to the well chips and reacted for different reaction times thereby inducing phosphorylation. Also, in each well chip, 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. After incubation, 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. Finally, after washing and drying, 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. Meanwhile, at a concentration lower than 100ug/ml, although the saturation time was 60 minutes, the fluorescence intensity was low, thereby making it difficult to obtain an accurate value. In other words, although the enzyme reaction time varies against a substrate concentration, the preferable reaction time ranges from 30 to 60 minutes.

Meanwhile, this graph was used to calculate an initial rate, and the value and substrate concentrate were substituted in a Michaelis-Menten equation. However, Km was not obtained. It is assumed that this is because a substrate immobilized on a protein chip surface cannot satisfy an enzyme reaction rate derived from a conventional aqueous solution.

[Example 2-6] reactivity against a phospho-specific antibody reaction time

In this test, the 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.

As shown in FIG. 7, fluorescence intensity rapidly increased within 15 minutes, and then after 15 minutes, the intensity gently increased over time. In other words, although fluorescence intensity increased as phospho- specific antibody reaction time increased, the increase was not significant after 15 minutes. Thus, it was found that the reaction is sufficiently completed after 30 minutes or more. Therefore, in the following Examples, the phospho- specific antibody reaction time was adjusted to 30 minutes.

[Example 2-7] Reactivity against an ATP concentration In 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. In an enzyme reaction where MBP (substrate) immobilized on a well chip and MAPKl were used, 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. Then, after washing and spotting of a secondary antibody, final reactivity was examined. The reactivity was examined by varying only ATP concentration while maintaining other conditions. As a result, as shown in FIG. 8, it can be seen that at 50 and 10OuM, maximum values were shown, and at a high concentration of 30OuM, the reactivity was slightly decreased. An ATP/Mg buffer was commercially available, and it was determined that its most preferable concentration is lOOuM (the concentration used in conventional other methods) . Also, it was found that when ATP was not included, no reaction occurred despite the existence of MAPKl at the same concentration.

From the results, it can be seen that fluorescent images resulting from all Examples for the present invention indicate enzyme reactions by kinase.

[Example 2-8] Reactivity against a secondary antibody concentration

Enzyme reactivity against a secondary antibody concentration was examined. As a result, as shown in FIG.

9, it can be seen that maximum values were shown at dilution ratios of 1:50 and 1:100, and fluorescence intensity decreased as the concentration decreased.

Accordingly, it was found that the most preferable dilution concentration is 1:100.

[Example 2-9] Inhibitory effect by inhibitor (Staurosporine)

Through the above described Examples, 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.

When a substrate immobilized on the well chip prepared according to Example 1 was phosphorylated by an enzyme, an enzyme reaction solution including Staurosporine was spotted. After incubation, it was examined whether or not there was an inhibitory effect on substrate phosphorylation. First, a substrate was immobilized on a well chip, and washing, blocking, washing, and drying processes were carried out. Then, in the spotting of an enzyme, ATP and Staurosporine dissolved in DMSO were added in an enzyme reaction solution in such a manner that the final concentration is decreased in a stepwise manner from 10OuM, and the mixed solution was spotted into each well chip. The final content of DMSO was adjusted to be 10% in all wells, whereas in the last well as a negative control group, distilled water, instead of DMSO, was added. After an enzyme reaction through 1-hour incubation in a 30 ^°C incubator where humidity was maintained, the well chip was taken, and washed for 20 minutes and dried. Then, 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. Finally, a microarray scanner was used to obtain the results of fluorescent images.

Table 3

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.

[Example 2-10] Inhibitory effect by specific inhibitor (ERK inhibitor II)

Based on the results obtained by using Staurosporine which has been widely used as a kinase inhibitor in Example 2-9, an inhibitor which is known to specifically inhibit MAPKl (ERKl) was used for this test.

Detailed test methods were the same as described in Example 2-9, and an ERK inhibitor II (Cat. No. 328007, negative control: 328008) purchased from Merck, instead of Staurosporine, was added to examine the inhibitory effect. 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 (-NH₂) 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. As shown in graphs, at ATP concentration of lOuM, as the inhibitor concentration increased, relative fluorescence intensity decreased. Thus, IC50 was about 4uM. Meanwhile, in a negative control group, as described in product information, even at 10OuM, an inhibitory effect hardly occurred. Example 3 reaction of Histone H3-Aurora kinase A

[Example 3-1] specific reactivity

In order to examine specific reactivity of Histone H3 phosphorylated by Aurora kinase A, different conditions were applied to wells. 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. Also, since an anti-phospho Ser-10 antibody showed a more specific reaction, as compared to an anti- phospho Ser-28 antibody, it was possible to directly find out that Histone H3 phosphorylation by Aurora kinase occurred at ser-10 site as conventionally known in the art.

In FIG. lib, against a Histone proteins to be subjected to various post-translational processes (methylation, phosphorylation, acetylation, etc.), Anti- pan-Histone H3 Ab capable of binding to the proteins regardless of existence or non-existence of a post- translational process was used as a positive control group. As shown in fluorescent images, in the lower row using Anti-pan-Histone H3 Ab, fluorescence intensity was high regardless of the existence or non-existence of aurora kinase, while in the upper row using Anti-phospho Ser-10 H3 Ab, fluorescence intensity was shown in only wells including Aurora kinase. From the results, it was found that it is possible to obtain desired specific reaction results from only wells satisfying all conditions. In other words, when a desired enzyme reaction occurs in a substrate immobilized on a protein chip, 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.

The following examples were carried out for more accurate concentration/time conditions.

[Example 3-2] reactivity against an enzyme concentration

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

In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine a substrate concentration appropriate for a reaction, Histone H3 (a substrate) was diluted to stepwise decreasing concentrations, thereby inducing an enzyme reaction. This Example was carried out in the same manner as Example 2-3.

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 3-4] reactivity against a phospho-specific antibody concentration

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 In a kinase reaction, 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) .

[Example 3-6] Inhibitory effect by inhibitor (Staurosporine)

Through the above described Examples, 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 4

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

Aurora A than in MAPKl.

Example 4 reaction of Histone H3-Aurora kinase B [Example 4-1] specific reactivity

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. As 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. 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. 16a were obtained. Herein, only the first well satisfying all conditions showed fluorescence intensity. Also, since an anti-phospho Ser-10 antibody showed a more specific reaction, as compared to an anti-phospho Ser-28 antibody, it was possible to directly find out that Histone H3 phosphorylation by Aurora kinase occurs at ser-10 site as conventionally known in the art.

In FIG. 16b, in Histone proteins to be subjected to various post-translational processes (methylation, phosphorylation, acetylation, etc. ) , Anti-pan-Histone H3 Ab capable of binding to the proteins regardless of existence or non-existence of a post-translational process was used as a positive control group. As shown in fluorescent images, in the lower row using Anti-pan-Histone H3 Ab, fluorescence intensity was high regardless of the existence or non-existence of aurora kinase B, while in the upper row using Anti-phospho Ser-10 H3 Ab, fluorescence intensity was shown in only wells including Aurora kinase B. Accordingly, it was found that the test results by Aurora B were almost the same as that of Aurora A, except that processes slightly varied.

The following examples were carried out to find more accurate concentration/time conditions.

[Example 4-2] reactivity against an enzyme concentration

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.

[Example 4-3] reactivity against a substrate concentration

In determining enzyme activity on the protein chip prepared according to Example 1, in order to determine a substrate concentration appropriate for a reaction, 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 4-4] reactivity against a phospho-specific antibody concentration

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.

As a result, as shown in FIG. 19, it can be seen that the fluorescence intensity increased as antibody concentration increased. Although 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. Thus, in the following Examples, the concentration was adjusted to about 10ug/ml.

[Example 4-5] reactivity against an enzyme reaction time

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.

As shown in FIG. 20, it can be seen that phosphorylation of a substrate gradually increased over time. It can be seen that substrate concentration had little effect unlike MBP-MAPKl as shown in FIG. 6, and the reactivity was saturated after about 60 minutes. The concentration of a commercially available substrate (MPB) was sufficiently high (unit: mg/ml) , while the maximum concentration of Histone H3 in this Example was 250ug/ml. Thus, compared to the maximum amount of substrates capable of being bound in a well, the saturated amount of the substrates was small.

[Example 4-6] Reactivity against an ATP concentration

In a kinase reaction, in order to introduce 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 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 4-7] Inhibitory effect by inhibitor ( Staurosporine )

Through the above described Examples, 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]

Table 5 shows a phosphorylation extent against a a Staurosporine concentration as numerical values by calculating values resulting from fluorescent images. As the Staurosporine concentration increased, the phosphorylation reaction of an enzyme was reduced. Quantitatively, 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 5 specific reactivity of another substrate and kinase

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.

[Table 6]

In the case of Histone Hl phosphorylated by Cdkl,2, even the negative control groups showed high fluorescence intensity values of 6000 or more unlike the other above mentioned Examples. However, since such values were about 10 times smaller than that of a positive control group, there was no problem in this Example.

Especially, in the case of Iκ-Bα phosphorylated by IKKβ, fluorescence intensity of the first well satisfying all conditions was 300 or more times higher than those of negative control groups.

In specific reactivity in most substrates-kinases, such as LATl - ZAP-70, the fluorescence intensity value of the positive control group was 10 times higher than those of the negative control groups.

Also, in these substrates-kinases tests, tests as described in Examples 2 to 4 were carried out to find more accurate concentrations. The results were similar to the above .

Example 6 reaction of GS-Phosphatase-GSK3α/β

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.

In this Example, in determining the activity of GSK3 using Glycogen Synthase (GS) as a substrate, tests on problems and solutions were carried out.

[Example 6-1] specific reactivity of GSK3α/β In order to examine specific reactivity of GS phosphorylated by GSK3α, different test conditions were applied to respective wells. Detailed test processes were the same as described in Example 2-1.

Under the same conditions, the examination was carried out by varying some conditions, such as the existence/non existence of an enzyme, and a phosphorylation antibody. The results, as noted in Tables 7 (GSK3α) and Table 8 (GSK3β) , were obtained. The first well satisfying all conditions showed a fluorescence intensity value about 60 times higher than those of the negative control groups, and the third well excluding kinase showed a fluorescence intensity value about 40 times higher than those of the negative control groups.

[Table 7]

In other words, 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 6-2] dephosphorylation and phosphorylation of GS

As seen from 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 (GSK3α) and Table 10 (GSK3β) .

[Table 9]

[Table 10]

In the first well which was not treated with phosphatase as well as kinase, 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. Based on the results, it was found that when a substrate phosphorylated to the extent of the fluorescence intensity of the first well is dephosphorylated, the phosphorylation extent is reduced to the level observed in the third well, and then is increased again by kinase to the level observed in the fourth well. Accordingly, it can be seen that 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 In determining the kinase activity on a dephosphorylated substrate, in order to determine the enzyme concentration appropriate for a reaction, GS (substrate) was immobilized on each well chip, and was treated with λPP, thereby inducing dephosphorylation of the substrate. Then, GSK3α and β (enzymes) 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 GSK3α and β on a dephosphorylated substrate GS. Herein, at a kinase concentration of 0, fluorescence intensity corresponds to the phosphorylation extent of a substrate dephosphorylated by λPP treatment. When such a substrate was treated with kinase against a different concentration, it can be seen that the phosphorylation extent gradually increased as concentration increased.

[Example 6-4] repetition of phosphorylation and dephosphorylation

In order to confirm the results from Example 6-2, dephosphorylation by using λPP with dephosphorylation activity higher than PP2A, phosphorylation by treatment of GSK3α/β, and dephosphorylation by using λPP were repeatedly performed three times. The results as shown in FIG. 23 were caused by GSK3α, and showed that within an error deviation range, dephosphorylation-phosphorylation were repeated with the same fluorescence intensity.

Example 7 reaction of IR-PTPlB

As a test for another phosphatase on the protein chip prepared according to Example 1, the activity of PTPlB was examined by using IR (insulin receptor) as a substrate. [Example 7-1] examination of phosphorylation OF an IR substrate against a ATP treatment

In order to induce phosphorylation of an IR substrate prior to dephosphorylation of PTPlB phosphatase, the substrate was immobilized on a protein chip at different concentrations, and then 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 MnCl₂ with a concentration of 20 Mm, thereby inducing phosphorylation. Next, anti- phospho-IR (Tyrll58/Tyrll62/Tyrll63) antibody capable of recognizing a tyrosine residue (Tyrll58/Tyrll62/Tyrllβ3) 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.

In other words, it was found that ATP treatment induced phosphorylation of a tyrosine residue against a autophosphorylation of an IR substrate.

[Example 7-2] dephosphorylation of phosphorylated substrate against a PTPlB phosphatase concentration

First, 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

PTPlB phosphatase concentration, a phosphatase was treated with a concentration of 10 to 0.2ng/ul for 1-hour reaction. Next, in the same manner as Example 7-1, 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.

As shown in FIG. 25, as the phosphatase concentration increased, dephosphorylation extent of a phosphorylated substrate increased, while without phosphatase treatment, dephosphorylation was not induced. Also, in the control group where ATP and phosphatase were not treated, it can be seen that the substrate was not phosphorylated.

In other words, it is found that tyrosine phosphorylation of a substrate occurs by ATP treatment and dephosphorylation occurs by PTPlB phosphatase.

[Example 7-3] dephosphorylation against a substrate concentration

In the same manner as 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 .

As shown in FIG. 26, as the substrate concentration increased, phosphorylation extent by ATP increased. Also, it can be seen that phosphatase treatment induced dephosphorylation, thereby reducing fluorescent intensity at all substrate concentration levels. Example 8 determination on inhibitor of kinase

By using conditions optimized by Examples 3-1 to 3-6, a material having inhibitory activity against Aurora kinase

A was screened. Based on the result from Example 3-6, Staurosporine which has been widely used as a kinase inhibitor was used as the positive control group.

During the phosphorylation of a substrate immobilized on a well chip by an enzyme, Staurosporine and one kind of library material added in an enzyme reaction solution were spotted. Then, after incubation, the inhibitory effect on the phosphorylation of a substrate was examined. 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.

As a result, after the screening of the crude drug library, inhibitory activity on 26 samples was detected (see FIG. 27) . According to the results, the samples 1, 2, 18, 19, 21, 22, 23, 24, and 26 showed the highest inhibitory activity on Aurora kinase A, the samples 3, 8, 13, 14, 15, and 16 showed relatively low activity, and other samples showed no activity. By the above described results, it is possible to screen an inhibitor inhibiting the activity of a specific substrate and kinase from several thousands up to tens of thousands of libraries.

Claims

WHAT IS CLAIMED IS:

1. 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.

2. The protein chip as claimed in claim 1, wherein the cationic functional group comprises an ammonium group.

3. The protein chip as claimed in claim 1, wherein the substrate protein is not a fusion protein, and is directly immobilized on the calixcrown derivative without an additional linker connection.

4. The protein chip as claimed in claim 1, which is formed by immobilizing the calixcrown derivative on the chip base plate with uniform distribution, and attaching an adhesive tape thereon, the adhesive tape comprising well- forming holes with an average diameter of 0.1 to 5mm arranged therein.

5. The protein chip as claimed in claim 1, which is obtained by spotting a substrate protein-containing buffer into each well by 0.1 to bβi, and carrying out incubation with a humidity of 70 to 90%.

6. The protein chip as claimed in claim 1, wherein in the well, the substrate protein is capable of reacting with the kinase or phosphatase in an aqueous solution.

7. The protein chip as claimed in claim 6, wherein after reaction of the substrate protein with the kinase or phosphatase, it is possible to determine the kinase or phosphatase activity by directly determining phosphorylation extent of the substrate protein in the well.

8. The protein chip as claimed in claim 1, wherein the calixcrown derivative is a compound represented by Formula 1 or Formula 2 :

[Formula 1]

wherein n represents 1, each of Ri, R₂, R₃ and R₄ independently represents -CHO, -SH, or -COOH, each of R₅ and R₆ independently represents -H, -methyl, -ethyl, -propyl, - isopropyl, or -isobutyl; n represents 1, each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or each of Ri and R₃ represents -CH₂SH and each of R₂ and R₄ independently represents -H, and each of R₅ and R₆ independently represents -H, -methyl, -ethyl, propyl, -isopropyl, or -isobutyl; or n represents 2, each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or each of Ri and R₃ independently represents -CH₂SH and each of R₂ and R₄ represents -H, and each of R₅ and Rζ independently represents -H, -methyl, - ethyl, propyl, -isopropyl, or -isobutyl,

[Formula 2]

wherein each of Ri, R₂, R₃ and R₄ independently represents -CH₂SH, or two of Ri to R₄ are coupled together to form a group (-CH₂-S-S-CH₂-) .

9. The protein chip as claimed in claim 1, wherein the substrate protein is selected from the group consisting of Myelin basic protein, Histone H3, Forkhead box Ola, Histone Hl, Inhibitor-kappa-B alpha, Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor- alpha, Mitogen-activated protein kinase 2, Linker for activation of T cell-1, Phospho-lipase C gamma-1, Glycogen Synthase, Signal Transducers and Activators of Transcription 3, V-crk sarcoma virus CTlO oncogene homolog (avian) -like protein, B-cell lymphoma 2, c-Jun, and Insulin Receptor.

10. The protein chip as claimed in claim 1, wherein the substrate protein is in a phosphorylated state.

11. The protein chip as claimed in claim 1, wherein the kinase is selected from the group consisting of MAPKl, Aurora kinase A, Aurora kinase B, Akt kinase, Cdkl/cyclin B kinase, Cdk2/cyclin A kinase, IKKβ, IKKα, MEKl, ZAP-70, FGFRl, GSK3α, GSK3β, Janus kinase 3, AbI kinase, c-Jun N- terminal Kinase 1 and c-Jun N-terminal kinase 2, and the phosphatase is selected from the group consisting of

Bacterio-phage λ protein phosphatase, Protein Phosphatase 2A, Protein Phosphatase 1 and Protein Tyrosine Phosphatase IB.

12. A method of determining kinase or phosphatase activity, the method comprising the steps of: a) treating the protein chip as claimed in any one of claims 1 to 11 with kinase or phosphatase capable of reacting with a substrate protein; and b) measuring substrate-protein phosphorylation extent caused by the treatment.

13. The method as claimed in claim 12, further comprising, before the kinase is added to the protein chip in step a) , the step of dephosphorylating the substrate protein immobilized within the protein chip.

14. The method as claimed in claim 12, further comprising, before the phosphatase is added to the protein chip in step a) , the step of phosphorylating the substrate protein immobilized within the protein chip.

15. The method as claimed in claim 12, wherein step b) is carried out by using a phospho-specific antibody and a secondary antibody labeled with a marker.

16. The method as claimed in claim 15, wherein the marker is selected from the group consisting of a fluorescent substance, an enzyme, a radioactive material, a fine article, and a colorant.

17. The method as claimed in claim 12, further comprising, before step a) , the step of adding candidates for a kinase or phosphatase inhibitor so as to screen for potential inhibitors of the kinase or phosphatase.