WO2005071099A1

WO2005071099A1 - Enzyme chip and utilization thereof

Info

Publication number: WO2005071099A1
Application number: PCT/JP2004/019247
Authority: WO
Inventors: Daniel Petru Funeriu; Joerg Eppinger; Masato Miyake; Jun Miyake
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2003-12-24
Filing date: 2004-12-22
Publication date: 2005-08-04
Also published as: JPWO2005071099A1

Abstract

It is intended to provide an enzyme chip which makes it possible to simultaneously and efficiency assay a plural number of enzyme activities and a method of enzyme-specific and activity-dependent enzyme activity assay with the use of this enzyme chip. It is also intended to provide a high throughput method of screening an enzyme activity-controlling substance by which a substance capable of controlling enzyme activity can be efficiently searched for. It is further intended to provide an automated system for carrying out the above methods. A cysteine protease-immobilized chip for assaying enzyme activity wherein a plural number of cysteine proteases differing from each other are provided respectively at a plural number of positions differing from each other on a substrate; and a method of specifically and activity-dependently assaying the biological activity of a cysteine protease with the use of an affinity label.

Description

Specification

Enzyme chip and its use

Technical field

The present invention relates to an enzyme chip and a method for measuring the activity of an immobilized enzyme, wherein the enzyme activity of the enzyme immobilized on the enzyme chip is measured using an affinity label. TECHNICAL FIELD The present invention relates to a method for measuring kinetics based on the method for measuring activity, a method for screening a substance that regulates enzyme activity, and an automated system for performing these methods.

Background art

[0002] Enzymes are a type of protein having a catalytic function that is indispensable for the maintenance and activity of life and plays a role in the synthesis and decomposition of substances that make up living organisms in cells and the creation of energy. Therefore, enzymes are involved in almost all of a wide variety of biological reactions, and are essential elements in elucidating metabolic reactions in vivo and their regulatory mechanisms. Enzymes have extremely high selectivity (reaction specificity) to catalyze only specific reactions and selectivity (substrate specificity) to catalyze only specific substances!ヽ With catalyst specificity! Therefore, by utilizing the specificity of this enzyme, it becomes possible to selectively produce only the desired useful product in a high yield. Therefore, the measurement of the enzyme activity is to obtain knowledge on the effective amount of the enzyme, and is essential in the field of elucidation of biological functions and production of useful products.

[0003] Conventionally, the unit of enzyme activity is defined by the base mass that is converted into a product by a certain amount of enzyme per unit time. Therefore, the method of measuring the activity of the enzyme differs depending on the enzyme to be measured. Was measured by analyzing the amount of decrease in the substrate or the amount of increase in the product (see, for example, Non-Patent Document 1) .o For example, a reaction in which an enzyme and a substrate were mixed in a solution. By measuring the absorbance of the solution and analyzing the change in the amount of substrate or product, or by using a chromogenic substrate such as P-troia-phosphorus substrate or 4-methylcoumarin 7amide substrate, It has been practiced to measure the enzyme activity by analyzing the change in the amount of coloring. In the case of synthase, transferase, etc., the labeled substrate and enzyme After reacting and separating the products by electrophoresis or various types of chromatography, it has been widely practiced to analyze the amount of label in the products. For proteases, activity measurement by in situ zymography has been reported (for example, see Patent Document 1). O On an electrophoresis gel in which collagen or casein, etc., has been dissolved in advance.

After the enzyme is separated based on its molecular weight, etc., the enzyme separated on the gel melts collagen etc. by its decomposition action. The measurement is performed.

Patent Document 1: International Publication No. 97Z32035 pamphlet

Non-Patent Document 1: "Biochemical Dictionary," 1st Edition, Tokyo Chemical Dojin, 1984, p.452

Disclosure of the invention

Problems to be solved by the invention

[0004] However, the conventional method is supposed to measure the activity of one type of enzyme in one reaction system. We had to build a separate reaction system for each enzyme.

In some cases, multiple enzymes are reacted in the same reaction system, and the products are separated by electrophoresis for discrimination.However, when the enzymes to be measured belong to the same or similar families, It is difficult to discriminate products corresponding to each enzyme on a gel and discriminate and analyze the enzyme activity of each enzyme, because the properties and the like are similar. Similarly, when enzymes to be measured belong to the same or similar families, it is difficult to distinguish each enzyme on a gel because their molecular weights and properties are similar. there were.

[0005] In addition, when electrophoresis, chromatography, and the like are used, complicated steps are required, so that labor and time are required, and it is difficult to obtain experimental results quickly.

[0006] In addition, the conventional method requires a certain amount of sample and reagent, and thus has a problem that it is not suitable for handling expensive, minute samples and reagents.

[0007] Therefore, in recent years, the consumption of expensive and valuable samples and reagents has been reduced! / Therefore, it has been suggested that a low cost, high speed, and simple screening technique is highly useful, and that multiple proteins can be used on a substrate. Fixed protein chips have been studied for practical use. That is, DNA Attempts have been made to use a chip-making technology to produce a small chip that prints thousands of proteins on the same platform. However, proteins are generally very unstable to external factors such as heat, and protein chips are nowadays difficult due to their three-dimensional structure, which is much more complex than nucleic acids. However, there is a problem that it is a developing technology, and it has not been supplied to the factory and spread to the general public.

[0008] Further, as described above, enzymes play an important role in maintaining vital activities involved in various biological reactions, so that changes in enzyme activities are involved in the pathology of various diseases. Therefore, a substance capable of modulating enzyme activity has potential value as a pharmaceutical candidate for treating or preventing a disease. It is extremely important in the development of preventive drugs. In addition, the screening method is based on the conventional enzyme activity method, and the reaction method is based on the combination of the type of enzyme to be assayed and the number of test substances that are candidate substances for the enzyme activity regulator. I had to build the power. In particular, in recent years, in the search for new drug conjugates, the advent of combinatorial chemistry has made it possible to synthesize a diverse group of compounds at once. There is a need for a high-throughput screening method that can be performed efficiently and quickly.

[0009] Accordingly, an object of the present invention is to provide an enzyme chip capable of simultaneously and efficiently measuring a plurality of enzyme activities, and an enzyme-specific and activity-dependent enzyme activity measurement method using the enzyme chip. Is to provide. It is a further object of the present invention to provide a high-throughput method for screening enzyme activity regulating substances that can efficiently search for substances capable of regulating enzyme activity. The present invention aims at providing an automated system for performing these methods.

Means for solving the problem

The present inventors have conducted intensive studies to solve the above problems, and as a result, have succeeded in constructing an enzyme chip in which an enzyme is immobilized on a substrate without impairing the biological activity of the enzyme. Further, as a result of further intensive studies, they succeeded in detecting the biological activity of the immobilized enzyme with good sensitivity and comprehensively using an affinity label, and completed the present invention.

[0011] In other words, the present invention measures an enzyme activity on a chip using an affinity label. Specifically, an affinity can be specifically and activity-dependently linked to an enzyme by linking with a substrate enzyme-affinity label-reporter molecule through a strong covalent bond. An object of the present invention is to provide a method for measuring enzyme activity at high throughput on a chip using a label.

Further, the present invention provides a method for measuring kinetics for the first time on a chip.

[0012] Specifically, a first characteristic configuration of the present invention is:

A plurality of different enzymes are separately arranged at a plurality of different positions on the substrate, and this is in the form of an enzyme chip for measuring enzyme activity. Preferably, the enzyme is a cysteine protease.

[0013] The second characteristic configuration of the present invention is as follows.

An enzyme chip preparation step in which a plurality of different enzymes are arranged at a plurality of different positions on a substrate to form an enzyme chip;

A reaction step of contacting the enzyme chip with an affinity label to which a reporter molecule is attached, and reacting the affinity label with the enzyme;

A detection step of detecting a signal of an affinity label attached to the enzyme immobilized on the enzyme chip and a revoter signal.

Analyzing the enzyme activity based on the signal intensity detected in the detection step; and measuring the enzyme activity. Preferably, the enzyme is a cysteine protease, and the enzyme whose activity is to be measured is a cysteine protease. In this case, preferably, the affinity labeling force attached to the reporter molecule is represented by the general formula (I): Epoxide compound Leu—Xaa—Lys—Supha single reporter molecule [where Xaa is Tyr, Lys Or Arg, and one spacer is Amino-hexanoic acid].

[0014] According to the above characteristic configuration, it is possible to provide an enzyme chip in which a plurality of different enzymes are arranged, and to examine a plurality of different enzyme activities in one reaction system based on the enzyme chip. In addition, experimental results can be obtained quickly and efficiently at high throughput without the need to construct many reaction systems according to the types of enzymes. Further, the affinity label of the present invention shows reactivity with the active site of an enzyme belonging to one enzyme family. Since it has no reactivity with any enzyme belonging to another enzyme family, it is possible to achieve a specific and activity-dependent measurement of enzyme activity. Further, a third characteristic configuration of the present invention for achieving the above object is as follows:

An enzyme chip manufacturing step of arranging a plurality of different fixed units of enzymes at a plurality of different positions on a substrate to form an enzyme chip;

A reaction step of contacting the enzyme chip with a constant concentration of an affinity label to which a reporter molecule is linked, and reacting the affinity label with the enzyme;

The reporter signal strength of the immobilized enzyme before contact with the affinity label is also a method of measuring the kinetics between the enzyme and the affinity label from the time change of the enhanced signal intensity.

This method is based on the kinetic data obtained by the powerful enzyme kinetic measurement method.

According to the above feature configuration, a kinetic analysis method using a chip can be provided for the first time, and it is possible to simultaneously, comprehensively, and at a high throughput grasp the kinetic dynamics of a plurality of enzymes. .

Furthermore, a fourth characteristic configuration of the present invention for achieving the above object is:

A step of preparing an enzyme chip for arranging a plurality of different enzymes at a plurality of different positions on a substrate to prepare an enzyme-immobilized chip;

A test substance contacting step, in which a test substance is brought into contact with the enzyme chip and pre-incubated,

A reaction step in which an affinity label attached with a reporter molecule is brought into contact with the enzyme chip after the test substance contacting step to cause the affinity label to react with the enzyme. An enzyme immobilized on the enzyme chip And a detection step of detecting a signal of the affinity label riboter,

The enzyme activity is regulated by observing a change in the signal intensity of the test substance for the different enzymes compared with the signal intensity of the enzyme without contacting the test substance. A screening method for a substance that regulates an enzyme activity, which is used for screening for a predetermined regulatory substance. Preferably, the enzyme is a cysteine protease, and In the case where the enzyme is cysteine protease, the affinity label attached to a reporter molecule is preferably represented by the general formula (I): Epoxide compound Leu-Xaa-Lys-spacer reporter molecule [wherein Xaa represents Tyr, Lys or Arg, and one spacer is Amino-hexanoic acid].

[0016] According to the above-mentioned characteristic configuration, the test substance can be adjusted only once by contacting (reacting) the test substance once with the enzyme-immobilized protein chip on which a plurality of different enzymes are arranged. Can be examined for its reaction characteristics with a plurality of different enzymes. In other words, it is possible to obtain experimental results quickly and efficiently without the need to construct many experimental systems according to the combination of the type of enzyme and the number of test substances as in the conventionally performed method.

Therefore, the present invention is capable of directly and rapidly evaluating a substance capable of regulating an enzyme activity, and is effective for screening a modulator capable of regulating an enzyme activity at a high throughput.

[0017] To achieve the above object, a fifth feature of the present invention is:

Contacting the enzyme chip with an enzyme activity inhibitor and preincubating the enzyme chip;

Contacting the enzyme chip with the reporter molecule-attached affinity label with the enzyme chip after the enzyme activity inhibitor contacting step, and reacting the affinity label with the enzyme;

A method for analyzing the inhibitory constant of an enzyme activity inhibitor, wherein the inhibitory constant of an enzyme activity inhibitor is analyzed by measuring a change in the signal intensity of an enzyme that does not come into contact with the test substance and a change in signal intensity. According to the above configuration, it is possible to simultaneously, comprehensively, and at a high throughput evaluate an enzyme activity inhibitor for a plurality of enzymes.

[0018] In order to achieve the above object, a sixth characteristic configuration of the present invention is as follows.

Means for preparing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate,

Reacting an affinity label with the enzyme chip and reacting the affinity label with the enzyme;

A detection means for detecting a revoter signal of an affinity label bound to the enzyme immobilized on the enzyme chip,

Means for analyzing enzyme activity based on the signal intensity detected by the detection means,

An automated system for measuring enzyme activity is provided. Preferably, the enzyme is cysteine protease.

In order to achieve the above object, a seventh characteristic configuration of the present invention is:

A reaction means for contacting the enzyme chip with an affinity label to which a reporter molecule is attached, and reacting the affinity label with the enzyme;

Measurement means for measuring the kinetics between the enzyme and the affinity label, from a time change in the signal intensity of the reporter signal of the immobilized enzyme before contacting the affinity label, and

A database for analyzing a force kinetics constant based on the kinetics obtained by the kinetics measuring means;

The point is that it is a system that automates the calculation of kinetic constants.

Further, in order to achieve the above object, an eighth feature of the present invention is:

Producing enzyme chips by arranging different enzymes at different positions on a substrate Means for making enzyme chips,

A test substance contact means for pre-incubating the enzyme chip by contacting the test substance with the enzyme chip;

A reaction means for bringing an affinity label attached with a revoluter molecule into contact with the enzyme chip contacted with the test substance by the test substance contact means, and reacting the affinity label with the enzyme;

A detection means for detecting a reporter signal of an affinity label attached to a reporter molecule bound to the enzyme immobilized on the enzyme chip,

An analysis means for analyzing a characteristic as a modulator regulating the enzyme activity by observing a change in the signal intensity due to the test substance for each of the plurality of different enzymes compared with the signal intensity of the enzyme not contacting the test substance. Have,

In this regard, an automatic screening system for the preparation of enzymatic activity was used, and preferably the enzyme is a cysteine protease.

And the ninth characteristic configuration of the present invention for achieving the above object is:

A reaction means for contacting the enzyme label contacted with the test substance by the test substance contact means with an affinity label attached with a revoluter molecule, and reacting the affinity label with the enzyme;

Detecting means for detecting a reporter signal of the affinity label bound to the enzyme immobilized on the enzyme chip,

A measuring means for measuring the change in the signal intensity of the enzyme derived from the enzyme which is not brought into contact with the test substance;

Having a database for analyzing the inhibition constant of the enzyme activity inhibitor based on the measurement value measured by the measurement means,

In other words, the system is an automatic system for calculating the inhibition constant of an enzyme activity inhibitor. [0021] Further, a tenth feature of the present invention for achieving the above object includes an affinity label to which a reporter molecule is attached, and is used for measuring the enzyme activity of the immobilized enzyme. When used as a reagent for measuring the activity of immobilized cysteine protease, it is preferable that the affinity label to which the reporter molecule is attached is generally used. Formula (I): Epoxide Leu—Xaa—Lys—Sposa One Reporter Molecule (where Xaa represents Tyr, Lys, or Arg, and one spacer is Amino-hexanoic acid) It is constituted as the compound shown.

[0022] An eleventh feature of the present invention for achieving the above object is that a reporter molecule is attached to an enzyme chip in which a plurality of different enzymes are arranged and fixed at a plurality of different positions on a substrate. A method of contacting an affinity label with a plurality of compounds to screen an enzyme activity inhibitor and calculating an inhibition constant of the enzyme activity inhibitor, wherein the calculation method comprises the following steps: 1. a step of measuring a kinetic constant between a plurality of different immobilization enzymes and the affinity label and calculating an inhibition constant based on the kinetic constant; 2. a plurality of different immobilizations A) contacting the affinity label with a plurality of the above-mentioned compounds with the enzyme enzyme; screening the enzyme activity inhibitor for the reporter signal power of the affinity label measured at a time; Further Calculating the inhibition constant; 3. searching for an optimal concentration of a powerful compound which cannot obtain a reporter signal within a predetermined range among a plurality of the compounds, and searching for the optimal concentration measured at the optimal concentration. Calculating the inhibition constant from the reporter signal of the futility label; a method for calculating the inhibition constant that includes at least one of the steps described in the step of:

[0023] A twelfth characteristic configuration of the present invention for achieving the above-mentioned object is that a calculation automation system for making a database of the inhibition constants calculated by the method for calculating the inhibition constants.

[0024] A thirteenth feature of the present invention for achieving the above object is that the method of calculating the inhibition constant is such that the method of calculating the inhibition constant is different between a plurality of the immobilized enzymes and a plurality of the different enzyme activity inhibitors. The method used to calculate the inhibition constant calculated by an algorithm that can simultaneously calculate each concentration of multiple types of enzyme-inhibitor complexes formed on the enzyme chip is there.

[0025] A fourteenth feature of the present invention for achieving the above object is a calculation automation system for making a database of the inhibition constants calculated by the inhibition constant calculation method described in the twelfth feature. On the point.

The invention's effect

[0026] According to the present invention, it is possible to provide an enzyme chip in which an enzyme is immobilized on a substrate without impairing the biological activity of the enzyme. Further, by utilizing an affinity label, one reaction system can be provided. Thus, it is possible to provide a method for measuring the enzymatic activity in which the biological activity of the immobilized enzyme can be detected specifically and activity-dependently, with good sensitivity, and can be detected wirelessly. It will provide useful information in the field of elucidation and production of useful products.

Further, the present invention provides a method for measuring kinetics on a chip based on the method for measuring enzyme activity of the present invention.There is no method for measuring kinetics on a chip, and there is no method for measuring kinetics on a chip. It can provide useful information in the analysis of kinetic properties.

Also, based on the enzyme activity measurement method of the present invention, a substance that regulates the enzyme activity can be searched with a high throughput, and a simple and rapid screening method for the enzyme activity regulating substance can be provided. Useful information can be provided. Further, according to the method for calculating an inhibition constant of the present invention, it is possible to search for an inhibitor quickly and in a large amount (high throughput), and it is possible to accurately calculate the inhibition constant. As a result, the obtained information (inhibition constants, etc.) can be converted into a database as electronic data, which can be shared by pharmaceutical companies and various research institutions, so that duplicate research can be avoided and the reliability of the data itself can be improved. And drug development can advance dramatically.

According to the present invention, the enzymatically active substance screened is useful for treating and preventing diseases caused by abnormal enzyme activity.

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] The present invention provides a novel method for measuring enzyme activity, a method for screening a substance that regulates enzyme activity based on the measurement method, and an automation system for performing the method. To do. More specifically, a new measurement principle of preparing an enzyme chip in which an enzyme is immobilized on a substrate and detecting the activity of the immobilized enzyme immobilized on the enzyme chip using an affinity label. It is based on.

[0028] The enzyme chip according to the present invention is based on the principle of a protein array. The protein array has a plurality of protein or peptide fragments fixed to the surface of a substrate. The enzyme is immobilized on the surface of the substrate.

[0029] Then, like DNA microarrays, protein arrays are low cost, consume little valuable reagents and reagents, and are highly useful as a rapid and simple screening technique. A great deal of effort is currently being made for this purpose, and the key to the practical application of protein arrays is to solve issues such as immobilization of functional proteins that retain biological activity on substrates and reduction of knock ground noise. .

The present invention can be applied to, for example, the activity measurement of enzymes derived from animals including humans, plants, or microorganisms, and includes oxidoreductases, transferases, hydrolases, lyases, Although it can be applied to both a synthetase and a synthase, a protease, which is one of hydrolases, is particularly preferably used. Here, protease is a general term for proteolytic enzymes, which are classified into several groups according to residues, metals, and the like necessary for the expression of activity. And cysteine proteases including inn, cathepsin B, etc., aspartic proteases including pepsin, etc., collagenase, metallo-oral proteases including matrix meta-oral protease and the like.

[0031] These enzymes may be natural extract products extracted and purified from cells, microorganisms, etc.! /, Or may be prokaryotic or eukaryotic hosts (for example, cultured Bacterial cells such as Escherichia coli and Bacillus subtilis, yeast cells, higher plant cells, insect cells, mammalian cells, etc.) and commercially available purified products may be used. Extraction of enzymes from cells and microorganisms is known (see, for example, Shinsei Kagaku Kenkyusho 1 Protein 1 Separation 'Purification' Properties (Tokyo Kagaku Dojin), etc.). This is done by selecting. In addition, the production of recombinants is also known (eg, Molecular Cloning; 2nd Ed., Cold Spring Harbor Laboratory, etc.). See also. ), Which can be prepared according to a conventional gene recombination technique. Specifically, a recombinant DNA capable of expressing a gene encoding a desired enzyme protein in a host cell is prepared, the host cell is transformed with the recombinant DNA, and the transformant is cultured. By extracting the desired enzyme. In addition, an enzyme to which a tag is added, such as an oligohistidine tag, can also be suitably used, and can be prepared by adding a desired tag when expressing a protein. By immobilizing the protein on the substrate via the tag, it becomes possible for the protein to align its orientation on the substrate, and the immobilization reaction on the substrate is performed at a location separate from the main body of the protein. As it happens, the effect on enzyme activity can be minimized.

[0032] Here, the affinity label used for detection of the enzyme activity has a portion having an affinity for a protein, and utilizes the fact that the protein is specifically modified due to the affinity. In the present invention, affinity label is a type of chemical modification of protein. In the present invention, affinity label binds to an enzyme active site specifically and in an enzyme activity-dependent manner, and the enzyme activity is analyzed based on the amount of the bound affinity label. Is what you use for Therefore, the following three parts of the affinity label used here:

1. The part that reacts with the active site of the enzyme

2. The part that binds to the active site of the enzyme

3. Reporter molecule and the part connecting reporter molecule and the above 1 and 2

It is necessary to be structured and composed.

Next, each of the above components will be described in detail.

1. The part that reacts with the active site of the enzyme

Preferably, it is configured to contain an active chemically reactive group as a chemical modification reagent, and epoxides and the like are exemplified. However, it is appropriately designed according to the enzyme whose activity is to be measured.

2. The part that binds to the active site of the enzyme

Preferably, it is configured to have a substrate-like structure showing a specific affinity for the active site of the enzyme. In other words, a force that is specific to all enzymes belonging to one family belongs to another family! / Although exemplified as a peptide, it can also be configured as having a configuration other than a peptide as appropriate depending on the enzyme whose activity is to be measured.

3. A reporter molecule and a portion having a spacer group connecting the reporter molecule and the above 1 and 2

The reporter molecule is a marker for detecting the amount of the affinity label bound to the active site of the enzyme, and is linked to the above-mentioned portions 1 and 2 via a spacer. These can be either newly designed according to the enzyme to be measured or use of a known construct.

The reporter molecule means a molecule that can be detected by spectroscopic, biochemical, immunochemical, chemical means, and the like.For example, useful reporter molecules include fluorescent dyes such as biotin and Cy3. , ³² P, ³⁵ s radioactive materials, metal particles such as nano gold, hapten

Known substances that can be used as reporter molecules such as DNA, PNA, RNA, proteins and antibodies for which monoclonal antibodies are available can be appropriately selected and used.

[0034] These reporter molecules can be detected directly or indirectly. When the reporter molecule itself is composed of a detectable substance, it is directly detected by spectroscopic, biochemical, immunochemical, chemical means or the like as appropriate according to the type of the reporter molecule. When the reporter molecule is indirectly detected, for example, one reporter molecule is recognized indirectly by recognizing it by a substance having a detectable portion.

Specifically, when the reporter molecule is biotin, it can be detected using streptavidin labeled with a radioactive substance, a fluorescent dye, or the like.When the reporter molecule is a protein, it can be detected. Detection can be performed using a monoclonal antibody labeled with a fluorescent dye, and when the reporter molecule is DNA, it can be detected by hybridization with a labeled nucleic acid molecule. However, it is not limited to these.

Here, in measuring the enzyme activity of cysteine protease, an affinity label having the following structure can be suitably used.

General formula (I): Epoxide Leu— Xaa— Lys—one spacer A reporter molecule (where Xaa is selected from Tyr, Lvs and Arg, one spacer is Amino— hexanoic and the reporter molecule is configured as a fluorescent substance, and an affinity label can be suitably used.

In the affinity label for detection with cysteine protease, the chemical reaction group is composed of an epoxide. Epoxides have the property of reacting well with SH groups present in the enzyme active site, but have low reactivity with SH groups other than the enzyme active site, and therefore react specifically with the enzyme active site. , Can be combined. Accordingly, there are a wide variety of reactive groups capable of reacting with the SH group, but the use of epoxide is preferred from the viewpoint of specific reaction to the enzyme active site and the like.

Specifically, as used in Example 1,

[Chemical 1]

Is preferably used.

The affinity label described above can specifically detect the activity of each enzyme belonging to the cysteine protease family. In other words, the cysteine protease family It specifically binds to the active site of an enzyme belonging to one family, but does not recognize the active site of an enzyme belonging to another family, for example, one of the serine mouth thease families.

[0037] By using such an affinity label, the enzyme whose activity is to be measured erroneously recognizes the recognition group as a substrate and incorporates the affinity label into the active site. By attacking the functional group near the site to form a covalent bond, the affinity label is specifically accumulated at the active site of the enzyme. Since this reaction occurs only when the enzyme is active, the activity of the enzyme can be measured by measuring the amount of an affinity label that forms a covalent bond with the enzyme.

In addition, since the affinity label and the enzyme active site are covalently bonded, they are based on non-covalent bonding modes such as protein-protein, antibody-antigen, and ligand-receptor! In comparison with known protein chips, signals can be detected with higher sensitivity. In particular, in the post-reaction washing step described later, it is possible to wash under high stringency conditions, effectively reducing knock ground noise, which is particularly problematic for protein chips. This enables highly sensitive detection of the enzyme activity signal to be detected.

Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. However, the scope of the present invention is not construed as being limited by these embodiments.

(First Embodiment of the Present Invention)

The first embodiment of the present invention is a method for measuring an enzyme activity.

More specifically, this is an enzyme activity measuring method having the following steps, which will be described with reference to FIG.

[0040] First step: an enzyme chip preparation step (hereinafter referred to as an enzyme chip preparation step) in which different enzymes are arranged at a plurality of different positions on a substrate to form an enzyme chip.

Second step: a blocking step of performing a blocking treatment to avoid a non-specific reaction on the enzyme chip (hereinafter, referred to as a blocking step),

Third step: a washing step of removing an unnecessary blocking agent on the enzyme chip (hereinafter, referred to as washing step 1).

Step 4: Step of pre-incubating the enzyme chip (hereinafter referred to as pre-incubation) This is referred to as a process step. )

Fifth step: a reaction step (hereinafter, referred to as a reaction step) in which an affinity label attached with a reporter molecule is brought into contact with the enzyme chip, and the affinity label is reacted with an enzyme. A washing step of washing and removing the affinity label which has not formed a covalent bond with the enzyme on the enzyme chip by washing the enzyme chip (hereinafter referred to as washing step 2);

Seventh step: a detection and analysis step in which a reporter signal of an affinity label bound to the immobilized enzyme on the enzyme chip is detected, and the detected signal is quantitatively analyzed. ).

Hereinafter, each step will be described in detail.

[0041] First step: Enzyme chip preparation step

The enzyme chip preparation step is performed by printing and immobilizing the enzyme on an appropriate substrate. The enzyme is immobilized on the substrate while maintaining the three-dimensional structure of the protein (that is, the secondary structure and tertiary structure) and the biological activity of the enzyme. Also, ideally, the enzyme is immobilized on the substrate with its orientation aligned to ensure the same reactivity of the immobilized enzyme. The ability to immobilize a functional enzyme, as described above, is an important point for enzyme chip development.

Here, as the type of the substrate used for the fixing device, for example, a glass substrate, a silicon substrate, a quartz glass substrate, or the like, particularly, glass can be preferably used. A slide glass having a 12 × 4 subarray divided by the fluororesin mask used in the examples is also preferably usable.

[0043] Printing of the enzyme is carried out on a suitable substrate using, for example, an ink-jet type microarrayer. After printing, the array is incubated under suitable conditions. Microarrayer robots for high-density printing are commercially available, and commercially available products such as High Precision ink—jet microarryer (manufactured by Artesian Technologies) can be used. However, simply adsorbing the enzyme on the surface of the substrate while applying force causes a problem of surface strength of the mechanical strength. Therefore, the enzyme is immobilized on the substrate through a chemical covalent bond. Surface treated. In particular, The substrate surface must have the following three chemical properties.

a) The ability to attach enzymes without reacting with active groups.

b) The enzyme can be kept in an active state in the folded structure.

c) Be able to attach as much enzyme as possible.

As a glass substrate having the above-mentioned properties, aldehyde functionalization is particularly preferred as a substrate surface having the above-mentioned property a), and N-hydroxysuccinimide (

Hereinafter, it is abbreviated as NHS. ) Functionalization can also be preferably used, and as the substrate surface having the property of the above b), there is an enzyme capable of retaining the enzyme activity only on the hide-mouthed gel-functionalized substrate, especially when the hide-mouthed gel is functionalized. I do. For the substrate surface having the property of c) above, the three-dimensional structure of the hide-opening gel increases the surface area of the substrate surface, thereby allowing more enzyme to be taken up. Mouth gel functionalization is particularly preferred. As a substrate surface having the above properties a), b) and c), for example, for measurement of the enzyme activity of cysteine protease, a aldehyde functionalized aldehyde is particularly preferred. NHS functionalization capability Can be used preferably.

When printing the enzyme, it is preferable to perform the printing under an atmosphere of 85% humidity. Under a high humidity atmosphere (90% or more), dew condensation may occur on the substrate surface, which is not preferable. In a low humidity atmosphere (60% or less), drying and enzyme denaturation may be caused. It is adjusted to. The printing buffer must use a buffer that can maintain enzyme activity, and must be suitable for the surface of the substrate to be used so as not to destroy the chemical functionality of the substrate surface. is there. For example, in measuring the enzyme activity of cysteine protease, an acetate buffer or a malonic acid buffer (pH 5.5) is preferably used. At a pH of 7, the enzyme activity is inactivated depending on the type of the enzyme (cathepsin L). Therefore, it is not suitable for use in the present invention.

[0045] When the concentration of the enzyme printed on the substrate is low, a detectable signal intensity cannot be ensured at a low concentration, and the detection spot is diffused at an undesirably high concentration. Not preferred. As a result of both considerations, it is preferable to adjust the range from 0.1 mgZml to 1mgZml. The shape and area of the spot are not particularly limited. Can be determined appropriately.

As a negative control, BSA printed on a substrate can be used. If the BSA spot is not confirmed to emit a signal in the detection step described below, Signal strength detected in the detection process It is possible to show that the signal is not due to the affinity label non-specifically bound on the substrate surface, and to prove the effectiveness of the reaction on the chip .

Incubation after printing is preferably performed in a high humidity atmosphere to prevent drying of the arrayed enzymes and at a low temperature, for example, at 4 ° C. In addition, depending on the type of enzyme, incubation can be performed at room temperature.

[0047] The enzyme once printed (fixed) on the substrate is stable under appropriate storage conditions. Although this observation confirms the importance of the choice of substrate surface and operating conditions, it contradicts the general perception of the fragility of proteins immobilized on a substrate, and therefore, is not This is very important as a reason for enabling the present invention.

[0048] Second step: blocking step

In the blocking step, non-specific binding to the non-printed area generates high background noise, so that non-specific binding should be inhibited in the area on the substrate where the enzyme is not immobilized. Is used to provide a substrate surface that is resistant to non-specific binding, which allows for a reduction in background noise. The blocking agent used in the blocking step needs to have the following properties.

a) Reacts quickly with chemical functional groups on glass substrates.

b) Do not modify the structure of the enzyme!

c) Do not denature the enzyme.

d) Low reactivity with the affinity label used.

e) It is fluorescent.

As a blocking agent having a powerful property, for example, in the measurement of the enzymatic activity of cysteine protease, Block Ace / malonic acid buffer 1/2 (pH 5.5) is particularly preferable. It can be used preferably. Also, 2% BSA is preferably used because it does not cause a high background. On the other hand, for example, in measuring the enzyme activity of cysteine protease, the use of Tris buffer alone, ethanolamine, or polyethylene glycol may cause inactivation of the enzyme activity or diffusion of the printed enzyme on the substrate surface. Not preferred. The blocking time, blocking temperature, calorie content of the blocking agent, and the like are appropriately set according to the type of the enzyme whose activity is to be measured, the type of the blocking agent used, and the like.

There are no particular restrictions on the means for adding and removing the blocking agent.For example, when the addition is performed, the array is immersed in the blocking agent, and the blocking agent is dropped on the array. The force exemplified by performing the suction removal of the blocking agent is not limited thereto.

Step 3: Cleaning process 1

In the washing step 1, after the blocking step, the array is washed with an appropriate washing solution to remove unnecessary blocking agents. The cleaning solution used here must have the following properties.

a) To effectively remove components contained in the blocking agent without reacting with the substrate surface.

b) The printed enzyme must not be modified in a danitorial manner.

c) Do not denature the printed enzyme

Above a) -. As a cleaning agent having a property of c), for example, in the enzyme Motokatsu ¾ measurement of cis Ting Protea Ichize, malonic acid buffer or Tris buffer, _(P H5 5) can be suitably used. The washing time, washing temperature, amount of washing solution to be added and the like are appropriately set according to the type of enzyme whose activity is to be measured, the type of blocking agent to be removed, the type of washing solution, and the like.

The means for adding the washing solution and the means for drying the array after washing are not particularly limited.For example, in the case of added calories, the array is immersed in the washing solution, and the washing solution is dropped on the array. For example, the drying may be performed by centrifugally drying the washed array, but is not limited thereto. However, for drying In this case, since the enzyme chip is maintained in a dry state, the enzyme is denatured. Therefore, the enzyme chip is applied in a short time without denaturing the enzyme. Preferably, it is 30 minutes or less.

[0050] Here, the enzyme chip on which the enzyme is immobilized can be stored until later use, and the preservation solution, preservation temperature, and preservation time for preservation are determined by the type of immobilized enzyme and the like. It is set appropriately in consideration of

[0051] Fourth step: pre-incubation step

It is necessary that the preincubation solution used in the preincubation step has a property capable of activating the enzyme. Further, those having a low background and having no reactivity with any component of the reaction system are preferable. Here, for example, when adapting to the cysteine protease used in Examples, a reducing agent is required, and for example, a preincubation solution containing 2 mM DTT can be suitably used. In addition, the preincubation time is important.If the preincubation time is short, the enzyme will not be sufficiently activated, and a long preincubation will inactivate the enzyme activity. Is not preferred. Preincubation for 20 minutes to 1 hour is particularly preferred. The incubation temperature, the amount of the incubation solution to be added, and the like are appropriately set according to the type of the enzyme whose activity is to be measured, the type of the incubation solution, and the type of the substance to be added as necessary. However, the pre-incubation step needs to be performed in a humid atmosphere.

There are no particular restrictions on the means for adding the incubation solution and the means for removing the incubation solution.For example, in the case of adding syrup, the immersion of the array in the washing solution or the dropping of the washing solution onto the array is performed. However, in the removal, a force exemplified to be performed by suction or the like is not limited to these. Since the enzyme activity is irreversibly inactivated by drying, it is necessary to avoid drying the surface of the microarray after the preincubation step.

[0052] Fifth step: Reaction step

In the reaction step, the enzyme immobilized on the substrate is brought into contact with an affinity label for detecting the enzyme activity, and the affinity label is reacted with the enzyme. Although a specific method is not particularly limited, for example, a test substance is dissolved in an appropriate solvent on a substrate on which an enzyme is immobilized. This is performed by immersing the detection solution in the soaked detection solution or by spotting the detection solution on the region where the enzyme is immobilized on the substrate. The reaction time is appropriately set according to the type of the enzyme to be immobilized. In this reaction, the enzyme immobilized on the substrate is modified by an affinity label in an activity-dependent manner, and the composition of the reaction solution used here has a low background and can be used in any of the reaction systems. The components have no reactivity.For example, the measurement of the enzyme activity of cystine protease requires the presence of a reducing agent such as DTT, and furthermore, the pH is adjusted to an acidic value such as 5.5. Need to be prepared on the side. The concentration of the added affinity label is such that the reaction with the knock ground progresses more slowly than the reaction with the arrayed enzymes, but the addition of a high concentration of the affinity label results in a rapid reaction of the affinity label. As a result, accurate enzyme activity cannot be detected. On the other hand, addition of an affinity label at a low concentration does not allow detection of a sufficiently detectable signal intensity. Therefore, accurate and, to achieve the detection of the detectable enzymatic activity signaling null, for example, you Itewa enzyme activity measurement cis Ting protease range § Fiyu tea label concentration from ^10-6 to 10- ⁸ M to be added It is preferable to prepare it. The reaction time, the temperature, the amount of the reaction solution to be added, and the like are appropriately set according to the type of the enzyme whose activity is to be measured.

There are no particular restrictions on the means for adding and removing the reaction solution.For example, in the case of adding caroten, the reaction solution is immersed in the array, and the reaction solution is dropped on the array. Examples of the method include suction, but the present invention is not limited thereto.

Step 6: Cleaning step 2

In washing step 2, the presence of an affinity label that is not covalently bonded to the enzyme immobilized on the substrate causes high background noise.Therefore, after the above-mentioned labeling reaction step, an appropriate washing solution is used. To wash the array to remove the affinity label that is not covalently bound to the enzyme immobilized on the array. The washing solution used here is preferably one that does not cleave the bond between enzyme affinity labels and can remove non-covalently bound molecules present on the substrate surface that cause knock ground noise. The attachment of the affinity label to the In addition, since the bond between the enzyme and the substrate and the bond between the affinity labels are also covalent, washing under highly stringent conditions becomes possible. In other words, since the enzyme and the affinity label are covalently bonded, the signal intensity is not affected even by washing under high stringent conditions, while the knock ground noise is effectively reduced. It is possible to achieve this objectively. In this regard, low reproducibility of the detected signal intensity due to the necessity of washing under low stringency conditions, and showing relatively high background noise! / It is different from other proteins that were previously assumed (protein-protein, antibody-antigen, ligand-receptor, etc.). The only limitation is that the application of the so- cation is not preferred, as it can destroy the surface substrate or the reporter molecules such as the fluorescent material. Therefore, for example, when measuring the enzyme activity of cysteine protease, pH 5.5 and 70 in a buffer containing SDS as a washing solution are particularly preferred. C, washing in 30 minutes is exemplified. The washing time, washing temperature, amount of washing solution to be added and the like are appropriately set according to the binding strength between the immobilized enzyme and the affinity label, the type of washing solution, and the like.

The means for adding the washing solution and the means for drying the array after washing are not particularly limited.For example, in the case of added calories, the array is immersed in the washing solution, and the washing solution is dropped on the array. In the drying, the force exemplified by centrifugally drying the washed array is not limited to these.

Step 7: Detection and analysis steps

The detection step is to detect an affinity label bound to the enzyme immobilized on the array, and the detection is performed according to the type of the label attached to the affinity label. It is preferably performed by fluorescence measurement.

For example, when a biotin label is introduced, streptavidin phycoerythrin (hereinafter sometimes abbreviated as SAPE) is used for detection. That is, the staining solution containing SAPE is brought into contact with the protein chip after the washing step, and after washing, the signal emitted by the label is detected using a microscanner or the like. The detection can be performed using a commercially available microarray scanner such as White light Scanner-Array Work (manufactured by Applied Precision). Subsequently, before the analysis step, the detected signal was quantified using commercially available software such as, for example, IMAGENE ™ software (manufactured by Bio Discovery), and the data was analyzed. To analyze. Here, the detected reporter signal corresponds to the amount of the affinity label bound to the enzyme. Only when the enzyme is active, the enzyme and the affinity label corresponding to the active site form a covalent bond.Therefore, by detecting the amount of the affinity label, the amount of the enzyme activity is dependent on the enzyme activity. It becomes possible to measure specifically.

(Second Embodiment of the Present Invention)

The second embodiment of the present invention relates to a method for screening a regulatory substance that regulates an enzyme activity. The method for measuring enzyme activity based on the present invention can be used for a method for screening a substance for regulating an enzyme activity.

[0057] Enzymes are involved in a variety of biological reactions in a living body. Therefore, an enzyme activity regulator capable of regulating enzyme activity is used as a therapeutic or prophylactic agent for diseases caused by abnormal enzyme activity. The enzyme activity-regulating substance of the present invention, which can act as a drug inhibitor, is a generic term for substances that inhibit or promote enzyme activity. The screening method of the present invention searches for and identifies a novel such substance.

[0058] Explaining by taking a specific enzyme as an example, in the examples of the present invention, the protease used as a target of activity measurement is involved in many physiological phenomena such as biological defense and information transmission mechanism in a living body. It is known that abnormalities in the balance between proteases and their regulatory mechanisms have a significant effect on the pathology of various diseases. Specifically, cancer invasion, metastasis, and infection It has been reported that it is closely related to the mechanism of inflammatory response, blood coagulation reaction, cell death, muscular dystrophy, rheumatoid arthritis, Alzheimer's disease and the like. Therefore, substances that regulate protease activity have potential value as therapeutic and prophylactic agents for various diseases, including cancer, infectious diseases, inflammation, and thrombosis.

[0059] The method for screening for a modulator that regulates enzyme activity according to the second embodiment of the present invention includes the following steps.

First step: enzyme chips are prepared by placing different enzymes at different positions on the substrate Enzyme chip preparation step (hereinafter referred to as enzyme chip preparation step),

Fourth step: a test substance contacting step in which the enzyme chip is pre-incubated in the presence of a test substance (hereinafter, referred to as a test substance contacting step (corresponding to a preincubation step in the first embodiment))

Seventh step: an analysis step of detecting the signal of an affinity label attached with a reporter molecule bound to the immobilized enzyme on the enzyme chip, quantitatively analyzing the detected signal, and analyzing the signal ( The signal intensity of the test substance for each of the plurality of different enzymes (E2) is compared with the signal intensity of the enzyme not contacted with the test substance. By examining the change in the activity, the characteristics of the regulatory substance that regulates the enzyme activity are examined, thereby screening for a predetermined regulatory substance.

Hereinafter, each of the steps will be described in detail, but steps that can be performed in the same manner as the enzyme activity measuring method of the first embodiment will not be described.

[0061] In the incubation step, this step is carried out by adding a substance that regulates the amount of enzyme activity and a rate of enzyme reaction, or a candidate substance thereof, as a test substance. Here, the test substance applicable to the screening method of the present invention is derived from an organic compound synthesized organically, a library of proteins, or animals, plants and bacteria such as cells, bacterial extracts, and culture supernatants. Components.

In addition, the screening method of the present invention is carried out by using a test substance as a substance having poor solubility in water. When applying to an enzyme chip, prepare a high-concentration solution dissolved in an appropriate organic solvent such as DMSO, and dilute it to a predetermined concentration with an incubation solution. It is also possible to apply. At the time of dilution, the DMSO concentration is adjusted so as not to affect the enzyme activity.

Further, in the subsequent reaction step without bringing the immobilized enzyme spot into contact with the test substance, the one that has been brought into contact with the affinity label in the same manner as the test spot can be suitably used as a control. Due to the presence of the control spot, the ability of the test substance to regulate the enzyme activity is evaluated by comparing the signal intensity detected at the control with the signal intensity detected at the contact spot with the test substance. be able to.

[0062] After the detection, the test substance which changes the signal intensity as compared with the signal (control) in the enzyme spot without contacting the test substance is selected as an enzyme activity regulating substance capable of controlling the enzyme activity. In other words, when the affinity label is not detected at the position where the enzyme is immobilized on the substrate, or when the detection amount is reduced compared to the control, the test substance inhibits the enzyme activity, which inhibits the enzyme activity. If it is determined that the substance is a substance and the amount of detection increases compared to the control, the test substance is determined to be an enzyme-activating substance that activates the enzyme activity. The modulator screened in this way can be a potential therapeutic or prophylactic agent for diseases caused by abnormal enzyme activity.

(Third Embodiment of the Present Invention)

The third embodiment of the present invention combines the first and second embodiments described above to screen inhibitors (regulators) capable of regulating enzyme activity at high throughput (HTS), and at the same time, In this method, the inhibitory constant of each inhibitor for each enzyme is calculated.

More specifically, for each of a plurality of enzymes immobilized on an enzyme chip, the enzymatic potency-initiative constant for each of the affinity labels is measured mainly in accordance with the method described in the first embodiment. Then, in the manner described in the second embodiment, a regulator (eg, an inhibitor) capable of regulating the enzyme activity of the enzyme is screened, and the inhibitor of the inhibitor is combined with the enzyme kinetics constant described above. Calculate the constant.

FIG. 10 summarizes the third embodiment of the present invention. The process is configured. Step (a): The reaction mechanism between the affinity label and each enzyme immobilized on the enzyme chip and the kinetic constants of each enzyme are analyzed. Step (b): A test substance capable of functioning as an inhibitor is screened by applying a plurality of test substances to each subarray, and the inhibitory constant of the inhibitor is measured. Step (c): In step (b), an appropriate signal can be obtained by changing the concentration of the test substance for a powerful test substance that cannot obtain a signal of appropriate strength during screening. The concentration is searched, and the inhibition constant is measured again based on the result. Hereinafter, the steps (a) to (c) will be specifically described.

Step (a) is a step of analyzing the reaction mechanism between the affinity label and each enzyme fixed on the enzyme chip and the kinetic constants of each enzyme according to the first embodiment. .

As shown in step (a) of FIG. 10, according to the first embodiment, the enzyme chip is subjected to a blocking treatment and a pre-incubation treatment (preferably for 5 to 90 minutes), and then to a plurality (preferably four kinds). The above-mentioned) is contacted with the affinity label-containing solutions having different affinity label concentrations to initiate the reaction. That is, for example, as shown in Fig. 12, when 48 (4 columns x 12 rows) subarrays are present on an enzyme chip, each column has a different concentration (in this case, 4 types) of affinity. Use the tea label solution. Next, when a predetermined time comes for each line, the column is washed with an SDS-containing buffer, and the signal intensity (preferably, a fluorescent signal) generated by the affinity label is detected using a microscanner or the like. In other words, the change in signal intensity over time is measured for each spot.

In the next step, the detected signal is converted into the concentration of the reaction product using a commercially available software such as IMAGENE ™ software (manufactured by BioDiscovery). Here, the reaction product refers to an enzyme to which an affinity label is covalently bonded (the same applies hereinafter).

[0069] Data analysis is performed on the concentration (actually measured value) of the reaction product obtained at each reaction time. For data analysis, for example, using the DYNAFIT ™ program or the like, plotting the measured values on the vertical axis as the reaction product concentration and the horizontal axis as elapsed time It is performed by fitting a progress curve (time change curve) that is automatically derived (curve fitting).

[0070] In brief, a progress curve is basically a Michaelis-Menten (

Michaelis-Menten). That is, the following [Formula 2]

From the enzyme reaction model shown in

[0072] [Formula 3] Ichi

However,

max

It is well known that the Michaelis-Menten reaction rate equation shown in the following equation is derived.

That is, the reaction rate equation according to the Michaelis-Menten equation is determined by determining the rate constant (k + l, kl, k2), and furthermore, the reaction rate equation A curve can be calculated.

Therefore, by optimizing each rate constant, it is possible to derive a progress curve that fits the actually measured value. [0075] However, the enzyme reaction on the enzyme chip of the present invention is similar to the Myelis-Menten equation, but strictly different. In other words, in the original Myelis-Menten equation, the enzyme (E) and the reaction product (P) are produced from the enzyme-substrate complex (ES) as shown in the above [Chemical Formula 2]. Only the reaction product (P) is produced from the enzyme-substrate complex (ES) in the enzyme chip of the present invention. Therefore, in the present invention, the formula is hereinafter referred to as Michaelis-Menten derived formula for convenience. Also, in the present invention! / In some cases, non-enzymatic side reactions can occur. In such cases, curve fitting must be performed by assuming a new enzyme reaction model (enzyme reaction modeling).

FIG. 14 shows an example of enzymatic reaction modeling, in which three reaction equations (113) are assumed. In detail, Reaction Schemes 1 and 2 follow the equation derived from Michaelis-Menten, while Reaction Scheme 3 shows a non-enzymatic side reaction, for example, when the substrate (affinity label) is an enzyme. This reaction is intended to covalently bond with a reactive group that may be present in a portion other than the active center (for example, an enzyme surface). In FIG. 14, E: enzyme, S: substrate (affinity label in the present invention), ES: enzyme-substrate complex (in the present invention, the affinity label is in the active site of the enzyme). ), P: reaction product (in the present invention, an affinity label is covalently bound to an enzyme), k (+ l, -1, cat, side): rate constant of each reaction formula (Kinetics constant).

[0077] The modeling of an enzymatic reaction refers to assuming a mechanism of a reaction occurring between a certain enzyme and a substrate (affinity label in the present invention), and may differ for each enzyme (for example, of course, For some enzymes, non-enzymatic side reactions such as those shown in Reaction Scheme 3 may occur.

As described above, in the curve fitting of the present embodiment, modeling of the enzyme reaction is performed for each enzyme so as to fit the actually measured value data, and at the same time, the rate constant (kinetic constant) (k + l, k-1, kcat, kside, etc.) are calculated. From the results obtained in this step (a), the various conditions for carrying out the following step (b) (the concentration of the affinity label to be used (A) and the predetermined reaction time for the reaction (T)) Is determined. The determination of the affinity label concentration (A) and the prescribed reaction time (T) is preferred. In other words, the range in which the initial velocities of all kinds of enzymes fixed in the same subarray can be calculated (for example, the progress curves of all kinds of enzymes are in the initial stage, ie, the relationship between the reaction time and the signal intensity is proportional. (A range close to (straight line)).

In the step (b), according to the second embodiment described above, a plurality of test substances (candidate substances) are screened at a high throughput, and a modulator (inhibitor) capable of controlling (suppressing) the enzyme activity is discovered. This is a step of analyzing the inhibitory constant Ki of the inhibitor to the enzyme.

As shown in step (b) of FIG. 10, according to the second embodiment, an enzyme chip is subjected to a blocking treatment, and then each is subjected to a predetermined concentration (preferably 80 nM-300 / zM, for example, If the concentration of the inhibitor is 3 μ K, the range of Kf ^PP can be accurately calculated to be 80 η 2-2 μ）). In other words, if there are 48 (4 columns x 12 rows) subarrays on an enzyme chip as shown in Figure 12, for example, a maximum of 48 test substances can be screened per enzyme chip. it can. Next, the reaction is initiated by contacting the solution containing the affinity label having the concentration determined in step ( _a ). Then, when the predetermined reaction time (T) determined in step (a) comes, the plate is washed with an SDS-containing buffer, and the signal intensity (preferably, the fluorescent signal) generated by the affinity label is measured using a microscanner or the like. Is detected. The detected signal is converted into the concentration (d) of the reaction product by using a commercially available software such as IMAGENE ™ software (manufactured by Biodiscovery Inc.).

First, as shown in FIG. 10, by comparing the results obtained in step (a) with the results obtained in step (b) (comparison of signal intensities), each test was performed. The nature of the substance is analyzed. In other words, if each test substance shows a decrease in signal intensity for all enzymes (a substance that nonspecifically inhibits the reaction of any enzyme (nonspecific inhibitor)), conversely, all the enzymes No change in signal intensity is observed (substance that does not cause any reaction inhibition by any enzyme), or a decrease in signal intensity is observed only in spots where a specific enzyme is immobilized ( An enzyme (Specific inhibitors)).

[0082] From the viewpoint of pharmaceutical potential, it is desirable to screen for a specific inhibitor. Therefore, the inhibition constant (Ki) is calculated in order to evaluate the degree of inhibitory effect of the specific inhibitor.

[0083] To calculate the inhibition constant, the kinetic constant obtained in step (a) and one actual measurement value measured in step (b) (the reaction product generated in the presence of the candidate substance) Concentration) is calculated using either! Of two types of algorithms (hereinafter referred to as algorithm 1 and algorithm 2).

[0084] Algorithm 1 is based on the case where the initial concentration (total number) I of the inhibitors used in each subarray is larger than 5 times the concentration (total number) E of the whole enzyme immobilized on each subarray.

total

Used for I> 5E. Algorithm 2, on the other hand, is used when I <5E.

total total

It is. In the case of K5E, some of the enzymes immobilized on the subarray

total

Since there may be an enzyme having a very high affinity, a case where the inhibitor in the subarray during the reaction is insufficient may be assumed. Therefore, in that case, it is necessary to correct the calculated value using algorithm 2.

[0085] 1. Details of Algorithm 1

Algorithm 1 is implemented when I> 5E.

total

First, the kinetic constants (k + l, k_l, kcat, kside) obtained in step (a) and the reaction at a predetermined reaction time (T) obtained in step (b) or step (c) From the product concentration d, the apparent inhibition constant for each enzyme (assuming that N enzymes are immobilized in one subarray) is determined by the one-point curve fitting method described below or the initial velocity method ( Ki, n ^app ; n = l—N) is calculated.

[0086] <One-point curve fitting method>

The one-point curve fitting method performed in this algorithm is slightly different from the curve fitting method performed in step (a) described above.

As shown in Fig. 15 (a), the enzyme reaction model in the presence of the inhibitor is assumed to consist of four reaction equations, and k + i, ki, and ヽぅ kinetic constants are newly added. Is done.

[0087] In the curve fitting performed in step (a) described above, each force kinetics is set so that the progress force is fitted to a plurality of measured data. The constants are optimized, but only one measured data is obtained in step (b).

Therefore, in the curve fitting method performed in this algorithm, among the six kinetic constants, four kinetic constants (k + 1, k-1, kcat, kside) are obtained in step (a). Then, assuming that k + l = k + i for convenience, ki is optimized so as to fit the single measured value data obtained in step (b). From the two kinetic constants (k + i and ki) obtained in this way, an apparent inhibition constant Ki, _n ^app = k + i, n / ki, n is calculated.

[0088] Initial velocity method>

FIG. 15 (b) schematically shows a progress curve 1 in the absence of the inhibitor and a progress curve 2 in the presence of the inhibitor. For progress curve 1, the force that can be derived by the curve fitting method in step (a) For producer curve 2, only one actual measurement data is obtained in step (b), ) Cannot be derived by the curve fitting method. Therefore, the progress curve 2 shows a virtual curve by a dotted line.

Note that, originally, the initial speed is also calculated by calculating the inclination force of the tangent to the progress curve at the start of the reaction. However, in the initial speed method implemented in this algorithm, a predetermined reaction time (T) is used for convenience. The initial product V (0) (= D / T) and V (I) are the values obtained by dividing the concentration of the reaction product at T (D for progress curve 1 and d for progress curve 2) by T. (= d / T), and the apparent inhibition constant Ki, n ^app = {I—En (l—V (I, n) / V (0,11))} / {¥ (1,11) / Calculate ¥ (0,11) -1} (11 = 1-?), Where En (n = l-N) is the initial concentration of each enzyme.

[0090] Then, determine the inhibition constant Ki apparent calculated as described above, the inhibition constant Ki for inhibitors against each enzyme using the n ^app like, n and (n = 1- N).

Inhibition constant Ki, n (n = 1—N) of inhibitor for each enzyme in Algorithm 1 The solution is shown below.

[0091] 0) Algorithm 1 is performed when the following [Formula 4] is satisfied.

[0092] [Formula 4]

[0093] 1) The start conditions are shown in the following [Dani 5].

[0094] [Formula 5]

Ε _η · Ι

' ⁰ one ι + κζ ^ρ ! ~ Ν)

[0095] 2) According to the following formula shown in [Chemical formula 6], calculation is performed from k = 1, and λ and Ν

The vector% k consisting of the components of k is sequentially calculated. The calculation (iteration) is repeated until the value of k sufficiently converges (k = l, 2, 3,...).

[0096] [Formula 6]

: = +1:

3) The vector% corresponding to λ that converged sufficiently in 2) is the solution to be found.

k k

As shown in [Chemical formula 7], each of the N components of the vector X is the concentration of each complex (n =, N) formed between each enzyme and the inhibitor.

[0098] [Formula 7]

[0099] 4) Next, the% _n (n = l-N) for each enzyme calculated in 3) was substituted into the following [Idani 8], and the inhibition constant Ki, n (n = 1) N) is calculated.

[0100] [Formula 8]

_V , L _n ~ Xn) '(~ n)

λ η

[0101] Here, Εη (η = 1-Ν); initial concentration of each enzyme, I; initial concentration of inhibitor used in each subarray, X (η = 1-N); each enzyme and inhibitor Concentration of the complex formed during Ki, n ^app (n = l-Ν); apparent inhibition of each enzyme to the inhibitor calculated by the one-point curve fitting method or the initial velocity method described above. constant.

[0102] 2. Details on Algorithm 2

As for Algorithm 2, the apparent inhibition for each enzyme (assumed to be the type of enzyme fixed in one subarray) by the one-point curve fitting method or the initial velocity method as in Algorithm 1 described above. Calculate the constant (Ki, n ^app ; n = l—N).

Then, determine the inhibition constant Ki of the calculated apparent inhibition constant Ki of Inhibi coater for each enzyme using n ^app like, n and (n = 1 one N).

The solution of the inhibitory constant Ki, n (n = 1-N) of the inhibitor for each enzyme in Algorithm 2 is shown below.

[0103] 0) Algorithm 2 is implemented when the following [Formula 9] is satisfied.

[0104] [Formula 9]

v = 1

[0105] 1) Solve the equation shown in the following [Chemical Formula 10] using a computer, and obtain N + 1 solutions for λ. [0106] [Formula 10]

[0107] 2) Next, among Ν + 1 solutions obtained for λ, I or 解 = (= Ε)

total

Solutions are excluded.

[0108] 3) Next, with respect to the remaining solution λ that was not removed in 2)! /, And shown in the following [Formula 11]

k

Thus, the vector% of the N component powers is calculated as follows.

[0109] [Formula 11]

[0110] 4) Next, all the Ν components of the vector% are within the range of [0 ... min (En, 1)].

k

Judge whether the power is good or not. If any of the N components does not fall within the range [0 ... min (En, 1)], its vector% (λ)

kk is excluded.

[0111] 5) There is always only one λ that satisfies 2) and 4) above, and the vector corresponding to that

k k

1% is the solution sought, and as shown in [Chemical Formula 12] below, each of the N constituents of the vector% is replaced by the concentration of each complex formed between each enzyme and the inhibitor. (n = 1-N).

[0112] [Formula 12]

6) Then,% _n (n = l−N) for each enzyme calculated in 5) was substituted into the following [Chemical Formula 13], and the inhibition constant Ki, n (n = 1 One N) is calculated.

[0114] [Formula 13]

[0115] Here, = η (η = 1- 濃度); initial concentration of each enzyme, I; initial concentration of inhibitor used in each subarray, X (η = 1-N); each enzyme and inhibitor Concentration of the complex formed during Ki, n ^app (n = l-Ν); apparent inhibition of each enzyme to the inhibitor calculated by the one-point curve fitting method or the initial velocity method described above. constant.

[0116] <Step (c)>

In step (c) shown in FIG. 10, in step (b), for a test substance that cannot obtain an appropriate signal intensity (concentration), the concentration of the test substance is variously changed, and This is the step of searching for the concentration (optimal concentration) at which a high signal can be obtained, and measuring the inhibition constant again based on the results.

[0117] Since step (b) is performed using a test substance-containing solution having a predetermined concentration arbitrarily set by an operator, the set concentration may not always be appropriate. For example, setting If the concentration is too low, a sufficient inhibitory effect cannot be obtained and the inhibitor does not have an effect! /, Or if the set concentration is too high, the inhibitory effect becomes excessive and the inhibition constant is calculated. This is the case when the signal intensity required to perform the measurement cannot be obtained. In view of such a situation, the present inventors have set a certain standard for convenience. That is, if the signal intensity (concentration) obtained in step (b) is within a certain predetermined range, the inhibition constant is calculated based on the result using the above-described algorithm. However, if the signal intensity (concentration) obtained in step (b) is not within the predetermined range, the concentration of the test substance may be varied as shown in step (c) of FIG. After changing, search for the concentration (optimal concentration) that can obtain an appropriate signal, and then re-measure the inhibition constant based on the result.

[0118] Specifically, first, in step (b), it is determined whether or not a signal intensity force obtained for each spot (enzyme) is within a predetermined range.

[0119] For example, a case where the determination is made for enzyme I will be described. The signal intensity obtained in step (b) is the signal intensity obtained at the specified affinity label concentration (A) and the specified reaction time (T), and the signal intensity at this time is assumed to be f. . Also, since various kinetic constants (ie, progress curves) have already been determined for enzyme I in step (a), the specified affinity label concentration (A) and the specified reaction The signal intensity F at time (T) can be calculated.

Here, the predetermined range refers to a range satisfying 0.9F> f> 0.IF. Therefore, if 0.9F≤f or 0.lF≥f, perform step (c) to search for an appropriate test substance concentration.

[0121] In step (c), as shown in Fig. 10, the enzyme chip was subjected to a blocking treatment, and then the concentration was changed for each line (typically, 120 / z M-O. LlnM). The pre-incubation treatment is carried out by contacting the contained solution (in Fig. 10, the concentration of the left side force is also reduced in order of the direction force). In other words, as shown in Fig. 10, when there are 48 (4 columns x 12 rows) subarrays on an enzyme chip, for example, up to 12 kinds of up to 4 kinds of test substances per enzyme chip (The number of test substances to be performed and the measured concentrations are not limited to this). For the enzyme to be tested, if a total of eight enzymes were immobilized on the subarray, For example, in step (b), out of the eight enzymes, two of them are used to obtain an appropriate signal intensity! /, Na! /, And in the case of step (c), the remaining six enzymes are used. Is performed.

[0122] Next, in the same manner as in step (b), the reaction is initiated by contacting an affinity label-containing solution having the concentration determined in step (a). Then, when a predetermined reaction time (T) comes, the plate is washed with an SDS-containing buffer, and the signal intensity (preferably, a fluorescent signal) generated by the affinity label is detected using a microscanner or the like.

FIG. 16 schematically shows the results that can be obtained for one type of enzyme when step (c) is performed. The curve shown in FIG. 16 is an inverse sigmoid curve, and shows that the signal intensity of the affinity label decreases as the test substance concentration increases.

[0124] Contact the test substance and set the signal intensity detected at the ヽ spot (test substance concentration OnM) to 1, and sequentially increase the signal detected when the test substance concentration was increased. Test lot concentration (N-Ν ') with signal intensity in the range of 0.1-0.9 is judged to be appropriate test substance concentration. Next, in the same manner as described above, any of the signal intensities within this concentration range (Ν-Ν ') was analyzed using a commercially available software such as IMAGENE ™ software (manufactured by Biodisc Company). The inhibition constant is recalculated by the above algorithm 1 or 2 using the measured value. FIG. 17 shows a flowchart summarizing the above-described third embodiment of the present invention.

(Other Embodiments of the Present Invention)

1. The present invention provides an automated system for measuring enzyme activity, comprising means for automatically performing each of the above steps sequentially using the enzyme activity measurement method as described above. . By constructing such a system, enzyme activity measurement can be performed easily and quickly. In addition, by connecting to a computer, it becomes possible to construct a database on enzyme activity, to elucidate biological functions and to provide useful information in the production site of useful products.

2. The present invention provides a method for controlling an enzyme activity, comprising means for automatically performing each of the above steps sequentially using the method for screening a substance for regulating an enzyme activity as described above. An automated system for screening nodal substances is provided. By constructing such a system, screening for a modulator of enzyme activity can be performed easily and quickly. In addition, by connecting to a computer, the measured values of the signals from each spot are analyzed by the computer to create an entire signal pattern, and the signal from the control spot is compared with the signal from the test spot contacted with the test substance. Then, since it is possible to output the determination of the possibility as an enzyme activity regulator, it is possible to provide useful information in the field of drug development.

3. The present invention provides a method for measuring enzyme kinetics between an enzyme and an affinity label by applying the method for measuring enzyme activity as described above. Specifically, a fixed unit of enzyme is immobilized on a substrate, and a constant concentration of an affinity label is brought into contact with the enzyme chip, and the time change of the signal intensity is detected on the enzyme chip to measure the kinetics of the enzyme. Is what you do. Conventionally, there is no technique for measuring enzyme kinetics on a chip, and this is a problem first achieved by the present invention. By constructing a method for measuring enzyme kinetics on such a chip, a plurality of enzymes can be immobilized to simultaneously, comprehensively, and at a high throughput. It becomes possible to measure.

The affinity label has specificity and activity dependency on the enzyme. When the enzyme and the affinity label react, the reaction proceeds at a constant rate in the initial stage of the reaction, and thereafter, the reaction rate gradually decreases. The Michaelis-Menten Kinetics model, which is well known to those skilled in the art, is best fitted between the reaction rate in the initial stage of the reaction and the affinity label, and the affinity label is determined according to the kinetics of the powerful enzyme. From the relationship between the concentration [S] and the reaction rate (V), Km and Vmax can be obtained. In other words, the enzyme reaction speed

By examining the dependence of the degree on the affinity label concentration, the kinetic constant can be calculated. Therefore, the present invention also provides a method for calculating a kinetic constant.

Further, the present invention provides a method for measuring kinetics, which has a means for automatically performing each of the above steps sequentially using the kinetics measuring method as described above. Provide an automation system. Furthermore, by connecting to a computer, it is also possible to construct a database for automatically analyzing the kinetic constants by automatically inputting the measured values measured above, and to provide an automatic kinetic constant analysis system. Becomes. This makes it possible to elucidate biological functions and provide useful information at the production sites of useful products.

4. The present invention provides a method for calculating the inhibition constant of an enzyme activity inhibitor by applying the enzyme activity measurement method as described above. For example, a fixed unit of enzyme is immobilized on a substrate, pre-incubated with inhibitors prepared at different concentrations, then contacted with an affinity label at a fixed concentration, and the change in inhibitor concentration of signal intensity is measured on the array. Upon detection, the inhibition constant of the inhibitor can be calculated. The method of calculating the inhibition constant is known, and it can be calculated based on the change in the inhibitor concentration of the signal intensity, which changes the signal intensity observed at the enzyme spot without contacting the test substance with the signal intensity. Is possible,

By constructing a method for calculating the inhibition constant of an enzyme activity inhibitor using an enzyme chip, it becomes possible to simultaneously and comprehensively calculate the inhibition constants for a plurality of enzymes. High-throughput enzyme activity inhibitors can be evaluated.

Further, the present invention has a means for automatically performing each of the above steps sequentially using the method for measuring the inhibition constant of an enzyme activity inhibitor as described above, and is connected to a computer. It is also possible to construct a database for automatically inputting the measured values measured above and analyzing the inhibition constant, and to provide an automated system for analyzing the inhibition constant of enzyme activity. This makes it possible to provide useful information in the field of drug development.

5. The present invention provides a reagent for measuring immobilized enzyme activity, which includes an affinity label to which a reporter molecule is attached, and is used for measuring the enzyme activity of the immobilized enzyme. The reagent for measuring the activity of the immobilized enzyme of the present invention contains an affinity label and is dissolved or suspended in an appropriate solvent. The composition ratio of the reagents, but is variously suitably selected according to the intended purpose, in a suitable buffer in one, 10- ⁶ - 10- ⁸ M concentration in preparation by § off I - containing tea label, fixed I匕酵containing An activity measuring reagent can be exemplified. 〔Example〕

[0126] Hereinafter, experiments performed to demonstrate the effectiveness of the enzyme activity measurement method of the present invention will be described. The cysteine protease activity was measured by a suitable protocol.

[0127] (Material used)

1.enzyme

Cathepsin B from human liver (Canolebiochem)

Recombinant human cathepsin B (E. Coli: MTA, Canada)

カテ Cathepsin B from calf spleen (Calbiochem)

Cathepsin C

Cathepsin H from human liver (Canolebiochem)

Cathepsin H from kidney

His—Tag human cathepsin K (E. coli)

Cathepsin L derived from kidney

Cathepsin L from human liver

His—Tag human cathepsin L

Recombinant human cathepsin L (E. coli: MTA, Canada)

カテ Cathepsin S from calf spleen (Calbiochem)

Cathepsin S from human spleen (Calbiochem)

Human erythrocyte-derived calpain I (Calbiochem)

Recombinant rat calpain II (E. coli)

Human calpain II

Papain derived from papaya (Merck)

Human procatebcin L (E. coli: MTA, Canada)

BSA (manufactured by Wako Pure Chemical Industries) may be used as a negative control.

2. Substrate [0128] A glass aldehyde glass slide modified by No Ab Diagnostics (Canada) was used for a glass slide manufactured by Erie Scientific (USA). The glass slide used in this experiment was a fluororesin. It was configured to form 48 12 × 4 sub-arrays separated by a mask.

[0129] 3. Microarrayer

A microarrayer manufactured by Castesian Scientific was used.

[0130] 1. Composition of buffer

The composition of the buffer used in the following experiments is as follows.

'Malonic acid buffer: 20 mM malonic acid, 50 mM NaCl, pH 5.5

'Tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris) buffer: 50 mM Tris, pH 5.5

'' Imidazole buffer: 20 mM imidazole, 30% glycerol

[0131] (Examination example)

The following is an example of a study performed to optimize the implementation of the present invention. In the following examination examples, optimization of the optimal conditions for detecting cysteine protease activity was examined.

[0132] (Examination example 1)

Printing buffer optimization experiment

The effect of the printing buffer on the detection of the enzyme activity signal when printing the enzyme on the substrate was compared with various printing buffers, and the optimal printing buffer was selected.

[0133] The printing buffers studied are shown below.

Malonic acid buffer ( _P 5.5)

Malonic acid buffer ZPBSlZl (pH 7.0)

[0134] Using each of the above printing buffers, cathepsin L and cathepsin B were printed on a solid substrate under the same conditions except for the printing buffer, and then the respective enzyme activity signals were obtained under the same conditions. Detected using affinity label.

[0135] The results are shown in FIG. Cathepsin B was a signal that a good signal was detected by using any of the printing buffers, and in cathepsin L, the enzymatic activity signal disappeared in a pH 7.0 printing buffer. Therefore, Printy It was found that the use of a malonic acid buffer (pH 5.5) was the most suitable as the buffering buffer.

[0136] (Examination example 2)

Optimization experiment of enzyme printing concentration

The effect of the concentration of the enzyme printed on the substrate on the detection of the enzyme activity signal was compared and examined by immobilizing the enzyme prepared at various concentrations on the substrate. Attempted selection.

[0137] The printing concentrations of the studied enzymes are shown below.

0.7 mgZ ml,

丄. 4mgZ ml

[0138] Cathepsin B prepared at each of the above concentrations was printed on a substrate surface-treated with a noided gel NHS under the same conditions except for the above concentrations, and then the respective enzymes under the same conditions. The activity signal was detected using an affinity label.

[0139] The results are shown in FIG. With 1.4 mg Zml, it was found that with 0.7 mg Zml, a detectable enzyme activity signal could be detected with 0.7 mg Zml (Fig. 3, right). In addition, when the same examination was performed using papain, signal diffusion was confirmed at 1.4 mgZml (Fig. 3, left). Although not shown here, by preparing the enzyme within the range of 0.1 mg / ml to 1 mg / ml and printing it on the substrate, a sufficiently detectable signal intensity is ensured and the spot diffusion is achieved. It was also found that a good signal could be detected with a minimum amount of noise.

[0140] (Examination example 3)

Experiments on optimization of substrate surface treatment

The effect of the surface treatment of the immobilized substrate on which the enzyme is immobilized on the detection of the enzyme activity signal was examined by comparing the various chemical surface treatment methods described below and trying to select the optimal substrate surface treatment. Was.

[0141] The substrate used was a glass slide having a 12 x 4 sub-array divided by a fluorine resin mask, and was manufactured by Matsunami Glass Industries, ERIE Scientific (USA)! . The surface treatment methods studied this time are as follows.

Hide mouth gel aldehyde functionalization treatment

Hide mouth gel BSA-NHS functionalization treatment

Hyde mouth gel NHS functionalization treatment

[0142] Papain, cathepsin B and cathepsin L, respectively, were prepared in each printing buffer of malonic acid buffer (pH 5.5), malonic acid buffer / PBS 1/1 (pH 7.0), and After printing under the same conditions as above and under the same conditions, the respective enzyme activity signals were detected under the same conditions using affinity labels.

FIG. 4 shows the results. In Study 1 above, in the study with malonic acid buffer (pH 5.5), which was found to be the most suitable printing buffer, the aldehyde-functionalized aldehyde treatment and the N- and ido-functionalized gel BSA-NHS functionalization treatment were performed. A good enzyme activity signal was detected on the substrate subjected to the treatment. On the other hand, on the substrate on which the hide-mouth gel NHS functionalization treatment was applied, it was possible to detect the enzyme activity signal derived from cathepsin B and cathepsin L. Therefore, it was found that the functionalization treatment of the gel aldehyde at the mouth and the gelation of BSA-NHS at the mouth were the most suitable as the surface treatment of the substrate.

[0144] (Study example 4)

Experiment to study optimization of blocking agent

The effect of the blocking agent on the enzyme activity signal and the background signal was compared with various blocking agents described below, and an attempt was made to select an optimal blocking agent for use in the present invention.

[0145] The blocking agents studied this time are shown below.

Block ace

Tris buffer ethanolamine

Polyethylene glycol

[0146] After printing of the enzyme under the same conditions, a blocking treatment was performed with each of the above-mentioned blocking agents, and then under the same conditions, the respective enzyme activity signals were detected using affinity labels. [0147] The results are shown in FIG. When Block Ace was used as a blocking agent, the background was clearly reduced, and a good enzyme activity signal was detected, compared to the case where Tris Knofer, ethanolamine, or polyethylene glycol was used. confirmed. Therefore, it was found that the use of Block Ace was optimal as a blocking agent.

[0148] (Examination Example 5)

Pre-incubation buffer optimization experiment

The effect of the pre-incubation buffer on the enzyme activity signal during pre-incubation was compared with DTT (+) and DTT (-) containing buffers, and an attempt was made to select the optimal pre-incubation buffer for use in the present invention. Was.

[0149] The cysteine proteases shown in Fig. 6 were printed on the surface of the NHS hydrid gel-functionalized substrate, and pre-incubation was performed using a pre-incubation buffer containing DTT or not containing DTT. After that, each enzyme activity signal was detected under the same conditions using an affinity label.

[0150] The results are shown in Figure 6 (subarrays 10, 11, and 12). When using a preincubation buffer containing DTT, a good enzyme activity signal was detected, whereas when using a preincubation buffer containing no DTT, an enzyme activity signal was detected. Power Therefore, it was found that the presence of a reducing agent was necessary for the activity of the arrayed enzymes, and that the addition of DTT was particularly desirable.

[0151] (Examination example 5)

Optimization experiment of pre-incubation time

Based on the influence of the preincubation treatment on the enzyme activity signal, various treatment times were compared and examined, and an attempt was made to select an optimal preincubation time for application of the present invention.

[0152] The time of the pre-incubation treatment examined was 11 points in 5-minute intervals between 5 minutes and 60 minutes. After printing the enzyme under the same conditions, pre-incubation was performed in the pre-incubation buffer for each of the above times, and the enzyme activity of papain and cathepsin L when incubated with the affinity label for 8 minutes. The course of the signal was followed.

FIG. 7 shows the results of the study. It was found that the enzyme activity signal could be detected well in the preincubation treatment time of 20 minutes to 1 hour.

[Examination Example 8]

Experiment on optimization of additive concentration of affinity label

The effect of the concentration of the affinity label on the enzyme activity signal in the reaction process was compared and examined for various concentrations, and an attempt was made to select an optimum concentration for application of the present invention.

[0155] The amount of addition of the affinity label was as follows, and was examined at various reaction times.

20 X 4.8 X 10

8 X 4.8 X 10

3 X 4.8 X 10

0.5 X 4.8 X 10

FIG. 8 shows the results. The hydrogenation mosquito卩high concentrations of § Fiyu tea label, Bruno Tsu increased click ground sheet Gunaru is observed, the § Fiyu tea label 10 ⁶ - by the addition child in a range of 10 ⁸ M, by reducing the knock ground, It turned out that a good signal could be detected. Example 1

[0157] Activity measurement of protease

Among the enzymes described above, the activities of the seven enzymes shown in FIG. 9 were measured.

Then, all enzymes except calpain I were diluted with malonic acid buffer to a concentration of 0.7 mgZml, while calpain I was diluted with imidazole buffer to a concentration of 0.7 mgZml. However, enzymes provided at concentrations lower than the above concentrations were used without dilution.

[0158] The enzyme prepared at the predetermined concentration as described above was printed on a hide-mouthed gel aldehyde glass slide substrate using a microarrayer at 23 ° C under 70% humidity, and the enzyme was printed on the substrate. An arrayed enzyme chip was prepared. At this time, the enzymes were arranged such that each of the 48 subarrays formed the same enzyme sequence pattern. Enzyme sequence pattern Figure 2 (right, top).

[0159] Then, the enzyme chip was incubated at 25 ° C under 90% humidity for 1 hour. After immersion in Block Ace Z Malonic Acid Buffer 1Z2 at 4 ° C for 2 minutes to perform the blocking treatment, immediately wash in 50 ml Malonic Acid Buffer: Wash Solution 1 three times, The enzyme chip was completely dried by centrifugation at 2000 rpm for 2 minutes at 23 ° C. After drying, 6 μL of Tris buffer containing 2 mM DTT, 5 mM Ca ²⁺ and 5 mM Mg ²⁺ was applied to each subarray.

[0160] At this time, a test substance at a desired concentration can be optionally present in the mixture, and up to 3% DMSO can be similarly present in the mixture, and 30 minutes can be used. Incubation can be carried out for a variable period within a range not exceeding. Here, the test substance to be added is a candidate substance for the enzyme activity regulating substance, and it is possible to evaluate the influence of the test substance on the enzyme activity.

Next, 3 μl of Tris buffer containing the selected tag affinity label was added to the subarray. At this time, the concentration of the affinity label shall be adjusted within the range of 10-6 ⁶ and 10-8 ⁸ . Then, the mixture was incubated at 23 ° C., preferably for a fixed time selected from a period of 20 minutes to 12 hours. After incubation for a predetermined period of time, the subarray was washed under running water of 200 1 detergent-containing washing solution (preferably, Tris buffer containing Tween20, particularly preferably 13%): washing solution 2.

[0162] After the above treatment, the entire enzyme chip was washed with washing solution 2 at 80 ° C for 5 minutes, and then washed with distilled water at room temperature for 5 minutes, and the enzyme chip was washed at 2000 rpm at 23 ° C. After the enzyme chip was completely dried by centrifugation for 2 minutes, the signal pattern on the enzyme chip was scanned with a microarray scanner. After analyzing the obtained signal image, the data was quantified by a computer program. A typical image is shown in FIG. 2 together with the reaction conditions (left shows the reaction conditions, middle shows the whole array, and lower right shows the sub-array). It was found that target and activity-dependent measurements could be made.

Example 2

[0163] Evaluation of protease inhibitors The protease inhibitors were evaluated according to the method for measuring protease activity described in Example 1 above. The enzymes used for the evaluation of inhibitors were the seven enzymes shown in Fig. 6 among the enzymes described above.

E64 was used as a protease inhibitor. E64 (irreversible inhibitor of cysteine protease) prepared at 40 / z g / ml was serially diluted and incubated on the enzyme chip at the concentrations shown in FIG. 6 for 15 minutes each. Subsequently, the enzyme activity signal was detected using affinity labels.

[0164] The results are shown in FIG. As a result, it was confirmed that when the concentration of the inhibitor was high, the detection amount of the enzyme activity signal was decreased, and when the concentration of the inhibitor was low, the enzyme activity signal was increased. It was confirmed that the enzyme activity was inhibited.

Therefore, it has been found that the present invention can favorably evaluate an enzyme activity inhibitor.

Example 3

[0165] Example of the third embodiment

As shown in FIG. 11, eight cathepsin enzymes belonging to the cysteine protease family (CatB, bov, spleen-derived cathepsin B (CatB, bov), human liver-derived cathepsin B (CatB, hum), recombinant human cathepsin B (CatB , rec), catfish spleen-derived cathepsin C (CatC, bov), catfish kidney-derived cathepsin H (CatH, bov), human liver-derived cathepsin L (CatL, hum), recombinant human cathepsin S (CatS, rec), and Recombinant human cathepsin K (CatK, rec)} force Two spots (duplicate) were fixed in each of the S1 subarrays, for a total of 16 spots. As shown in FIG. Row), a glass slide functionalized with hydrogen aldehyde (hereinafter referred to as HGS) was used.

Three HGSs were prepared, and HGS 1 was used in step (a), HGS 2 was used in step (b), and HGS 3 was used in step (c).

[0166] 1. Step (a)

First, HGS1 is subjected to a blocking treatment as described in the first embodiment according to step (a) to remove unnecessary blocking agents by washing, and then a pre-incubation solution (8 μL) containing 2 mM DTT is used. )) To preincubate each subarray. Next Then, four different concentrations (0.5 M, 1.5 M, 4.5 M, 13.5 M) of a fluorescently labeled affinity label (above [Chemical Formula 1]) solution (4 μL) are brought into contact with each row of the subarray. And reacted with each enzyme. Then, for each row, SDS-containing at the specified reaction time (0 min, 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 7 min, 10 min, 13 min, 17 min, 22 min) Washing was performed under high stringent conditions with a buffer. Next, the fluorescence intensity of each spot was measured with a microarray scanner, and the detected fluorescence signal was converted to the concentration of the reaction product by IMAGENE ™ software (manufactured by Bio Discovery).

[0167] Using the DYNAFIT ™ program, the concentration of the reaction product obtained at each reaction time (actually measured value) was measured for four enzyme label concentrations for each enzyme as shown in Fig. 13. Curve fitting was performed. In detail, the enzyme reaction is modeled for each enzyme so as to fit each measured data, and at the same time, the rate constants (force kinetics constants) (k + l, kl, kcat, kside) for each reaction are The optimal value has been calculated. The results are shown in Table 1 below. Note that FIG. 13 is a progress curve (time change curve) obtained by converting the signal obtained in FIG. 12 into the concentration of the reaction product, with the ordinate representing the reaction product concentration and the abscissa representing the elapsed time. The four curves correspond to different affinity label concentrations (0.5 M, 1.5 / zM, 4.5 μΜ, 13.5 μΜ). FIG. 13 shows the results for only one type of enzyme, and is obtained according to the number of types of enzymes immobilized on the enzyme chip (therefore, for example, in this example, eight types of cathepsin enzymes were used). 8 results will be obtained.

[0168] [Table 1]

Power Tef ° Shin k _{+ 1} koat ^ side

[yM ^ s- ¹ ] [s- ¹ ] [s- [M ^ s- ¹ ]

B (bov) 1. 4 10. 4 0. 0465 0. 0102

B (hum) 3.7 31.0 0.00994 10.7

B (rec) 1. 0 19.3 0. 0207 3.18

C (bov) 2.9 3.46 0.006655 3.12

H (bov) 3.4 54.7 0.00461

K (rec) 3.8 56.6 0.06.009 7.1 1 1

L (hum) 6. 8 36. 1 0. 0229 101

S (rec) 3.0 16.2 0.0606 29.9

[0169] Based on the results shown in Table 1 above, conditions (affinity label concentration (A) and predetermined reaction time (T)) for carrying out step (b) were set. In this embodiment, the above 8 o

The conditions for being able to support all types of cathepsin enzymes were a predetermined CM affinity label concentration (A) = 1.0 M and a predetermined reaction time (T) = 120 seconds.

[0170] 2. Process (b)

176 types of HGS2 were prepared according to the conditions set in step (a) according to step (b) (specified affinity-label concentration (A) = 1.0 M, specified reaction time (T) = 120 seconds). Inhibitor screening was performed for the test substances. Eight known cysteine protease inhibitors were randomly mixed in advance with 176 test substances.

[0171] Blocking treatment was performed in the same manner as in step (a), unnecessary blocking agents were washed away, and then a test substance-containing solution (8 µL) containing each test substance (3 M) and DTT (2 mM) Was contacted with each subarray and pre-incubated for 90 minutes. Next, 1 ml of an affinity label solution (4 L) was added to the mixture, and the mixture was further incubated for 120 seconds to react. After 120 seconds, each subarray was washed with a buffer containing SDS under high stringency conditions. Next, the fluorescence intensity of each spot was measured using a microarray scanner, and the detected fluorescence signal was converted to the concentration of the reaction product using IMAGENE ™ software (manufactured by Bio Discovery). Compared with the fluorescence intensity in the absence of the test substance obtained in step (a), all eight types of known cysteine protease inhibitors could be screened.

[0172] 3. Step (c)

Using HGS3, four of eight known cysteine protease inhibitors [E-64 (Powers, J.C., Asgian, J 丄., Ekici, O.D., James, K.E.

Irreversible inhibitors of serine, cysteine, and threonine proteases.Chem. Rev. 102, 4639-4750 (2002).), Leu (Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase.Analytical Biochem. 237, 260-273 (1996); and Azaryan A., Galoyan A. Human and bovine brain cathepsin L and cathepsinH: purification, physico-chemical properties, and specificity. Neurochem. Res. 12, 207-13 (1987). .), ALLN (Sasaki, T. et al. Inhibitory effect of di-- and tripeptidyl aldehydes on calpains and cathepsins.J. Enzyme Inhibition 3, 195-201 (1990)), CNI (Rydzewski, RM et al. Peptidic 1- cyanopyrrolidines: synthesis and SAR of a series of potent, selective cathepsininhibitors. Bioorg. Med. Chem. 10, 3277-3284 (2002).

[0173] The test was performed in the same procedure as in the above step (b) except that the test was performed using a test substance-containing solution in which the concentration of the test substance (inhibitor) was variously changed. FIG. 18a) and FIG. 19a) show the results (signal (fluorescence) images) obtained by varying the concentrations of the inhibitors E-64 and CM, respectively. Figures 18b) and 19b) show the results obtained by varying the concentrations of the inhibitors E-64 and CNI for the six types of cathepsin enzymes (B, C, H, K, L, S) in step (c). 11 is a result obtained by plotting a change in signal intensity when the measurement is performed. Each curve was an inverse sigmoid curve, and an appropriate inhibitor concentration (optimal concentration) having a signal intensity in the range of 0.1-0.9 was selected.

[0174] From the above results, the inhibition constant Ki was calculated by Algorithm 1 or Algorithm 2, and the results are shown in Table 2 below. The values in parentheses in the table are described in the literature It is a known inhibition constant, and it has been confirmed that it is in good agreement with the result measured by the present invention.

[Table 2] Comparison of measured inhibition constants (K i) with those reported in the literature

Example 4

[0176] Method robustness of the present invention

The present inventors have varied the operating conditions (surfactant (SDS) concentration and pH) to determine whether the present invention can be properly performed under a wide variety of operating conditions. The enzyme immobilized on the substrate was reacted with the affinity label.

[0177] As shown in Fig. 20 (a), using one row of HGS (12 subarrays), the subarrays were subjected to blocking treatment, and unnecessary blocking agents were washed away. A pre-incubation solution (8 μL) containing DTT (2 mM) with different SDS concentrations (0-35 mM, cmc (SDS) = 6-8 mM) was then pre-incubated for 90 min by contacting each subarray. did. Next, 1 mL of an affinity label solution (4 L) was added to the mixture, and incubated for 120 seconds to react. After 120 seconds, each subarray was washed with a buffer containing SDS under high stringency conditions. Next, use microarray scanner for each spot. The fluorescence intensity was measured, and the detected fluorescent signal was converted to the concentration of the reaction product using IMAGENE ^1M software (manufactured by Bio Discovery), and as shown in Fig. 20 (b), the enzyme activity was determined for each SDS concentration. PiZPO was calculated and plotted (Pi: the concentration of the reaction product at each SDS concentration, PO: the largest of the reaction product concentrations at each SDS concentration, the reaction product concentration (maximum value)).

As shown in FIG. 20 (b), the activity of cathepsin S and cathepsin H rapidly decreases as the SDS concentration increases. On the other hand, the enzymatic activity of cathepsin K sharply increases when the SDS concentration is around 6-8 mM (cmc (SDS)). Cathepsin B, cathepsin C and cathepsin L also showed a tendency similar to that of catabsin K described above, and were more remarkable than cathepsin K.

As shown in FIG. 21 (a), using one row of HGS (12 subarrays), the subarrays were subjected to blocking treatment, and unnecessary blocking agents were washed away. Then, a preincubation solution (8 μL) containing DTT (2 mM) having various different pHs (3.5-9) was brought into contact with each subarray and preincubated for 90 minutes. Next, 1 ml of an affinity label solution (4 L) was added to the mixture, and the mixture was further incubated for 120 seconds to react. After 120 seconds, each subarray was washed with a buffer containing SDS under high stringency conditions. Next, the fluorescence intensity of each spot was measured using a microarray scanner, and the detected fluorescence signal was converted to the concentration of the reaction product using IMAGENE ™ software (manufactured by BioDiscovery), as shown in Fig. 21 (b). Then, PiZPO was calculated and plotted for each pH as enzyme activity (Pi: reaction product concentration at each pH, P0: largest among reaction product concentrations at each pH! /, Reaction product concentration (maximum value)).

As shown in FIG. 21 (b), it was confirmed that all cathepsins exhibited high and high enzyme activity in a low pH range. In particular, the enzymatic activity of cathepsin L decreases sharply with increasing pH, and becomes virtually inactive when pH is greater than 6.5. In addition, in contrast to cathepsin, cathepsin S and cathepsin C maintain significant enzyme activity even at a slightly alkaline pH, which is shown in other literatures (Kirschke, H., Barrett , AJ, awlings, ND Proteinases 1: Lysosomal Cysteine Proteases. Protein Profile, 2, 1587-1643 (1995).). Affiliate Since the epoxide group on one label is inherently more reactive at low pH, it is expected that non-enzymatic side reactions will increase at low pH. The method described in the aforementioned document (Kirschke, H., Barrett, AJ, Rawlings, ND Proteinasesase: Lysosomal Cysteine Proteases. Protein Profile, 2, 1587-1643 (1995).) The fluorescence signal of most cathepsin enzymes at low pH obtained by this example was slightly higher than the fluorescence signal expected from the pH curve curve of It is likely that the proportion of fluorescent signal from non-enzymatic side reactions may increase, and that the prediction is probably correct.

[0181] In the present invention, the fact that the kinetics of each enzyme immobilized on the enzyme chip was similar to the expected kinetics of the enzyme in the solution was confirmed, and in this example, an affinity label was used. The enzymatic activity studies carried out underscore the possibility of applying microarrays under a wide range of operating conditions. Thus, the present invention may be particularly robust under typical operating conditions, such as those found in organisms (organisms).

Industrial applicability

[0182] The present invention is extremely useful in searching for novel pharmaceutical compounds that can regulate enzyme activity, and can contribute to further development of the medical industry, pharmaceutical industry, and the like.

Brief Description of Drawings

[FIG. 1] Diagram showing the general principle of the enzyme activity measuring method of the present invention.

[Figure 2] Figure showing the results of examining the effect of printing pH on enzymes.

[Figure 3] Figure showing the results of examining the effect of the printing concentration of the enzyme.

[Figure 4] Diagram showing the results of examining the effect of substrate surface treatment

[Figure 5] Diagram showing the results of examining the effects of blocking agents

[Figure 6] Diagram showing the results of examining the effects of preincubation and inhibitors

[Figure 7] Diagram showing the results of examining the effect of preincubation time

[Figure 8] Diagram showing the results of examining the effect of affinity label concentration

FIG. 9 is a diagram showing a signal pattern image obtained by the enzyme activity measuring method of the present invention. [10] Diagram showing each step (a)-(c) of the third embodiment of the present invention

FIG. 11 shows an example of a subarray in the enzyme chip of the present invention.

FIG. 12 is an example of the enzyme chip of the present invention, showing a signal (fluorescence) image of each subarray after the treatment in step (a).

[Figure 13] The signal obtained in step (a) is converted into the concentration of the reaction product, the vertical axis represents the reaction product concentration, and the horizontal axis represents the progress curve (time change curve) obtained.

[Figure 14] Diagram showing an example of modeling of an enzyme reaction

FIG. 15 is a diagram showing an enzyme reaction model in the presence of an inhibitor

[16] A diagram schematically showing the results that can be obtained for one type of enzyme when step (c) is performed

FIG. 17 shows a flowchart summarizing a third embodiment of the present invention.

[FIG. 18] (a) shows the results (signal (fluorescence) image) obtained by changing the concentration of inhibitor E-64 in various ways, and (b) shows the results of the six types of cathepsin enzymes (B , C, H, K, L, S) plotting the change in signal intensity when performing step (c) with various concentrations of inhibitor E-64.

[FIG. 19] (a) shows the results (signal (fluorescence) image) obtained by changing the concentration of the inhibitor CNI variously, and (b) shows the results of the six types of cathepsin enzymes (B and C). , H, K, L, S) plotting the change in signal intensity when step (c) was performed with various changes in the inhibitor CM concentration.

[FIG. 20] (a) shows the results (signal (fluorescence) image) obtained by changing the concentration of SDS variously, and (b) shows the results of six types of cathepsin enzymes (B, C, H). , K, L, S) plotting the change in enzyme activity when the SDS concentration was changed variously

[FIG. 21] (a) shows the results (signal (fluorescence) images) of various pH changes, and (b) shows the results of six types of cathepsin enzymes (B, C, H, and K). , L, S) plotting the change in enzyme activity when carried out with various pH changes

Claims

The scope of the claims

[1] An enzyme chip for measuring enzyme activity, in which a plurality of different enzymes are individually arranged at a plurality of different positions on a substrate.

[2] The enzyme chip according to claim 1, wherein the enzyme is a cysteine protease.

[3] an enzyme chip preparation step of preparing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate;

A reaction step of contacting the enzyme chip with an affinity label to which a reporter molecule is attached, and reacting the affinity label with the enzyme; and

A detection step of detecting a reporter signal of the affinity label bound to the enzyme immobilized on the enzyme chip,

A method for measuring enzyme activity, comprising analyzing enzyme activity based on the signal intensity detected in the detection step.

[4] The method according to claim 3, wherein the enzyme is a cysteine protease.

[5] The affinity label to which the reporter molecule is attached is a compound represented by the general formula (I): Epoxide Leu—Xaa—Lys—spacer reporter molecule [where Xaa is Tyr, Lys, or Wherein Arg represents one spacer and Amino-hexanoic acid is a group represented by the following formula:

[6] an enzyme chip production process in which an enzyme chip is produced by arranging a plurality of different constant units of enzyme at a plurality of different positions on a substrate;

Contacting the enzyme chip with an affinity label to which a reporter molecule of a certain concentration is attached, and reacting the affinity label with the enzyme; a reaction step; and the enzyme immobilized on the enzyme chip. Detecting a reporter signal of the affinity label bound to

The kinetic between the enzyme and the affinity label is measured from the time change of the signal intensity of the reporter signal of the immobilized enzyme before contacting the affinity label. Kinetics measurement method.

[7] A method for measuring kinetics between an enzyme and an affinity label according to claim 6. A kinetic constant analysis method that analyzes kinetic constants based on kinetic constants.

[8] an enzyme chip preparation step of preparing an enzyme immobilization chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate;

A test substance contacting step, in which a test substance is brought into contact with the enzyme chip for preincubation.

Contacting the enzyme chip after the test substance contacting step with an affinity label to which a reporter molecule has been attached, and reacting the affinity label with the enzyme; and

Compared with the signal intensity of the enzyme that is not brought into contact with the test substance, the change in the signal intensity due to the test substance for each of the plurality of different enzymes is examined, and the characteristics as a regulator that regulates the enzyme activity are examined. Used for modulator screening

A method for screening a substance that regulates an enzyme activity.

[9] The method for screening a substance regulating an enzyme activity according to claim 8, wherein the enzyme is a cysteine protease.

[10] Affinity label strength with a reporter group General formula (I): Epoxide Leu—Xa a—Lys—a spacer group Reporter molecule [where Xaa is Tyr, Lys, or Arg Wherein the spacer is Amino-hexanoic acid.] The method for screening a substance regulating an enzyme activity according to claim 9, wherein the compound is represented by the following formula:

[11] an enzyme chip preparation step of preparing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate;

An affinity label attached with a reporter molecule is brought into contact with the enzyme chip after the enzyme activity inhibitor contacting step, and the affinity label is allowed to react with the enzyme. Reaction step, and

A method for analyzing the inhibitory constant of an enzyme activity inhibitor, comprising: measuring an inhibitory change of the enzyme activity inhibitor by measuring a change in the addition of an inhibitor of a signal intensity that changes, the signal intensity being derived from the enzyme that is not brought into contact with the test substance.

[12] means for producing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate,

Detecting means for detecting a reporter signal of the affinity label bound to the enzyme immobilized on the enzyme chip, and

Automated enzyme activity measurement system.

13. The enzyme activity measurement automatic filtration system according to claim 12, wherein the enzyme is a cysteine protease.

[14] means for producing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate,

A database for analyzing a force kinetics constant based on the kinetics obtained by the kinetics measuring means; Automatic calculation system for kinetic constants.

[15] means for preparing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate,

Analysis means for analyzing the characteristics of a modulator as a regulator that regulates enzyme activity by observing a change in the signal intensity of the test substance with respect to each of the plurality of different enzymes in comparison with the signal intensity of the enzyme that is not brought into contact with the test substance. Having,

An automatic screening system for preparation of enzyme activity.

[16] The automatic screening system for a preparation of an enzyme activity according to claim 15, wherein the enzyme is a cysteine protease.

[17] means for producing an enzyme chip by arranging a plurality of different enzymes at a plurality of different positions on a substrate,

Inhibition constant of the enzyme activity inhibitor based on the measurement value measured by the measurement means Having a database for analyzing

An automated system for analyzing the inhibition constants of enzyme activity inhibitors.

[18] A reagent for measuring an immobilized enzyme activity, which comprises an affinity label to which a reporter molecule is attached, and is used for measuring the enzyme activity of the immobilized enzyme.

[19] Affinity label force attached to the reporter molecule General formula (I): Epoxide Leu—Xaa—Lys—Spacer group Reporter molecule [where Xaa is Tyr, Lys, or Arg Wherein one of the spacers is Amino-hexanoic acid.] The reagent for measuring the activity of an immobilized enzyme according to claim 18 for measuring the activity of immobilized cysteine protease.

[20] An enzyme chip in which a plurality of different enzymes are arranged and fixed at a plurality of different positions on a substrate is contacted with an affinity label attached with a reporter molecule and a plurality of compounds to form an enzyme activity inhibitor. Screening, and calculating the inhibition constant of the enzyme activity inhibitor, wherein the calculation method comprises the following steps:

1. a step of measuring a force kinetics constant between the plurality of different immobilized enzymes and the affinity label and calculating an inhibition constant based on the kinetic constants;

2. Contacting the affinity label with a plurality of types of the compounds to a plurality of different immobilized enzymes and inhibiting the activity of the reporter signal of the affinity label at a time! Screening the agent and calculating its inhibition constant,

3. Search for the optimal concentration of a compound that cannot obtain a reporter signal within a predetermined range among a plurality of types of the compounds, and determine an inhibition constant from the reporter signal of the affinity label measured at the optimal concentration. The process of calculating,

A method for calculating an inhibition constant that includes at least one of the steps described in 1.

[21] A calculation automation system for making a database of inhibition constants calculated by the method for calculating inhibition constants according to claim 20.

[22] The method for calculating an inhibition constant according to claim 20, wherein the plurality of immobilized enzymes formed on the enzyme chip between the plurality of different immobilized enzymes and the plurality of types of the enzyme activity inhibitors. A method for calculating an inhibition constant calculated by an algorithm capable of simultaneously calculating each concentration of a kind of enzyme-inhibitor complex.

An automatic calculation system for converting the inhibition constant calculated by the method for calculating an inhibition constant according to claim 22 into a database.