JP2007068509A - Method for detecting phosphorylation inhibition activity by using radioactive material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluating system for protein kinase inhibiting activity in On-chip examination in a simple method which corresponds to a high through put for drug design screening. <P>SOLUTION: The invention relates to the method for detecting phosphorylation inhibition activity comprising detection of the phosphorylation by a radio isotope (RI) on a peptide or a protein by acting a solution containing a protein kinase or a solution considered to contain the substance on a chip composed of the peptide or the protein immobilized on the chip, wherein the detection is carried out in the coexistence of a compound having protein kinase inhibiting activity or a compound considered to have the activity in the solution. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting phosphorylation inhibitory activity using a radioactive substance (hereinafter also referred to as RI). More specifically, when detecting phosphorylation of the peptide or protein by RI on a chip on which the peptide or protein is immobilized, by acting a solution containing or thought to contain protein kinase, the protein The present invention relates to a method for detecting phosphorylation inhibitory activity, which comprises allowing a compound that inhibits or is thought to inhibit kinase activity to coexist in the solution.

  In recent years, biochips used for biomolecule interaction analysis, profiling of expressed molecules, or diagnosis have attracted attention. The operation is facilitated by immobilizing the biomolecule on the substrate, and in some cases, the interaction of a very large number of substances can be analyzed. In particular, peptide arrays in which peptides with relatively small molecular weights are immobilized on a substrate have relatively few problems of denaturation such as proteins, and have strong combinatorial chemistry aspects. It has come to be widely used for searching and the like. In particular, in the post-genome, post-translational modification of proteins, especially analysis of phosphorylation, is important for elucidating in detail the regulation of protein activity and function.

  As a related technique that has already been reported, for example, a technique in which a peptide is directly synthesized on a cellulose membrane by the SPOT technique and then immobilized on a chip is known. It has been reported that a p60 tyrosine kinase substrate was immobilized on a chip using this technique, and kinase activity was evaluated using a fluorescent substance or a radioactive substance (see, for example, Non-Patent Document 1). In addition, the activity of protein kinase A (hereinafter sometimes referred to as PKA) and NIMA-related kinase 6 (NEK6) was assayed using a radioactive substance using a peptide array prepared by the same technique. There is also a report about this (for example, see Non-Patent Documents 2 and 3). In addition, there is an example in which a biotin-bonded peptide is immobilized on a substrate coated with avidin and PKA activity is examined (for example, see Non-Patent Document 4). However, in any of the above methods, the background at the time of detection has an adverse effect on observing the spot, and its reduction is a major issue. In the case of a method using an antibody, there are problems such as insufficient cost and required characteristics, and it is difficult to say that the method is always satisfactory.

Curr. Opin. Biotechnol. 13,315,2002 Angew. Chem. Int. Ed. 43,2671,2004 Nature Methods 1,27,2004 J. et al. Biol. Chem. 277, 27839, 2002

  An object of the present invention is to provide an evaluation system for protein kinase inhibition test with an on-chip, which is capable of high throughput for drug discovery screening by a simple method.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention. That is, the present invention has the following configuration.
(1) A method using a radioactive substance to phosphorylate a peptide or protein by allowing a solution containing or considered to contain a protein kinase to act on a chip having a gold surface on which the peptide or protein is immobilized A method for detecting phosphorylation inhibitory activity, wherein a compound that inhibits or is thought to inhibit the protein kinase activity is allowed to coexist in the solution.
(2) method for detecting a phosphorylation inhibitory activity, wherein the species of radioactive material is ^{32 P} or ^{33 P} (1).
(3) The method for detecting phosphorylation inhibitory activity according to (1) or (2), wherein the method using a radioactive substance is autoradiography.
(4) The method for detecting phosphorylation inhibitory activity according to any one of (1) to (3), wherein the peptide or protein is immobilized on a chip via a thiol group.
(5) The method for detecting phosphorylation inhibitory activity according to any one of (1) to (4), wherein a peptide having 30 or less amino acids is immobilized on a chip.
(6) The method for detecting phosphorylation inhibitory activity according to any one of (1) to (5), wherein the compound that inhibits or is thought to inhibit the protein kinase activity is a peptide or a protein.
(7) The method for detecting phosphorylation inhibitory activity according to any one of (1) to (6), wherein the compound that inhibits or is thought to inhibit the protein kinase activity is a low molecular weight compound having a molecular weight of 2000 or less.

  By the array analysis using RI in the present invention, it is possible to obtain a measurement system for protein kinase inhibition test with On-chip. It is also useful to be able to cope with high throughput by performing analysis with an array. In particular, the pharmaceutical industry is expected to be very useful as an evaluation system for novel drug discovery screening such as kinase inhibitors.

  The present invention is characterized by conducting an on-chip protein kinase inhibition test by analysis using an array having a gold surface using RI. By using RI, a phosphorylation reaction can be directly monitored, and a detection system excellent in both sensitivity and specificity can be obtained. Furthermore, the use of a gold surface has the advantage that surface modification is easier and non-specific influences can be reduced more easily than using a general glass surface.

  The material of the substrate surface used in the present invention is preferably gold that is very stable in acid, alkali, organic solvent, and the like. In fact, gold is a material frequently used in the above optical detection method. Further, the material supporting gold is preferably transparent, and more preferably transparent glass. Alternatively, plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic are also included. Transparent glass is easily available and is advantageous in terms of versatility. When a glass substrate is used, the type of glass and the thickness of the substrate are not particularly limited, but the thickness is preferably about 0.1 to 20 mm, for example, a substrate of about 1 to 2 mm is used. Although it does not specifically limit regarding a magnitude | size or a shape, You may use things like the slide glass marketed normally.

  As a method of forming the metal substrate, a method of coating a thin gold layer is preferable. The method for coating gold is not particularly limited, but generally a vapor deposition method, a sputtering method, an ion coating method or the like is selected. In order to provide an optical detection method, it is necessary to control the thickness of the thin metal layer at the nano level. The thickness of the thin metal layer is not particularly limited, but is generally selected in the range of 30 nm to 80 nm. In order to suppress peeling of the thin metal layer, a chromium layer or a titanium layer of 0.5 nm to 10 nm may be coated on the substrate in advance.

  In the present invention, an array in which peptides or proteins are immobilized on a substrate as described above is used. As the peptide or protein, at least one containing an amino acid sequence capable of functioning as a protein kinase substrate, preferably a plurality of amino acid sequences that are phosphorylated by different types of protein kinases are used. Furthermore, it is preferable that a phosphorylated amino acid residue (positive control) or a negative control is also immobilized on the same substrate. When a negative control is used, it is preferable that the phosphorylation site is a serine residue, a threonine residue is substituted with an alanine residue, and a phosphorylation site is substituted with a phenylalanine residue when the phosphorylation site is a tyrosine residue. In terms of ease of synthesis, ease of handling, storage stability, etc., it is preferable to use a peptide having a relatively low molecular weight. Here, the peptide refers to a commonly used meaning, in which two or more amino acids are linked by peptide bonds. The number of amino acid residues is not particularly limited, but is usually about 5 to 60 residues, preferably 30 residues or less, and more preferably about 10 to 25 residues.

  The method for immobilizing a peptide in the peptide array of the present invention is not particularly limited, and examples thereof include a method via an amino group or thiol group in the peptide sequence, a method using a His tag, and the like. Among these, the method of immobilizing a peptide via a thiol group is particularly preferable from the viewpoints of specificity and sensitivity.

  It is preferable that at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used in order to increase the efficiency of interaction with a substance that acts by increasing the degree of freedom of the immobilized peptide. As an amino acid sequence of 1 to several residues may be further added. The amino acid sequence of the spacer portion is not particularly limited, but it is particularly preferable to add a sequence having 1 to several glycine residues and / or alanine residues or serine residues.

  Moreover, you may use the state in which the compound which has a thiol group is chemically combined in the amino acid residue in one or more places with respect to the peptide fix | immobilized. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

  In the present invention, when a peptide is immobilized via a thiol group, a heterobifunctional cross-linking agent having a succinimide (NHS) group or a sulfated succinimide group and a maleimide (MAL) group is introduced after introducing an amino group to the surface in advance. It is particularly preferable to use it. As such a heterobifunctional cross-linking agent, it is possible to use a hydrophilic polymer such as PEG in which both ends are modified with NHS groups and MAL groups, but the immobilization yield of the peptide is improved. In order to achieve this, a lower molecular weight may be used. Specifically, the compound Succinimidyl 4- [N-maleimidomethyl] 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC) or the compound Sulfosuccinimidyl 4- [N-maleimidocylimide] represented by the formula (II) -1-carboxylate (hereinafter referred to as SSMCC). It should be noted that not only compounds having the same structure as the compound represented by formula (I) or formula (II) but also compounds analogized within a range not impairing the function thereof are included. In the present invention, both SMCC and SSMCC can be applied. However, from the viewpoint of solubility in water, it is more preferable to use SSMCC when the reaction is carried out in an aqueous system such as a buffer solution.

  By applying low molecular weight compounds as described above, the efficiency of immobilizing peptides on the chip is significantly higher than when high molecular weight substances are used as cross-linking agents. Thus, there is an effect that the signal due to the binding becomes clearer. Further, there is almost no problem with non-specific influences, which is advantageous in that the so-called S / N ratio can be increased. However, the type of the crosslinking agent in the present invention is not particularly limited to these.

  In order to introduce the SMCC or SSMCC onto a chip to form a maleimide surface, a succinimide group at the other end of the SMCC or a functional group reactive with the succinimide sulfate group at the other end of the SSMCC, Specifically, it is necessary to introduce an amino group on the chip in advance. The means for introducing an amino group on the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing an amino group using a cross-linking agent starting from the functional group introduced on the surface.

  The peptide array of the present invention is characterized in that the non-immobilized portion (background portion) of the peptide is coated with a compound as shown in formula (III). In formula (III), m represents an integer of 1 to 20, and n represents an integer of 1 to 10. The value of m is more preferably in the range of 2 to 10, and still more preferably in the range of 4 to 8. As for the value of n, the range of 2-8 is more preferable, and the range of 3-6 is still more preferable. By coating the background portion with this compound, non-specific adsorption in the background portion can be very effectively suppressed. In addition, the peptide array of the present invention improves the reproducibility of data depending on various fluctuation factors such as the substrate production lot and processing conditions, and can obtain very stable measurement data.

  In an ELISA method or an interaction analysis method using a label substance, physical adsorption by bovine serum albumin or casein is generally selected as a blocking method. Although the physical adsorption method is easy, it is not stable and may be detached from the chip surface over time. In the above optical detection method, it is preferable to perform blocking by covalent bond in order to detect even the elimination of the blocking agent. In particular, when blocking the surface of the unreacted maleimide group, a compound having a thiol group is preferably used, and a PEG (polyethylene glycol) derivative is particularly preferably used.

The object of the present invention is to detect phosphorylation inhibitory activity using RI. As the nuclide of RI, ³² P or ³³ P can be mentioned, and ³³ P is more preferable from the viewpoint of the stability of the reagent itself with slightly longer sensitivity and half-life. When a solution containing or considered to contain a protein kinase is allowed to act on the array obtained as described above, a compound that inhibits or is thought to inhibit the protein kinase activity is allowed to coexist in the solution. become. The protein kinase to be subjected to profiling may be a commercially available reagent, but it is also possible to use a protein kinase already contained in or estimated to be contained in a cell-derived extract.

For example, by immobilizing a protein kinase reagent or nucleoside triphosphate (ATP), which is already contained in a buffer or cell extract or a mixture of both, and acting on the array directly, the immobilized substrate Can be phosphorylated. In this case, ATP labeled with RI is used. For example. Examples thereof include γ ³² P-ATP and γ ³³ P-ATP. From the viewpoint of the stability of the reagent, γ ³³ P-ATP is more preferable. The phosphorylation conditions vary depending on the type of protein kinase, but it is usually about 10 to 40 ° C., preferably about 20 to 40 ° C. for about 5 minutes to 8 hours, preferably about 10 minutes to 5 hours. Thus, the peptide or protein can be phosphorylated. If necessary, a substance that assists or promotes phosphorylation such as cAMP, cGMP, Mg ²⁺ , and Ca ²⁺ may coexist in the reaction solution.

  Autoradiography is effective for detecting phosphorylation on the array. After sufficiently washing the array that has undergone phosphorylation, it can be dried and exposed to a dedicated film or sheet and analyzed by a dedicated image reader. This method is a very useful technique in that phosphate uptake can be directly observed for a detection system using an antibody or the like. It is also the most excellent method from the viewpoint of sensitivity and specificity.

  The coexisting inhibitor is not particularly limited, but may be a peptide or protein, or other low molecular weight compound. Low molecular compounds include those having a molecular weight of 2000 or less, but are not particularly limited.

  As described above, exhaustive analysis by profiling of protein kinase activity can be performed by carrying out phosphorylation reaction with On-chip according to the present invention. In particular, by monitoring the inhibitory activity, it is possible to provide a technique useful for drug discovery screening.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to the examples.

[Example 1]
(Production of array)
As shown in the formula (IV), a linear thiol PEG reagent (SPSPT-0011 manufactured by SensoPath) having a terminal functional group as a thiol group was dissolved in 7 ml of ethanol at a concentration of 1 mM. The molecular weight of the linear thiol PEG is 336.54. In particular, it shows metal bondability to gold.

  A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the linear PEG thiol solution for 3 hours to bond PEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove PEG thiol in the UV irradiation part. The photomask has 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. The portion of the photomask with a hole is transparent to UV light and is irradiated onto the slide for patterning. PEG remains in the unirradiated portion, and functions as a reference portion as a background portion of the chip.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. SSMCC (Pierce) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 0.4 mg / ml and reacted with 8-AOT on the gold surface for 15 minutes. Since the amino group of 8-AOT and the NHS group of SSMCC reacted and the MAL group remained unreacted, the maleimide group could be introduced to the surface via PEG.

  On the surface obtained as described above, as shown in FIG. 1, a peptide consisting of an amino acid sequence serving as a substrate for PKA (protein kinase A) (SEQ ID NO: 1), a PKA substrate negative control (SEQ ID NO: 2; A serine residue is substituted with an alanine residue), a positive control of a PKA substrate (the serine residue of SEQ ID NO: 1 is phosphorylated), a peptide consisting of an amino acid sequence serving as a substrate of cSrc kinase (SEQ ID NO: 3), a cSrc kinase substrate The positive control (the tyrosine residue of SEQ ID NO: 3 was phosphorylated; SEQ ID NO: 5) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / ml, and MultiSPRinter (registered) A spotter (made by Toyobo Co., Ltd.) was used for spotting every 10 nl. Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the chip reacts with the thiol group possessed by the cysteine residue at the terminal of the substrate peptide, so that the substrate peptide can be covalently immobilized on the surface.

(Blocking of unreacted maleimide groups)
After washing the substrate peptide-immobilized surface with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (the functional group at one end is a thiol group and the other functional group is a methoxy group) NOF SUNBRIGHT MESH-50H) is dissolved in a phosphate buffer solution (20 mM phosphate, 150 mM NaCl; pH 7.2) to a concentration of 1 mM, 250 μl is poured onto the chip, and allowed to react at room temperature for 1 hour. It was. The molecular weight of PEG thiol used here is 5,000.

[Example 2]
(Detection of inhibitory activity of PKA phosphorylation by autoradiography)
Phosphorylation with PKA was performed on the array that had been blocked as described above. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. for 1 hour. The composition of the PKA solution was as follows: PKA catalyst subunit (Promega) 1 μl, 50 mM Tris-HCl buffer (pH 7.4) 378 μl, 1M magnesium chloride solution 20 μl, γ ³³ P-ATP (50 μCi / ml; manufactured by Amersham Bioscience) 1 μl. At that time, PKI 6-22 Amide, PKA Inhibitor (manufactured by Calbiochem) was coexisted as a PKA inhibitor peptide, and the phosphorylation inhibitory effect was examined. The coexistence concentration was examined at 10 ⁻⁸ M, 10 ⁻⁶ M, and 10 ⁻⁴ M. The amino acid sequence of the inhibitor is as shown in SEQ ID NO: 4.

  Thereafter, the array was washed three times with PBS and water, and the surface of the array was dried. Then, reading of RI uptake in each immobilized substrate by autoradiography was performed using BAS-1800II (manufactured by Fuji Photo Film). went. The exposure was carried out for 30 minutes using a sheet dedicated to the apparatus (SG imaging plate manufactured by Fuji Photo Film). The results are shown in FIG.

  As indicated by the arrows in FIG. 2, it has been confirmed that the coexistence concentration of PKI increases and only the RI uptake in the substrate peptide of PKA (No. 1 in FIG. 1) is weakened. FIG. 3 shows the result of quantification. Quantification was performed using the dedicated analysis software of the above apparatus. No. with no PKI added. The ratio of RI uptake in the substrate peptide when PKI coexists is graphed with the RI uptake of 1 substrate peptide taken as 100%, and phosphorylation is inhibited depending on the PKI coexistence concentration. It is recognized that

[Example 3]
An array similar to that in Example 1 was prepared, and detection of the inhibitory activity of PKA phosphorylation was examined in the same manner as in Example 2 except that the PKA inhibitor was changed to PKA Heat Stable Inhibitor, Isoform α (Calbiochem). did. This inhibitor is a protein, and the amino acid sequence is as shown in SEQ ID NO: 5. Coexistence concentration ^is 10 ^-4, 10 ^{-3 and} 10 -2 Unit / [mu] l. As in the case of Example 2, No. in the case where no inhibitor was added. FIG. 4 shows the results of graphing the ratio of RI uptake in the same substrate peptide when PKA Heat Stable Inhibitor, Isoform α coexists, with the RI uptake of 1 substrate peptide taken as 100%. Also in this case, it is recognized that phosphorylation is inhibited depending on the coexisting concentration of the inhibitor.

[Example 4]
Except that an array similar to that in Example 1 was prepared and the PKA inhibitor was changed to H-89 (N- [2- (p-Bromocinnamylamino) ethyl] -5-isoquinolinesulfonamide, Di-HCl Salt; manufactured by Biomol). In the same manner as in Example 2, detection of the inhibitory activity of PKA phosphorylation was examined. The structure of this inhibitor is as shown in Formula (V). The coexistence concentration was examined at 10 ⁻⁸ M, 10 ⁻⁶ M, and 10 ⁻⁴ M.

  As in the case of Example 2, No. in the case where no inhibitor was added. FIG. 5 shows the results of graphing the ratio of RI uptake in the same substrate peptide when H-89 coexists when the uptake of RI in one substrate peptide is 100%. Also in this case, it is recognized that phosphorylation is inhibited depending on the coexisting concentration of the inhibitor.

  By utilizing the present invention, it is possible to comprehensively analyze many types of protein kinase signals, and to effectively profile protein kinase dynamics in cells accompanying the introduction of genes with unknown functions or drug administration. it can. This is expected to expand into genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.

FIG. 3 is a diagram showing the arrangement of each immobilized peptide in the peptide array prepared in Example 1. In Example 2, it is a figure which shows the result of having observed the mode that On-chip phosphorylation by PKA was inhibited by coexistence of PKI by autoradiography. In Example 2, it is the figure which showed a mode that On-chip phosphorylation by PKA was inhibited by coexistence of PKI by the change in the amount of RI uptake by autoradiography. In Example 3, it is the figure which showed a mode that On-chip phosphorylation by PKA was inhibited by coexistence of PKA Heat Stable Inhibitor, Isoform alpha by the change in the amount of RI uptake by autoradiography. In Example 4, it is the figure which showed a mode that On-chip phosphorylation by PKA was inhibited by coexistence with H-89 by the change in the amount of RI uptake by autoradiography.

Claims (7)

  On a chip having a gold surface on which a peptide or protein is immobilized, phosphorylation of the peptide or protein is detected by a method using a radioactive substance by allowing a solution containing or considered to contain a protein kinase to act. In this case, a method for detecting phosphorylation inhibitory activity, wherein a compound that inhibits or is thought to inhibit the protein kinase activity is allowed to coexist in the solution. 2. The method for detecting phosphorylation inhibitory activity according to claim 1, wherein the nuclide of the radioactive substance is ³² P or ³³ P.   The method for detecting phosphorylation inhibitory activity according to claim 1 or 2, wherein the method using a radioactive substance is autoradiography.   The method for detecting phosphorylation inhibitory activity according to any one of claims 1 to 3, wherein the peptide or protein is immobilized on a chip via a thiol group.   The method for detecting phosphorylation inhibitory activity according to any one of claims 1 to 4, wherein a peptide having 30 or less amino acids is immobilized on a chip.   The method for detecting phosphorylation inhibitory activity according to any one of claims 1 to 5, wherein the compound that inhibits or is thought to inhibit the protein kinase activity is a peptide or a protein.   The method for detecting phosphorylation inhibitory activity according to any one of claims 1 to 6, wherein the compound that inhibits or is thought to inhibit the protein kinase activity is a low molecular weight compound having a molecular weight of 2000 or less.