JP2009236905A - Screening method of phosphate enzyme inhibitor using light emitting protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately, quickly and inexpensively screening a phosphate enzyme inhibitor. <P>SOLUTION: The screening method of the phosphate enzyme inhibitor uses an anti-phosphate antibody labeled by calcium-bonded type light emitting protein. A calcium-bonded type light emitting protein-labeled anti-phosphate antibody is used in screening of the phosphate enzyme inhibitor. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for screening a phosphorylating enzyme inhibitor using an anti-phosphorylated antibody labeled with a calcium-binding photoprotein, and a calcium-binding photoprotein-labeled anti-phosphorylation for use in screening a phosphorylating enzyme inhibitor Relates to antibodies.

  Calcium-binding photoprotein is a protein that specifically binds to calcium ions and emits light instantaneously by the binding. Currently, aequorin, oberin, clitin, mitrocomin, minneopsin, velvoin and the like are known as such. The sensitivity of these photoproteins to calcium ions is very high and the intensity of the emitted light is so strong that the detection limit of these photoproteins when detected using a commercially available detector is 1 picogram or less. That is, a high detection sensitivity can be realized (Non-Patent Document 1). For this reason, calcium-binding photoproteins are utilized for the detection and quantification of intracellular calcium and trace calcium ions in liquid specimens (for example, blood), taking advantage of the above-described characteristics.

  Usually, chemiluminescence reactions using alkaline phosphatase, horseradish peroxidase, etc. are used as detection methods in immunoassays, but the background during chemiluminescence becomes a problem in detection methods using them, and during the process before detection It is necessary to consider the degradation of the substrate. On the other hand, since the luminescence of the calcium-binding photoprotein is caused by specific binding with calcium ions, there is no background that poses a problem in the chemiluminescence reaction using alkaline phosphatase or horseradish peroxidase that is usually used. In addition, with a calcium-binding photoprotein, light emission occurs instantaneously upon binding of calcium ions by a simple process of adding calcium, and the light emission is completed within a few seconds. For this reason, it has an advantage that detection with a high signal-to-noise ratio (S / N) can be performed in a short time. In addition, since the luminescence depends on the binding with calcium, it is possible to generate luminescence only at the time of detection, and therefore it is not necessary to consider the degradation of the substrate during the treatment before the detection. Furthermore, the luminescence reaction of the calcium-binding photoprotein is an enzyme luminescence reaction called bioluminescence, and all the components necessary for the reaction are derived from living organisms, so harmful chemical substances (for example, radioisotopes, It does not contain carcinogenic compounds) and is therefore highly safe. For this reason, the photoprotein is applied as a label in diagnostic agents and the like.

  In living organisms such as humans, various response reactions are caused at the cellular level due to changes in the external environment. These responses include cell motility, cell death, production of physiologically active substances, etc. In some cases, other cells exhibit further response reactions upon stimulation of the produced physiologically active substances. Such a response reaction is deeply related to intracellular signal transduction through protein phosphorylation. It is widely known that gene transcription, protein translation, molecular transport, protein-protein interaction, and other various phenomena are controlled by information transmission via protein phosphorylation (Non-patent Document 2). In addition, it has been suggested that certain types of phosphorylase can be activated at the time of disease, and that the phosphorylase cannot be controlled due to gene mutation or the like can cause disease (Non-patent Document 3). .

William, W.C. Et al., Proc. Nat. Acad. Sci. USA. July 1975, Vol. 72, no. 7, p. 2530-2534 Yamamoto et al. (Edition), experimental medicine special edition "Signal transduction research 2005-'06", Yodosha, 2005 Kiyoi, H .; Et al., Leukemia. Sep 1998, 12 (9), p. 1333-1337

  Thus, protein phosphorylation plays an important role in cellular activities. As mentioned above, certain types of phosphorylase may be associated with diseases, so we develop therapeutic and preventive drugs for various diseases by screening for inhibitors that specifically inhibit specific phosphorylases. It becomes possible to do. Therefore, there is a need for a method for screening such inhibitors accurately, quickly and at a low cost.

  In order to solve the above problems, the present inventors have developed a means for effectively detecting a phosphorylation reaction and completed a screening method for a phosphorylase inhibitor using such means.

More particularly, the present invention has the following features:
[1] A screening method for a phosphorylase inhibitor, which comprises the following steps:
(A) a step of causing a phosphorylase to act on the substrate peptide under the condition that the substrate peptide can be phosphorylated in the presence of a candidate inhibitor;
(B) contacting an anti-phosphorylated antibody labeled with a calcium-binding photoprotein with a substrate peptide on which a phosphorylating enzyme has been acted in (a),
(C) adding calcium and detecting luminescence resulting from the labeled antibody-substrate peptide complex;
(D) performing the steps (a) to (c) in the absence of the candidate substance, wherein the anti-phosphorylated antibody is specific to a substrate peptide phosphorylated by the phosphorylase A substance capable of binding, and the candidate substance inhibits the activity of the phosphorylase when the luminescence signal obtained from step (c) is lower than the luminescence signal obtained from step (d) The method as described above.
[2] The method according to [1] above, wherein the substrate peptide is immobilized on a solid support.
[3] The method according to [1] or [2] above, wherein the calcium binding protein is aequorin.
[4] The method according to [3] above, wherein the aequorin is a recombinant aequorin.
[5] The method according to any one of [1] to [4] above, wherein the anti-phosphorylated antibody is capable of recognizing a phosphorylated peptide having the sequence shown in SEQ ID NO: 1.
[6] The method according to any one of [1] to [5] above, wherein the substrate peptide comprises the sequence represented by SEQ ID NO: 1.
[7] The phosphorylase is Aurora B, PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1, PAK1, IKKα, JIL-1, VRK1, Snf1, PBK / TOPK, MNK1, MNK2, MRCKα, PAK2 The method according to any one of [1] to [6] above, wherein the substrate peptide is selected from the group consisting of WNK2, and WNK3, and the substrate peptide comprises the sequence shown in SEQ ID NO: 1.
[8] An anti-phosphorylated antibody labeled with a calcium-binding photoprotein for use in screening for a kinase inhibitor.
[9] The antibody according to [8] above, wherein the labeling of the anti-phosphorylated antibody with the calcium-binding photoprotein is performed using a reaction between a maleimide group and a sulfhydryl group.
[10] The antibody according to [8] or [9] above, wherein the calcium binding protein is aequorin.
[11] A kit for screening for a phosphorylase inhibitor comprising the labeled antibody according to any one of [8] to [10] above and a substrate peptide of the target phosphorylase.
[12] The kit according to [11] above, wherein the substrate peptide comprises the sequence represented by SEQ ID NO: 1.

  According to the present invention, a phosphorylase inhibitor that can be applied as a therapeutic / prophylactic agent for diseases can be found.

FIG. 1 shows an electrophoretic analysis of aequorin labeled anti-phosphorylated antibody. FIG. 2 is a plot showing the substrate specific binding of AQ-S-H3S10P. FIG. 3 shows Aurora B activity detected using aequorin-labeled anti-phosphorylated antibody. FIG. 4 is a plot showing inhibition of phosphorylation activity of Aurora B by staurosporine detected using aequorin-labeled anti-phosphorylated antibody.

  In the present invention, the “phosphorylating enzyme” is a protein known to have an activity of phosphorylating a target protein in a cell. The phosphorylase in the present invention may be serine / threonine specific or tyrosine specific. Examples of the phosphorylating enzyme include FLT kinase, Aurora kinase, calmodulin kinase, PKC and the like.

  Suitable phosphorylases are Aurora B, PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1, PAK1, IKKα, JIL-1, VRK1, Snf1, PBK / TOPK, MNK1, MNK2, MRCKα, PAK2, MRCKα, PAK2, WNK2 and WNK3, particularly preferred phosphorylating enzymes are Aurora B, PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1, and PAK1.

  Aurora kinases such as Aurora B are enzymes that play an important role in cell division and are known to be up-regulated in various malignant tumors (Nakayama et al. (Ed.) The forefront of the cell cycle ", Yodosha, 2005, p. 105-109). Aurora B is known to phosphorylate serine in the sequence of histone H3.

  Phosphorylase enhanced activity has been shown to be involved in various diseases such as cancer and Alzheimer's disease, and can be a candidate for a molecular targeted therapeutic drug.

  The phosphorylase used in the method of the present invention may be extracted and purified from a biological tissue, or may be appropriately purified after recombinant expression in a host cell such as E. coli or mammalian cells.

  In the present invention, a “candidate substance” is any substance that is expected to have an activity of inhibiting phosphorylase, and includes, for example, a plurality of compounds such as animal / plant tissue extracts or microbial cultures. Mixtures containing, or preparations isolated therefrom; naturally occurring molecules (eg peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); synthetic analogs of naturally occurring substances Or derivatives (eg, peptide mimetics); non-naturally occurring molecules (eg, low molecular weight organic compounds made using combinatorial chemistry techniques); and mixtures thereof. In the present invention, the “phosphorase inhibitor” is a substance capable of inhibiting the activity of the target phosphorylase.

  In the present invention, the “calcium-binding photoprotein” refers to a protein that generates luminescence by the binding of calcium. The mechanism of luminescence includes the binding of calcium to the protein alters the conformation of the protein, thereby forming a luminophore. The luminophore may be formed by bonding between amino acid side groups of the protein, or may be formed by a chemical group other than the amino acid side group bonded to the protein. Examples of the calcium-binding photoprotein that can be used in the present invention include, but are not limited to, aequorin, oberin, clitin, mitrocomin, minneopsin, and velvoin. A preferred calcium-binding photoprotein is aequorin, and a more preferred calcium-binding photoprotein is recombinant aequorin. Recombinant aequorin can be obtained from Chisso Corporation (Tokyo, Japan), for example.

  Advantages of using a calcium-binding photoprotein as a label in the present invention are that luminescence is generated by a simple treatment of adding calcium, and that the luminescence of the entire molecule in the sample is shorter than the binding of calcium during luminescence and detection. In time, for example, reaching a peak within a few seconds. Luminescence preferably peaks within 1 second of calcium binding. The fact that light emission reaches a peak in a short time after the binding of calcium indicates that there is little variation in the timing of light emission between molecules, that is, almost all molecules emit light simultaneously. This increases the detection sensitivity.

  Other advantages of using a calcium-binding photoprotein as a label are: (1) Since detection is performed by luminescence, there is no leakage of excitation light at the time of detection, which is a problem when using a fluorescent dye, (2) Since the luminescence depends on the binding with calcium, there is no background that causes problems in the case of chemiluminescence using alkaline phosphatase or horseradish peroxidase. (3) The luminescence depends on the binding with calcium, Since it can emit light only at the time, it is not necessary to consider the decomposition of the substrate during the treatment before detection, and (4) not to use dangerous substances such as radioisotopes and carcinogenic dyes.

  In the present invention, the term “anti-phosphorylated antibody” means an antibody that specifically binds to a specific phosphorylated peptide or protein, but does not substantially bind to the same non-phosphorylated peptide. In such an antibody, the binding strength when the target peptide sequence is phosphorylated is 10 times or more, 50 times or more, 100 times or more, 1,000 times that when the peptide is not phosphorylated. Or more than 10,000 times. Anti-phosphorylated antibodies are commercially available for various target sequences. For example, anti-phosphorylated CREB (serine) rabbit antibody, anti-phosphorylated Jak2 (tyrosine) rabbit antibody, and the like. In addition, as an anti-phosphorylated antibody in the present invention, a monoclonal antibody raised using the peptide K9P shown below as an antigen can be used.

The anti-phosphorylated antibody in the present invention may be any of a polyclonal antibody (eg, IgG, IgM, IgA, IgD, or IgE), a monoclonal antibody, or an antibody fragment (eg, Fab, F (ab ′) ₂ , Fv, scFv). It may be. As the anti-phosphorylated antibody, a commercially available one may be purchased, or a new one may be prepared. Methods for producing antibodies of each antibody type are known to those skilled in the art. In particular, methods for producing monoclonal antibodies are known to those skilled in the art, and immunization of mice with an appropriate immunogen, production of hybridomas between spleen cells from immunized mice and syngeneic mouse myeloma cells, antibody production Includes cloning of the hybridoma and purification of the antibody from the cloned hybridoma.

  The labeling of the anti-phosphorylated antibody using the calcium-binding photoprotein in the present invention may be performed by any means as long as stable binding between them can be realized. Examples of such means include a glutaraldehyde one-step method, a glutaraldehyde two-step method, a periodic acid method, a maleimide method, and a succinimide method. A preferred means is a bond between a maleimide group and a sulfhydryl group. When the antibody is labeled using a bond between a maleimide group and a sulfhydryl group, a preferred calcium-binding photoprotein is cysteine-introduced aequorin. In this case, a sulfhydryl group, which is a side chain of cysteine introduced into aequorin, is bound to a maleimide group conjugated to an antibody.

As a method for detecting the phosphorylation state of a protein, a method using ³² P which is a radioisotope is well known, but a special facility is required to carry out a method using a radioisotope, In addition, the reagents must be strictly controlled. In the method of the present invention, such a problem can be avoided by detecting the phosphorylation state of the protein using an anti-phosphorylated antibody.

  In the method of the present invention, “a condition under which the substrate peptide can be phosphorylated” is a condition under which the substrate peptide can be phosphorylated by a target phosphorylase. For example, a target phosphorylase and ATP are added to a buffer containing a reagent usually used in a phosphorylation assay such as glycerophosphate and orthovanadate, and this buffer is allowed to act on a substrate peptide to be room temperature to 37 Incubate at 0 ° C.

  In the method of the present invention, the substrate peptide may be obtained by chemical synthesis using an appropriate peptide synthesizer, or may be obtained by appropriately purifying after recombinant expression in a host cell such as E. coli. Good.

  As used herein, the term “peptide” includes those obtained by chemical synthesis, or naturally occurring and recombinantly expressed proteins and their degradation products.

  The sequence of the substrate peptide is determined depending on the target phosphorylase. Specific phosphorylase target sequences are known to those skilled in the art. A preferred substrate peptide sequence for use in the present invention comprises ARTKQTARKSTGGKAPRKQC (SEQ ID NO: 1). This sequence is known as a partial sequence of histone H3, and the 10th serine of the sequence shown in SEQ ID NO: 1 is phosphorylated by a wide range of phosphorylases. The phosphorylation enzymes Aurora B, PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1 and PAK1 phosphorylate the 10th serine of the sequence is shown in the following examples. IKKα (Yang SR et.al, Am J Respir Cell Mol Biol. 2008 Jun; 38 (6): 689-98), JIL-1 (Ivaldi MS et.al, Genes Dev. 2007 Nov 1; 21 (21): 2818-31), VRK1 (Kang TH et.al, Mol Cell Biol. 2007 Dec; 27 (24): 8533-46), Snf1 (Lo WS et.al, EMBO J. 2005 Mar 9; 24 (5): 997-1008.), And PBK / TOPK (Park JH et.al, Cancer Res. 2006 Sep 15; 66 (18): 9186-95) phosphorylates the 10th serine in the sequence. Indicated in the literature listed in There. In addition, MNK1, MNK2, MRCKα, PAK2, WNK2, and WNK3 are described to be phosphorylated using the 10th serine of the sequence as a substrate in the manual of PerkinElmer's LANCE (registered trademark) Ultra kinase assay. ing.

  The method of the present invention makes it possible to rapidly and easily screen for inhibitors of disease-related phosphorylating enzymes such as Aurora kinase. In particular, in the method of the present invention, by using a calcium-binding photoprotein as a label, light emission occurs instantaneously upon binding of calcium ions by a simple treatment of adding calcium, and the light emission is completed within a few seconds. For this reason, it has an advantage that detection with a high signal-to-noise ratio (S / N) can be performed in a short time. In addition, since the luminescence depends on the binding with calcium, it is possible to generate luminescence only at the time of detection, and therefore it is not necessary to consider the degradation of the substrate during the treatment before the detection. Therefore, since it is possible to detect with high sensitivity in a short time with a simple operation, it is suitable for high-throughput screening of a phosphorylase inhibitor.

  In the method of the present invention, the substrate protein is preferably immobilized on a solid support. A preferred solid support is, but not limited to, a plastic measuring plate.

  In the method of the present invention, luminescence is detected using a detector that can detect the emission wavelength of the calcium-binding photoprotein used. The emission wavelength of the calcium-binding photoprotein is known to those skilled in the art. For example, in the case of aequorin, it is around 470 nm. Such a detector includes a commercially available plate reader. For example, the ARVO series manufactured by PerkinElmer can be used.

  The present invention includes an anti-phosphorylated antibody labeled with a calcium-binding photoprotein for use in screening for a kinase inhibitor.

  In addition, the present invention includes a kit for screening for a phosphorylase inhibitor comprising at least the above-mentioned labeled antibody and a substrate peptide of the target phosphorylase. The kit may further include an immobilization carrier, a buffer, a positive control sample and a negative control sample, instructions describing how to use the kit using the screening method of the present invention, and the like.

Example 1 Preparation of Immunogen Phosphorylated peptide K9P containing a partial sequence of histone H3 phosphorylated by Aurora B (SEQ ID NO: 1: Serine which is the 10th amino acid of peptide K9C represented by ARTKQTARKSTGGKAPRKQC is phosphorylated. Was obtained by synthesizing using an ordinary peptide synthesizer using previously phosphorylated serine.

  This K9P peptide was conjugated to KLH (Keyhole Limpet Hemocyanin) by Image (registered trademark) Maleimide Activated mcKLH Kit (PIERCE) and used as an immunogen. That is, in a sodium phosphate buffer (pH 7.2), 2 mg of maleimide active KLH and 2 mg of K9P peptide were allowed to react at room temperature for 2 hours. After the reaction, it was dialyzed sufficiently against phosphate buffered saline (PBS), adjusted to a concentration of 0.5 mg / mL with PBS, dispensed in 10 μL aliquots, frozen at −20 ° C. and stored.

Example 2 Immunization of Mice 400 μL of the peptide-bound KLH solution prepared in Example 1 and 400 μL of Freund's complete adjuvant were mixed well to prepare a suspension, and this suspension was used as an antigen for 3 mice ( A female, BALB / c) was intraperitoneally administered with 25 μg per mouse. Further, the same amount of antigen was administered 4 times every 2 weeks, and the spleen was removed 3 days after the last administration and used for cell fusion described in Example 3.

Example 3: Cell fusion, selection of fusion cells producing the desired monoclonal antibody (H3S10P antibody), and antibody spleen cells from which the antibodies were obtained and myeloma cells of syngeneic mice (P3X63Ag8.653) Cell fusion was performed using a mixture of 5: 1 and 50% polyethylene glycol 1500 as a fusion accelerator. The cells after the fusion were suspended in a GIT medium containing hypoxanthine, aminopterin and thymidine so that the cell concentration was 1 × 10 ⁶ cells / mL. The obtained cell suspension was dispensed at 100 μL per well into a 96-well microtiter plate (manufactured by Nunk, the same applies hereinafter).

The fused cells were cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.). The hybridoma was screened by exchanging the medium with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium) and growing the fused cells in the HAT medium. The resulting hybridoma of spleen cells and myeloma cells was conditioned with HT medium and further conditioned with GIT medium.

  Antibodies in the hybridoma culture supernatant were detected by ELISA using a microtiter plate on which the K9P peptide was immobilized. For wells that were found to be positive, cloning by limiting dilution was repeated twice to obtain cell clones producing antibodies having reactivity with the K9P peptide. A monoclonal antibody (H3S10P antibody) produced by the obtained cell clone was purified from a culture supernatant obtained by culturing the cell in a CD hybridoma medium using a HiTrap protein G column (GE Healthcare).

Example 4: Preparation of aequorin-labeled anti-phosphorylated antibody The aequorin-labeled anti-phosphorylated antibody was prepared by introducing a maleimide group into the antibody based on a chemical modification method, and then converting the antibody into an aequorin having a sulfhydryl group (cysteine-introduced type). The step of reacting with aequorin) was performed in two steps.

(1) Preparation of a maleimide group-introduced antibody that reacts with a sulfhydryl group A maleimide group-introduced antibody was prepared by introducing a cross-linking reagent having a maleimide group into an anti-phosphorylated monoclonal antibody.

  534 μg of a monoclonal anti-phosphorylated peptide antibody (H3S10P antibody) that specifically binds to the phosphorylated peptide K9P prepared above (serine phosphorylated from the 10th amino acid of K9C represented by SEQ ID NO: 1) (3 nmol) was dissolved in 200 μL of 66 mM phosphate buffer (pH 7.4, hereinafter referred to as “buffer A”) containing 0.1 mM EDTA. To this solution, 30 μL of sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, manufactured by PIERCE) prepared to 1 mM with buffer A was added (sulfo-SMCC 30 nmol, Antibody: Sulfo-SMCC = 1: 10) The mixture was reacted at 4 ° C. for 2 hours. After the reaction, 2 μL (100 nmol) of 50 mM Tris-HCl buffer (pH 7.6) was added, and the reaction was stopped by allowing to stand at 25 ° C. for 10 minutes or more. Centrifugation using Amicon Ultra-4 spin column (Millipore, molecular weight cut off 10,000) at 4 ° C. and 6000 rpm for 15 minutes (Hitachi, CR20B2) to remove unreacted sulfo-SMCC, maleimide An H3S10P antibody into which a group was introduced was obtained (hereinafter referred to as “maleimidated H3S10P antibody”). The recovery rate of the maleimidated H3S10P antibody calculated from E1% 280 nm = 14 was 84.6% (454 μg).

(2) Preparation of aequorin-labeled anti-phosphorylated antibody by reaction of cysteine-introduced aequorin with maleimidated H3S10P antibody Cysteine-introduced aequorin (chisso) dissolved in 66 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA 10 μL (7.5 nmol) manufactured by Co., Ltd. was added to 90 μL (1.5 nmol) of maleimidated H3S10P antibody and allowed to react at 4 ° C. overnight. After the reaction, 2 μL (20 nmol) of 10 mM cysteine aqueous solution was added to stop the reaction. Thus, an aequorin-labeled anti-phosphorylated antibody (hereinafter sometimes referred to as “AQ-S-H3S10P”) was obtained.

  The obtained AQ-S-H3S10P hardly decreased in luminescent activity, and all retained 96% of luminescent activity. The obtained AQ-S-H3S10P was subjected to SDS-PAGE analysis under reducing conditions using a 12% separation gel.

  The results are shown in FIG. A major band is observed at 75 kDa, which is thought to be formed by binding of cysteine-introduced aequorin (21 kDa) and H3S10P antibody-derived H chain (55 kDa), and AQ-S-H3S10P binds with aequorin: H3S10P antibody = 1: 1. It was shown that it was formed.

Example 5: Preparation of substrate peptide and detection of antibody reaction
(1) Preparation of substrate peptide Non-phosphorylated peptide K9C (SEQ ID NO: 1) and phosphorylated peptide K9P phosphorylated on serine, the 10th amino acid of K9C, were prepared in the same manner as in Example 1 above. Both synthetic peptides consist of 20 amino acid residues and have a cysteine residue that reacts with maleimide at the C-terminus.

  These K9C peptide and K9P peptide were combined with maleimide-activated bovine serum albumin (PIERCE) by chemical bonding. The binding was performed as follows. 1 mg (444 nmol) of phosphorylated peptide K9P was placed in a 1.5 mL tube, and 100 μL of 0.1 M phosphate buffer (pH 7.2) was added and dissolved. To 65 μL of this phosphorylated peptide K9P solution, 95 μL (16.1 nmol BSA, 241.7 nmol maleimide group equivalent) of maleimide activated bovine serum albumin (hereinafter referred to as maleimide-BSA, manufactured by PIERCE) was added and reacted at room temperature for 2 hours. Thus, phosphorylated peptide K9P-bound BSA (hereinafter referred to as K9P-BSA) was obtained. On the other hand, 1 mg of non-phosphorylated peptide K9C was put into another 1.5 mL tube, and 100 μL of 0.1 M phosphate buffer (pH 7.2) was added and dissolved (454 nmol / 100 μL). Thereafter, it was reacted with maleimide-BSA in the same manner as described above to obtain non-phosphorylated peptide K9C-bound BSA (hereinafter referred to as K9C-BSA).

(2) Immobilization of BSA-binding peptide substrate K9P-BSA and K9C-BSA stock solutions (each 1 mg / mL) were diluted to a concentration of 5 μg / mL with 50 mM carbonate buffer (pH 9.6). These solutions were dispensed into Maxisorp 96-well white plates (Nunc, # 437796) at 100 μL per well, shaken for 1 minute, and then incubated at 30 ° C. for 18 hours. The peptide solution was removed, and then 200 μL of post-coating solution (150 mM NaCl containing 1% BSA, 2 mM EDTA, 0.05% NaN ₃ , Tris-HCl buffer (hereinafter referred to as TBS)) was dispensed, and shaken for 1 minute After that, it was stored at 4 ° C. for 18 hours or more. In addition, the plate which gave only the post coating as a negative control was also produced.

(3) Binding of K9P-BSA, K9C-BSA, and BSA to AQ-S-H3S10P Aequorin-labeled anti-phosphorylated histone H3 antibody (AQ-S-H3S10P) was confirmed for reactivity with K9P-BSA. It was.

  The method was as follows. A Maxisorp 96-well white plate on which a K9P-BSA or K9C-BSA solution was immobilized was used. After removing the post-coating solution from the plate and thoroughly washed with TBS containing 2 mM EDTA and 0.05% Tween 20 (hereinafter referred to as TBST-E), 10% Block Ace (Dainippon Pharmaceutical) and 5 mM EDTA are contained. AQ-S-H3S10P diluted 1250 to 5000 times (2.1 to 0.53 μg / mL) with TBS (hereinafter referred to as a diluted solution) was dispensed at 100 μL / well. Then, it left still at 30 degreeC for 1 hour.

(4) After leaving the measurement of luminescence by aequorin , the reaction solution was discarded and washed with TBST-E, and then 50 mM CaCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.) was used with a luminescence measuring device ARVO (manufactured by PerkinElmer) A 50 mM Tris-HCl buffer solution (pH 7.6) (hereinafter referred to as calcium solution) containing 100 μL / well was injected, and the luminescence intensity was measured at 0.1 second intervals for 5 seconds.

  The maximum light emission intensity value (Imax) was calculated and shown in Table 1 and FIG. Data represent the average of three measurements. For the non-phosphorylated peptide K9C, no aequorin luminescence was detected at any antibody concentration, but for the phosphorylated peptide K9P, the aequorin luminescence intensity increased in proportion to the antibody concentration. You can see that.

  From the above results, it was shown that AQ-S-H3S10P reacts specifically and concentration-dependently with K9P-BSA.

Example 6: Examination of Aurora B Phosphorylase Activity Measurement The dose-dependent phosphorylation activity of the phosphorylation enzyme Aurora B (Upstate) against K9C-BSA solid-phased by the above-mentioned method was determined using an aequorin-labeled anti-phosphorylated antibody. AQ-S-H3S10P was used (Aurora B addition amount: 0 to 1000 ng / well). As the enzyme reaction solution, 25 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl ₂ , 5 mM β-glycerophosphate, 0.1 mM sodium orthovanadate (V), 2 mM dithiothreitol, 0.1% bovine serum albumin. 5) was used.

  The blocked peptide-immobilized 96-well plate was washed 3 times with TBST-E and then twice with pure water. Next, an enzyme reaction was performed. 100 μL of the enzyme reaction solution was dispensed into each well, and 25 μL of Aurora B diluted to a final concentration of 0, 1, 3, 10, 30, 100, 300, or 1000 ng / well was added and stirred. Next, 25 μL of 60 μM ATP prepared using the enzyme reaction solution was added. After stirring, phosphorylation reaction was performed at 30 ° C. for 1 hour. The reaction solution was removed, washed three times with TBST-E, and 150 μL of AQ-S-H3S10P diluted 1250 times (2.1 μg / mL) was added to each well. Then, it was made to react at 30 degreeC for 1 hour. After removing the reaction solution and washing 3 times with TBST-E, 100 μL of calcium solution was added to detect luminescence.

  The results are shown in Table 2 and FIG. Data represent the average of three measurements. As the concentration of Aurora B increases, it can be seen that the luminescence activity of aequorin increases. From this result, it is clear that the increase in the dose-dependent luminescence value of the Aurora B enzyme reflects the phosphorylation activity of Aurora B on the immobilized substrate peptide.

  From the above, it was shown that a quantitative phosphorylation activity measurement system was established in luminescence detection using an aequorin-labeled anti-phosphorylated antibody (AQ-S-H3S10P).

Example 7: Examination of the screening method of the present invention using an Aurora B kinase phosphorylation activity inhibitor Based on the phosphorylase activity detection method using the aequorin-labeled antibody described above, staurosporine, a protein kinase inhibitor The inhibition of Aurora B kinase phosphorylation activity by (Calbiochem) was examined.

  According to the above-mentioned method, the post-coated K9C solid phase plate was washed 3 times with TBST-E and twice with pure water, and the final concentration of the inhibitor staurosporine was 0, 1, 3, 10, Prepare a dilution series to be 30, 100, 300, 1000, or 3000 nM, and add 50 μL of enzyme reaction solution, 50 μL of staurosporine solution, and 25 μL of Aurora B kinase solution prepared to a final concentration of 100 ng / well to each well. And stirred. Finally, 25 μL of 60 μM ATP was added. After stirring, phosphorylation was performed at 30 ° C. for 1 hour. After the reaction, it was washed 3 times with TBST-E, 150 μL of 1250-fold diluted (2.1 μg / mL) AQ-S-H3S10P antibody was added, reacted at 30 ° C. for 1 hour, and again washed 3 times with TBST-E. Went. Subsequently, 100 μL of calcium solution was added to detect luminescence.

  The results are shown in Table 3 and FIG. Data represent the average of three measurements. When detected using an aequorin-labeled anti-phosphorylated antibody, it can be seen that the luminescence of aequorin decreases as the concentration of staurosporine increases.

  From the above results, a concentration-dependent decrease in the aequorin luminescence value in the protein kinase inhibitor staurosporine was observed, and quantitative detection of phosphorylase activity inhibition of Aurora B kinase was confirmed. That is, it was shown that this system is useful as a screening for a phosphorylase inhibitor.

Example 8: Use of the screening method of the present invention for various phosphorylating enzymes The detection method of phosphorylating enzyme activity using the above-mentioned aequorin-labeled anti-phosphorylated antibody and the screening method of a phosphorylating enzyme inhibitor using the same are described in Aurora. In order to demonstrate that it is applicable not only to B but also to other phosphorylating enzymes, the method of the present invention was used with respect to various phosphorylating enzymes predicted to use the peptide K9C used above as a substrate. .

  As protein kinases, PKA (PKAα; GenBank accession number NM_002730) and DYRK1A (GenBank accession number NM_001396.3; described in Japanese Patent Application No. 2008-190277, which is incorporated herein by reference in its entirety) In-house purified products, MSK1, PKCε, CRIK, NDR1, Rsk2, PIM1, and PAK1 were commercially available from Carna Biosciences (Hyogo). Moreover, staurosporine (Calbiochem) or Harmine (SIGMA) was used as an inhibitor for these phosphorylating enzymes.

  According to the above-mentioned method, the post-coated K9C solid phase plate was washed 3 times with TBST-E and twice with pure water. 0 μM or 30 μM inhibitor solution (in enzyme reaction solution) was prepared. In addition, in the phosphorylase solution, the final concentration of each phosphorylase is PKA: 800 ng / well, MSK1: 100 ng / well, PKCε: 300 ng / well, CRIK: 300 ng / well, NDR1: 300 ng / well, DYRK1A: The enzyme reaction solution was used to prepare 500 ng / well, Rsk2: 100 ng / well, PIM 1: 6 μg / well, and PAK1: 300 ng / well. To each well of the plate, 50 μL of the enzyme reaction solution, 50 μL of the inhibitor solution, and 25 μL of the phosphorylating enzyme solution were added and stirred, and finally 25 μL of 60 μM ATP was added. After stirring, the mixture was reacted at 30 ° C. for 1 hour. After phosphorylation, the plate was washed 3 times with TBST-E, 150 μL of 1250-fold diluted (2.1 μg / mL) AQ-S-H3S10P antibody was added, reacted at 30 ° C. for 1 hour, and again with TBST-E. Washed 3 times. Subsequently, 100 μL of calcium solution was added, and luminescence was detected.

  Table 4 shows the results using protein phosphorylating enzymes PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1, or PAK1. When phosphorylation is carried out in the absence of an inhibitor, the luminescence intensity increases compared to the case where no phosphorylase is added, and it is known that the phosphorylation has an inhibitory effect on each phosphorylase. It can be seen that the emission intensity is reduced in the presence of taurosporine or harmine.

  From the above results, it was shown that the method of the present invention is useful not only for Aurora B kinase but also as a screening method for inhibitors against a wide range of phosphorylases.

  INDUSTRIAL APPLICABILITY The present invention is useful for the development of a preventive / therapeutic agent for a disease involving a phosphorylase by making it possible to identify a phosphorylase inhibitor.

    SEQ ID NO: 1 substrate peptide

Claims (12)

A method for screening a phosphorylase inhibitor, comprising the following steps:
(A) a step of causing a phosphorylase to act on the substrate peptide under the condition that the substrate peptide can be phosphorylated in the presence of a candidate inhibitor;
(B) contacting an anti-phosphorylated antibody labeled with a calcium-binding photoprotein with a substrate peptide on which a phosphorylating enzyme has been acted in (a),
(C) adding calcium and detecting luminescence resulting from the labeled antibody-substrate peptide complex;
(D) performing the steps (a) to (c) in the absence of the candidate substance, wherein the anti-phosphorylated antibody is specific to a substrate peptide phosphorylated by the phosphorylase A substance capable of binding, and the candidate substance inhibits the activity of the phosphorylase when the luminescence signal obtained from step (c) is lower than the luminescence signal obtained from step (d) The method as described above.   The method according to claim 1, wherein the substrate peptide is immobilized on a solid support.   The method according to claim 1 or 2, wherein the calcium binding protein is aequorin.   4. The method of claim 3, wherein the aequorin is a recombinant aequorin.   The method according to any one of claims 1 to 4, wherein the anti-phosphorylated antibody is capable of recognizing a phosphorylated peptide having the sequence represented by SEQ ID NO: 1.   6. The method according to any one of claims 1 to 5, wherein the substrate peptide comprises the sequence shown in SEQ ID NO: 1.   The phosphorylating enzyme is Aurora B, PKA, MSK1, PKCε, CRIK, NDR1, DYRK1A, Rsk2, PIM1, PAK1, IKKα, JIL-1, VRK1, Snf1, PBK / TOPK, MNK1, MNK2, MRK2, WNK2, PNK2, WNK And the method of any one of claims 1 to 6, wherein the substrate peptide comprises the sequence shown in SEQ ID NO: 1.   An anti-phosphorylated antibody labeled with a calcium-binding photoprotein for use in screening for phosphorylase inhibitors.   The antibody according to claim 8, wherein the labeling of the anti-phosphorylated antibody with the calcium-binding photoprotein is performed using a reaction between a maleimide group and a sulfhydryl group.   The antibody according to claim 8 or 9, wherein the calcium binding protein is aequorin.   A kit for screening for a phosphorylase inhibitor comprising the labeled antibody according to any one of claims 8 to 10 and a substrate peptide of a target phosphorylase.   The kit according to claim 11, wherein the substrate peptide comprises the sequence shown in SEQ ID NO: 1.

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