JP4679870B2 - Kinase activity detection method - Google Patents

Description

  The present invention relates to a method for detecting kinase activity.

Protein phosphorylation and dephosphorylation are one of the most important in signal transduction in vivo, and are involved in various life activities from cell differentiation to proliferation to cell death. For example, it is well known that kinases such as Src and Raf are involved in cell tumorigenesis. In this way, it has been clarified that many kinases play an important role, but even an already identified kinase recognizes which protein is a substrate and what amino acid sequence is recognized and phosphorylated. There are many things that lack basic knowledge. The generality and importance of post-translational modification called “phosphorylation” is enormous, as evidenced by the results of the Human Genome Project that 2-3 percent of all genes encode kinases.
Many methods for detecting kinase activity have been reported, but can be classified into two types in principle. A method using ATP ([γ- ³² P] ATP) labeled with a radioisotope (RI) and a method using an antibody that recognizes phosphorylated amino acids. In the former, the kinase activity is quantified by measuring the radioactivity of ³² P incorporated into the substrate after phosphorylation of the substrate protein / peptide by the kinase. Although this method is widely used, there is a problem in that it uses a RI and requires a corresponding facility. The latter is a method for recognizing a phosphorylated peptide with an antibody. Although this method can be applied to safe and high-throughput screening (HTS), it is necessary to create an antibody that “specifically” recognizes “phosphorylated” amino acids, which causes problems in versatility and cost. It is believed that there is.
Recently, a method focusing on the reactivity of phosphate groups has been reported for the purpose of simultaneous detection of proteins phosphorylated in cells. The cell extract is chemically treated to chemically convert and separate the phosphorylated protein, and the phosphorylated site is estimated by mass spectrometry (Non-patent Documents 1 and 2). Although the usefulness of this method cannot be determined at this time, it is likely to require careful interpretation of the results because it targets a wide variety of proteins including various post-translational modifications other than phosphorylation. In addition, Hamachi et al. Recently found that a simple zinc complex recognizes a phosphate group and changes to fluorescence (Non-patent Document 3). However, the fluorescent probe responds strongly to ATP, which is one of the kinase substrates, and the sensitivity varies greatly depending on the peptide sequence to be phosphorylated. is there. These are few examples that focus on the chemical properties of phosphate groups, but none of them are aimed at detecting kinase activity.
Most recently, a fluorescent reagent for detecting a phosphorylated amino acid called “Pro-Q Diamond” was released by Molecular Probe (Non-patent Document 4). As long as the literature is referred to, it is considered to be a very excellent probe, but its structure, detection principle, etc. are all kept secret, so accurate evaluation of its usefulness is difficult at present.

H. Zhou, J. D. Watts, R. Aebersold, Nat. Biotechnol. 2001, 19, 375-378. Y. Oda, T. Nagasu, B. T. Chait, Nat. Biotechnol. 2001, 19, 379-382. A. Ojida, Y. Mito-oka, K. Sada, I. Hamachi, J. Am. Chem. Soc. 2004, 126, 2454-2463. K. Martin, T. H. Steinberg, L. A. Cooley, K. R. Gee, J. M. Beechem, W. F. Patton, Proteomics 2003, 3, 1244-1255. M. E. M. Noble, J. A. Endicott, L. N. Johnson, Science 2004, 303, 1800-1805.

  Under the above background, an object of the present invention is to provide a new method for detecting kinase activity.

In order to achieve the above object, the present inventors have repeatedly studied. First, we focused on the chemical properties of phosphorylated amino acids, which have not received much attention until now. In general, there are three types of amino acids that undergo phosphorylation: serine, threonine, and tyrosine, and it is known that serine threonine kinase accounts for the majority. Therefore, an attempt was made to develop a kinase activity detection method targeting serine threonine kinase. As a result, a detection method based on a new principle has been completed. The present invention is based on this result and provides the following configuration.
A method for detecting kinase activity comprising the following steps:
Allowing a kinase to act on a peptide;
Β-eliminating the introduced phosphate group;
Introducing a labeling substance into the double bond formed by β-elimination of the phosphate group by a Michael addition reaction; and detecting the introduced labeling substance.
In one aspect of the present invention, the kinase is a serine-threonine kinase.
In one embodiment of the present invention, the labeling substance is a fluorescent substance.
Furthermore, one embodiment of the present invention is characterized in that the labeling substance has a nucleophilic functional group such as an SH group.
Furthermore, one embodiment of the present invention is characterized in that the peptide is immobilized on an insoluble support.

  The detection method of the present invention has the advantage of high versatility because it detects the phosphorylated amino acid itself. Therefore, application to a wide range of uses such as identification of specific substrates of various kinases and high-throughput screening of specific inhibitors is expected. Moreover, it can also be determined whether the target kinase is serine threonine kinase or tyrosine kinase by using in combination with the existing kinase activity detection method. Furthermore, compared with existing methods for detecting kinase activity, the detection method of the present invention enables simple detection and reduces costs.

  Examples of the present invention (including experimental examples) will be described below.

<Experiment method>
1. Synthesis of fluorescent compounds Most of the reagents used were commercially available from TCI, WAKO, and nacalai, and DIPEA was commercially available from Watanabe Chem without any particular purification. A distilled solvent was used for the reaction and recrystallization.

1-1. Synthesis of compound 1

Resorcinol (1.00 g; 9.1 mmol) was placed in a 50 mL double-necked eggplant flask, and conc.H ₂ SO ₄ (10 mL) and 4-chloroacetoacetic acid ethyl ester (1.65 g; 10.0 mmol) were added. In the evening, it was stirred in the dark. The reaction mixture was added dropwise to ice-cooled water (55 mL), and the precipitate was collected by filtration and dried under reduced pressure to obtain 0.88 g of a white solid. Y. 46%
¹ H NMR (DMSO-d ₆ ): d 4.96 (s, 2H); 6.43 (s, 1H); 6.76 (d, 1H, J = 2.3 Hz); 6.84 (dd, 1H, J = 2.3, 8.8 Hz) ; 7.68 (d, 1H, J = 8.8 Hz).
MS FAB (NBA): 211 (M + 1)

Raw material (200 mg; 0.95 mmol) was placed in a 50 mL double-necked eggplant flask, and after Ar substitution, freshly dist.THF (8 mL), thioacetic acid (86 mg; 1.13 mmol), DIPEA (146 mg; 1.13 mmol) In addition, the mixture was shielded from light at room temperature and stirred for 90 min. The solvent was concentrated and the residue was dissolved in CH ₂ Cl ₂ and purified by column chromatography (EtOAc / n-hexane = 2/3). 230 mg of a white solid was obtained. Y. 97%
¹ H NMR (DMSO-d ₆ ): d 2.40 (s, 3H); 4.26 (s, 2H); 6.24 (s, 1H); 6.74 (d, 1H, J = 2.4 Hz); 6.81 (dd, 1H, J = 2.4, 8.8 Hz); 7.60 (d, 1H, J = 8.8 Hz); 10.64 (s, 1H).
MS FAB (NBA): 251 (M + 1)
Partial recrystallization with CH ₃ CN
Anal. Calcd for C ₁₂ H ₁₀ O ₄ S: C, 57.59; H, 4.03; N, 0.00. Found: C, 57.78; H, 4.09; N, 0.00.

Add the raw material (1.19 g; 4.75 mmol) to a 300 mL three-necked eggplant flask, dissolve in methanol (120 mL), degas, replace with Ar, add 0.25 g / mL NaOH aqueous solution (2.2 mL; 13.75 mmol). , Protected from light at room temperature and stirred for 50 min. Under ice cooling, 1N HCl was added to acidify the reaction solution. Methanol was concentrated, extracted with EtOAc (40, 30, 30 mL), brine wash (30 mL × 1), EtOAc was dried over anhydrous MgSO ₄ and concentrated. The residue was dissolved in methanol, applied to silica gel, and purified by column chromatography (5% methanol / CH ₂ Cl ₂ ). 0.84 g of a white solid was obtained. Y. 85%
¹ H NMR (DMSO-d ₆ ): d 3.19 (t, 1H, J = 7.9 Hz); 3.87 (d, 2H, J = 7.9 Hz); 6.28 (s, 1H); 6.73 (d, 1H, J = 2.2 Hz); 6.81 (dd, 1H, J = 2.2, 8.7 Hz); 7.71 (d, 1H, J = 8.7 Hz); 10.60 (s, 1H).
MS FAB (M + 1): 209 (M + 1)
Partial recrystallization with EtOAc
Anal.Calcd for C ₁₀ H ₈ O ₃ S: C, 57.68; H, 3.87; N, 0.00. Found: C, 57.43; H, 3.99; N, 0.00.

1-2. Synthesis of compound 2

3-Aminophenol (2.18 g; 20 mmol) was placed in a 100 mL double neck eggplant flask, and EtOAc (30 mL) was added and refluxed for 30 min. Phenyl chloroformate (1.72 g; 11 mmol) was added dropwise over 20 min, and the mixture was further refluxed for 1 h. The white precipitate generated during the reaction was removed by filtration, and the filtrate was concentrated. Drying under reduced pressure gave 2.30 g of a white solid. Y. quant
1H NMR (DMSO-d ₆ ): d 10.10 (s, 1H); 9.43 (s, 1H); 7.45-7.40 (m, 2H); 7.28-7.20 (m, 3H); 7.10-7.04 (m, 2H) ; 6.91 (d, 1H, J = 4.23 Hz); 6.44 (d, 1H, J = 4.23 Hz)
MS FAB (NBA): 230 (M + 1)

The raw material (3.00 g; 13.1 mmol) was placed in a 100 mL double-necked eggplant flask, 80% H ₂ SO ₄ (40 mL), 4-chloroacetoacetic acid ethyl ester (2.37 g; 14.4 mmol) was added, and 6.5 mL at room temperature. h Stir in the dark. The reaction mixture was added dropwise to ice-cooled water (50 mL), and the precipitate was collected by filtration. This was recrystallized from ethanol to obtain 1.92 g of a white solid. Y. 45%
¹ H NMR (DMSO-d ₆ ): d 4.99 (s, 2H); 6.55 (s, 1H); 7.25-7.30 (m, 3H); 7.42-7.49 (m, 3H); 7.61 (d, 1H, J = 2.0 Hz); 7.81 (d, 1H, J = 8.8 Hz); 10.80 (s, 1H).
MS FAB (NBA): 330 (M + 1)
Anal. Calcd for C ₁₇ H ₁₂ ClNO ₄ : C, 61.92; H, 3.67; N, 4.25. Found: C, 61.67; H, 3.75; N, 4.38.

Raw material (330 mg; 1.00 mmol) was placed in a 50 mL double necked eggplant flask, and after Ar substitution, freshly dist.THF (10 mL), thioacetic acid (84 mg; 1.1 mmol), DIPEA (142 mg; 1.1 mmol) were added. In addition, it was protected from light at room temperature and stirred for 2 h. The solvent was concentrated and the residue was dissolved in CH ₂ Cl ₂ and purified by column chromatography (EtOAc / n-hexane = 2/3). 354 mg of white solid was obtained. Y. 96%
¹ H NMR (DMSO-d ₆ ): d 2.39 (s, 3H); 4.29 (s, 2H); 6.35 (s, 1H); 7.25-7.30 (m, 3H); 7.42-7.46 (m, 3H); 7.59 (d, 1H, J = 2.0 Hz); 7.68 (d, 1H, J = 8.8 Hz); 10.77 (s, 1H).
MS FAB (NBA): 370 (M + 1)
Partial recrystallization with CH ₃ CN
Anal. Calcd for C ₁₉ H ₁₅ NO ₅ S: C, 61.78; H, 4.09; N, 3.79. Found: C, 61.55; H, 4.05; N, 4.03.

Add the raw material (790 mg; 2.14 mmol) to a 50 mL double-necked eggplant flask, add Ar, and then add freshly dist.THF (50 mL) and 10 M NaOH aqueous solution (860 μL; 8.6 mmol). And stirred for 45 min. Under ice cooling, 1N HCl was added to acidify the reaction solution (pH 4-5). THF was concentrated and neutralized with 0.2 M pH7 sodium phosphate buffer. After extraction with EtOAc (70 mL × 3), brine wash (50 mL × 1), EtOAc was dried over anhydrous MgSO ₄ and concentrated. The residue was dissolved in a mixed solvent of methanol / EtOAc, then applied to silica gel and purified by column chromatography (5% methanol / CH ₂ Cl ₂ ). 240 mg of a yellow solid was obtained. Y. 54%
¹ H NMR (DMSO-d ₆ ): d 3.11 (t, 1H, J = 8.2 Hz); 3.79 (d, 2H, J = 8.2 Hz); 6.05 (s, 1H); 6.17 (bs, 2H); 6.42 (d, 1H, J = 2.0 Hz); 6.56 (dd, 1H, J = 2.0, 8.7 Hz); 7.50 (d, 1H, J = 8.7 Hz).
MS FAB (NBA): 208 (M + 1)

1-3. Synthesis of compound 3

Raw material (500 mg; 2.40 mmol) was placed in a 100 mL double necked eggplant flask.After Ar substitution, freshly dist.THF (50 mL), methyl vinyl ketone (190 mg; 2.76 mmol), 0.25 M NaOH / methanol (0.95 mL) 0.24 mmol) was added, protected from light at room temperature, and stirred for 4 h. Under ice cooling, 1N HCl was added to acidify the reaction solution. The solution was concentrated, the residue was dissolved in EtOAc (200 mL), brine wash (40 mL × 1), EtOAc was dried over anhydrous MgSO ₄ and concentrated. The residue was dissolved in a mixed solvent of methanol / EtOAc, applied to silica gel, and purified by column chromatography (5% methanol / CH ₂ Cl ₂ ). 521 mg of a white solid was obtained. Y. 78%
¹ H NMR (DMSO-d ₆ ): d 2.07 (s, 3H); 2.61 (t, 2H, J = 7.0 Hz); 2.76 (t, 2H, J = 7.0 Hz); 3.89 (s, 2H); 6.25 (s, 1H); 6.72 (d, 1H, J = 2.3 Hz); 6.80 (dd, 1H, J = 2.3, 8.8 Hz); 7.68 (d, 1H, J = 8.8 Hz); 10.58 (s, 1H)
MS FAB (NBA): 279 (M + 1)

1-4. Synthesis of compound 4

Raw materials (30 mg; 0.14 mmol) and potassium tert-butoxide (1.6 mg; 0.014 mmol) are placed in a 50 mL double-necked eggplant flask.After Ar substitution, THF (5 mL), methyl vinyl ketone (11 mg; 0.15 mmol) Was shielded from light at room temperature and stirred for 17 h. Under ice cooling, 0.2 M pH 7 sodium phosphate buffer (10 mL) was added to neutralize the reaction solution (about pH 7-8). The THF was concentrated, extracted with EtOAc (30, 20, 20 mL), brine wash (20 mL × 1), and the EtOAc was dried over anhydrous MgSO ₄ and concentrated. The residue was dissolved in a mixed solvent of methanol / EtOAc, then applied to silica gel, and purified by column chromatography (EtOAc / n-hexane = 1/1). 25 mg of a yellow solid was obtained. Y. 64%
¹ H NMR (DMSO-d ₆ ): d 3.11 (t, 1H, J = 8.2 Hz); 3.79 (d, 2H, J = 8.2 Hz); 6.05 (s, 1H); 6.17 (bs, 2H); 6.42 (d, 1H, J = 2.0 Hz); 6.56 (dd, 1H, J = 2.0, 8.7 Hz); 7.50 (d, 1H, J = 8.7 Hz).
MS FAB (NBA): 278 (M + 1)

2. Fluorescence measurement The fluorescence of the synthesized compounds was measured using a Hitachi F4500 fluorometer. A 5 mM DMSO solution of each compound was prepared, diluted in glycine / NaOH buffer (pH 9.4) or sodium phosphate buffer (pH 2-12) and used for measurement. The excitation wavelength was 360 nm, the fluorescence wavelength was 465 nm, and measurements were performed at room temperature.

3. Peptide synthesis The peptides used in the experiments were synthesized according to the general Fmoc peptide solid phase synthesis method as shown in the following scheme. Whether or not the coupling of Fmoc amino acid completely progressed was confirmed by a Kaiser test. If it was incomplete, the coupling time was extended or the reagent was added again.

Fmoc amino acids (Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser ( ^t Bu) -OH, Fmoc-Ser {OP (OBzl) OH} -OH , Fmoc-Thr ( ^t Bu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Asn (Trt) -OH) and peptide synthesis reagents are commercially available from Watanabe Chem, WAKO unless otherwise specified. Was used. The PKA used in the assay was Sigma's cAMP dependent protein kinase catalytic subunit from bovine heart, and the other reagents were commercially available from WAKO.

The following resins were used for peptide synthesis depending on the purpose.
Peptide preparation resin cleaved from the bead by amidating the C-terminus;
TentaGel S RAM (0.3 mmol / g); Advanced ChemTech
Resin used for synthesis of peptide immobilized on beads
TentaGel S NH ₂ (0.3 mmol / g); Advanced ChemTech
Amino PEGA Resin (PEGA ₈₀₀ ; 0.4 mmol / g); Novabiochem
PL-PEGA Resin (PEGA ₁₉₀₀ ; 0.2 mmol / g, 300-500 mm, 30-50 mesh); Polymer Laboratories

4). Investigation of β-elimination conditions under solution conditions Conditions were examined using solid-phase synthesized Ac-LRRASLG-NH ₂ and Ac-LRRApSLG-NH ₂ . The analysis was performed by HPLC after changing the base conditions such as controlling the pH with the type, concentration or buffer of the base to be added and terminating the reaction with acetic acid. The analysis conditions of HPLC are as follows.
Column used: Inertsil ODS-3
Developing solvent 1; 0.1% TFA aqueous solution Developing solvent 2; 0.1% TFA acetonitrile solution
Gradient; Developing solvent 1 / Developing solvent 2 [90/10 → 65/35 (25 min)]
Flow rate; 1mL / min

5. 5. Examination using bead-carrying peptide 5-1. Enzymatic reaction
Take beads prepared by solid-phase synthesis of Ac-LRRASLG peptide in a 1.5 mL Eppendorf tube, water (10 min × 3), kinase reaction buffer (KB; pH 6.8, 30 mM MES, 10 or 15 mM MgCl ₂ , 0.4 mg / After washing with (mL BSA) (10 min × 3), it was preincubated in KB for 12 h at room temperature. After washing with KB (10 min × 2), adjust the final concentrations of ATP and PKA to 200 or 300 μM, 30, 50 or 70 units / mL, respectively, and shake on a shaker for 10 h at room temperature (25 ° C). Shake with. In the case of adding an inhibitor, a predetermined concentration of the inhibitor was added to the reaction solution, and similarly shaken at room temperature for 10 hours. After completion of the reaction, the beads were washed (10 min × 2, 1 h × 2, 5 min × 2) with a washing buffer (WB; 50 mM pH 7.2 PBS, 0.68 M NaCl, 13 mM KCl, 0.05% Tween20). Thereafter, it was further washed with water (10 min × 2, 1 h × 2, 5 min × 2).

5-2. β Desorption Washed beads were shaken in a H ₂ O / DMSO / EtOH = 4/3/1 solution at a NaOH concentration of 0.1 M at room temperature (25 ° C.) for 1 h on a shaker. After removing the reaction solution, ice-cooled 10% AcOH / H ₂ O was added to terminate the reaction. Thereafter, it was washed with water (1 min × 5).

5-3. Michael addition reaction After β-elimination reaction, NaOH in H ₂ O / DMSO / EtOH = 4/3/1 solution was added to the washed beads to a concentration of 25 mM and Compound 1 to a concentration of 0.5 to 3.0 mM. And shaken on a shaker for 1 h. The reaction solution was removed, and ice-cooled 10% AcOH / DMF was added to terminate the reaction. After that, the beads were DMF (10 min × 2, 2 h × 1, 10 min × 2), methanol (5 min × 1, 1 h × 1, 5 min × 1), H ₂ O (10 min × 3), Washed with 6 N guanidine hydrochloride (1 h × 1, 10 h × 1), H ₂ O (5 min × 2). The sample was observed in a pH 9.4 Glycine / NaOH buffer solution with a fluorescence microscope (Hamamatsu Photonics ARGUS / HiSCA; excitation wavelength 360 nm, fluorescence wavelength 425 nm).

<Experimental result>
Of the three amino acids that are phosphorylated, phosphorylated serine and threonine have similar chemical properties. Therefore, the following kinase activity detection method was planned.
Phosphorylated serine and threonine have similar chemical properties, and it is known that the phosphate group is easily β-eliminated under basic conditions. This phenomenon has been a big problem when chemically synthesizing phosphorylated peptides. The synthetic problem was solved by the development of a new protecting group, but the applicant thought that this chemical property could be applied to the detection of kinase activity. As shown in FIG. 1, it is considered that a double bond formed by β-elimination of a phosphate group becomes a Michael reaction acceptor and reacts quickly with a nucleophile having an SH group or the like. Therefore, we thought that if a fluorescent molecule is used as a nucleophile, it is possible to introduce a fluorophore selectively with an amino acid residue having a phosphate group (FIG. 1).
First, we started to synthesize fluorescent molecules essential for this detection method. Compounds 1 and 2 were designed and synthesized as fluorescent substances having SH groups (Fig. 2). The synthesis was performed according to the scheme shown below.
Subsequently, it was decided whether these compounds would react with the Michael reaction acceptor or remain fluorescent after the reaction, as per the working hypothesis. When reacted with methyl vinyl ketone as a model compound of the Michael reaction acceptor, the reaction proceeded rapidly to give the target compounds 3 and 4. These compounds 3 and 4 showed an increase in fluorescence intensity as compared to compounds 1 and 2 before the reaction (Scheme 2, FIG. 3).
The property that the fluorescence intensity increases after the reaction leads to a relative decrease in fluorescence caused by non-specific adsorption of the fluorescent substance on the beads, and an improvement in the reliability of the assay system can be expected. That is, it became clear that all the compounds have properties suitable for this assay.
As shown in FIG. 1, this detection method consists of two steps: 1) β elimination and 2) Michael addition. All of these are reactions under basic conditions where the peptide is unstable. Especially recently, several side reactions under β-elimination conditions have been reported, so the reaction conditions for each step should be optimized. It was. At this time, since it is difficult to perform instrumental analysis under the solid phase conditions where the peptide is supported on the beads, each reaction is performed as a peptide solution, not a solid phase using beads, and the progress of the reaction is determined by HPLC, MS (MALDI ). Ac-LRRASLG-NH ₂ and Ac-LRRApSLG-NH ₂ were used as peptides. Β-elimination of the phosphate group of pS was attempted using 0.1 M Caps buffer in an environment of pH 10 and 11, but β-elimination could not proceed efficiently even when the temperature was raised. In order to allow β-elimination to proceed efficiently (within 1 to 2 hours as the reaction time at room temperature), it was necessary to add NaOH or LiOH at a certain concentration (50 mM or more). When LiOH and NaOH were compared, there was no significant difference in reaction efficiency. In addition, a small amount of Ba ²⁺ , which is known to promote β-elimination of phosphate groups, was added, but the concentration of the base itself had a greater effect on the reaction efficiency than the presence of Ba ²⁺ . Next, the stability of non-phosphorylated Ac-LRRASLG-NH ₂ was analyzed using HPLC under conditions of 100 mM NaOH in which β elimination of the phosphate group proceeded efficiently. As a result, under the condition of 100 mM NaOH, Ac-LRRASLG-NH ₂ was unstable and caused some reaction. By changing the reaction solvent from H ₂ O to H ₂ O / DMSO / EtOH (4: 3: 1), side reactions could be suppressed. In this way, various reaction conditions are studied, and conditions under which β elimination and Michael addition proceed efficiently, ie, Step 1) β elimination Solvent: 0.1 M NaOH / DMSO / EtOH = 4/3/1
Reaction temperature: 25 ° C
Step 2) Michael addition Solvent: 25 mM NaOH / DMSO / EtOH = 4/3/1
Fluorescent compound concentration: 1 mM
Reaction temperature: 25 ° C
Succeeded in finding out.
Next, optimization was performed for two types of beads: beads carrying a peptide containing phosphorylated serine (LRRApSLG) in the sequence and beads carrying a peptide containing serine not phosphorylated (LRRASLG). Β-elimination and Michael addition reaction were performed under the reaction conditions. As beads, two types of beads, TentaGel and PEGA ₈₀₀ , which are considered to be suitable for enzyme reaction and are actually very well used, were used. When the beads were observed using a fluorescence microscope (excitation wavelength: 360 nm, fluorescence wavelength: 425 nm), a significant difference in fluorescence was observed between the two types of beads, and the peptide having a phosphate group was selectively fluorescent. It was suggested that a team was introduced. Similar results were obtained using either synthesized molecules 1 or 2, but the difference in fluorescence between the two types of beads was slightly greater when 1 was used. It was decided to use (FIG. 4). When the pH dependence of the fluorescence of compound 3 was examined, stable and strong fluorescence was given over pH 9-10 (FIG. 5). Therefore, a pH 9.4 buffer was used when measuring fluorescence. Subsequently, as a result of examining the concentration of the fluorescent compound, the reactivity between the positive beads and the fluorescent compound was low at a low concentration, and nonspecific adsorption of the fluorescent substance to the negative beads was observed at a high concentration. As shown in FIG. 6, the clearest difference was observed around 1 mM (about 0.5 to 3 mM).
Subsequently, the assay was performed according to FIG. The well-studied commercially available protein kinase A (PKA) was selected as the kinase, and its specific substrate peptide (Kemptide; LRRASLG) was solid-phase synthesized on the two kinds of beads described above. As a result, as shown in FIG. 7, no difference was observed in the fluorescence intensity, suggesting that the enzyme reaction did not proceed. Even though various conditions such as enzyme, ATP concentration, reaction temperature, etc. were examined, improvement was not seen, but when using beads PEGA ₁₉₀₀ that permeates even larger molecules, chemically synthesized phosphorylated serine-containing peptide (LRRApSLG ), The same fluorescence difference was observed (FIG. 8). Furthermore, an increase in fluorescence intensity depending on the enzyme reaction time was observed, suggesting the possibility of detecting serine / threonine kinase activity on the beads. When the enzyme reaction conditions were optimized using peptides carried on PEGA ₁₉₀₀ , it was found that the conditions shown below were optimal.
・ Enzyme reaction:
PKA 30 units / mL, ATP 300 μM in KB, 25 ℃, 10-12 h
(Note: When the kinase concentration was increased to 100 units / mL, the reaction was completed in 3-4 h. The optimal reaction temperature was 30-35 ° C, but it was difficult to warm in the experimental environment. Therefore, room temperature conditions were selected)
Β desorption:
0.1 M NaOH / DMSO / EtOH = 4/3/1, 25 ℃, 1 h
・ Michael addition:
3 mM compound 1, 25 mM NaOH / DMSO / EtOH = 4/3/1, 25 ℃, 1 h
For the purpose of confirming whether the difference in fluorescence intensity of this detection method reflects the enzyme activity, a change in fluorescence intensity was observed using a strong inhibitor peptide of PKA (IP: Ac-GRTGRRNAI-NH ₂ ). We examined whether it was possible. As a result, as shown in FIG. 9, the fluorescence intensity decreased depending on the inhibitor concentration, indicating that this detection method can be applied to the kinase inhibitor assay. In the future, it is considered that further improvement in reliability and quantitativeness can be expected by using fluorescent molecules whose fluorescence wavelength changes after the Michael reaction.

<Summary>
It was revealed that this detection system can easily detect fluorescence of a kinase activity and can be used for an inhibitor assay. This detection method can be applied as follows.
1. Determination of Kinase Recognition Sequence Using Peptides Supported on Beads Many kinases have no known recognition sequence. Since methods for synthesizing a variety of bead-carrying peptides have already been established, it is considered that simultaneous screening of target kinase recognition sequences can be performed using bead-carrying peptides containing peptides of various sequences.

2. Screening for Kinase Inhibitors Using Peptides Loaded on Beads As shown here, this detection method can be used for kinase inhibitor assays. Since it is a simple assay based on the detection principle of fluorescence, it is highly versatile and applicable to various kinases. Since many kinase inhibitors have been targeted for pharmaceuticals, the utility value of this method applicable to HTS is considered to be high.

3. Determination of Kinase Recognition Sequence and Screening for Inhibitors Using Microarray Peptides can be carried on the microarray, although somewhat specialized equipment is required compared to bead assays. If a fluorescent dye having a longer excitation wavelength and fluorescence wavelength is used, use of a commercially available microarray reader developed for DNA can be expected. By using a microarray, it is possible to integrate a high level of information, and there is a possibility that determination of a kinase recognition sequence as described above and screening of an inhibitor can be performed on a smaller scale.
In addition, in the case of diseases in which specific kinase activity is characteristically increased or decreased, information relating to kinase activity can be easily obtained by using this method, and application to diagnosis of disease states can be expected. In recent years, it has become clear that many enzymes control their activity in a complex and strict manner by various post-translational modifications. This method, which can easily detect the enzyme activity itself, is considered to be highly reliable and is expected to have a wide range of applications.

4). Induction of fluorescence of phosphorylated proteins in gels In recent years, proteomics research has been actively conducted. The central role is played by two-dimensional electrophoresis of proteins and identification by mass spectrometry. If this detection principle is used, there is a possibility that the phosphorylated protein contained in the gel after electrophoresis may be selectively fluorescence-induced, and phosphorylation and desorption that play an important role in intracellular signal transduction. It is expected that much information can be obtained regarding phosphorylation. In addition, it is known that proteins and peptides having a phosphate group are generally very insensitive to mass spectrometry, but this is thought to be due to the negative charge of the phosphate group. When derivatized by this detection method, phosphorylated amino acids do not have a negative charge, and thus a large increase in sensitivity is expected.
As described above, this detection system provides a general principle that can easily detect biochemically and medically important kinase activity, and can be said to have high versatility and wide applicability.

As described above, existing methods for detecting kinase activity are roughly classified into RI methods and antibody methods, and any of them can be said to apply existing biochemical methods to kinases. There are few kinase assay methods that utilize the chemical properties of phosphorylated amino acids. This detection method is unique in that it uses a simple chemical method rather than a biochemical method that is problematic in terms of danger and versatility.
This detection method provides the basic principle of detecting phosphorylated serine and threonine, and has the feature that it is very versatile. Assays can be performed in Eppendorf tubes or microplates using peptides supported on beads, and can also be applied to peptide arrays in which peptides are supported on membranes or glass plates. Furthermore, it may be applicable to proteins. If the phosphorylated protein in the gel after being subjected to SDS-PAGE can be derivatized to fluorescence, the utility value of this detection method will be extremely high.
Specific kinase inhibitors are expected as various pharmaceuticals including anticancer agents, and development research is progressing (see Non-Patent Document 5). HTS is indispensable for drug development using combinatorial chemistry, which is now becoming the mainstream, but the characteristics of this assay system exactly meet that requirement.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

FIG. 1 shows the principle of this assay. FIG. 2 shows fluorescent materials (1, 2) designed and synthesized in the examples. FIG. 3 is a graph showing the fluorescence spectra of compounds 1 and 3 (5 μM (0.1% DMSO) in glycine / NaOH buffer (pH 9.4), Ex: 360 nm). FIG. 4 shows the results of studies using bead-carrying peptides. FIG. 5 is a graph showing the pH dependence of the fluorescence of compound 3. FIG. 6 shows the results of studies on the concentration of Compound 1. FIG. 7 shows a method for detecting kinase activity using a peptide carried on a solid phase and the result (1). FIG. 8 shows a method for detecting kinase activity using a peptide carried on a solid phase and the result (2). FIG. 9 shows the effect of kinase inhibitors on this assay system.

Claims (4)

Each of the following steps:
Allowing a kinase to act on a peptide;
Β-eliminating the introduced phosphate group;
The double bond moiety of the phosphate group is generated apart β removal step to introduce fluorescent substance by Michael addition reaction; detecting a and introduced fluorescent material,
Comprise becomes, the presence and solvent NaO H is _{H 2 O / DMSO / EtOH (} 4: 3: 1) have a row the β elimination and the Michael addition reaction under the conditions of,
NaOH concentration at the time of β desorption is 0.1M,
NaOH concentration in the Michael addition reaction is 25 mM,
Kinase activity detection method. The detection method according to claim 1, wherein the kinase is a serine-threonine kinase. The detection method according to claim 1 or 2 , wherein the labeling substance has a nucleophilic functional group such as an SH group. The detection method according to any one of claims 1 to 3 , wherein the peptide is immobilized on an insoluble support.