KR101034884B1 - Inhibition Method of CK2? Activity Using Mitoxantrone - Google Patents

Abstract

The present invention relates to a method for measuring the interaction between CK2α and Nopp140, to a method for screening interaction modulators of CK2 and Nopp140 using the above method, and to a method for inhibiting CK2 activity, and more particularly to multi-well GST-CK2α protein. securing to the plate; Treating the Nopp140 protein phosphorylated on the material; And measuring the amount of Nopp140 bound to the GST-CK2α; a method for measuring interaction between CK2α and Nopp140, candidates for controlling the interaction of CK2α, CK2α and Nopp140 (human nucleolar phosphoprotein), and InsP ₆ ( inositolhexakisphosphate); And a method for screening interaction modulators of CK2α and Nopp140, comprising treating Nopp140 with the reactants, and a method for inhibiting CK2 activity using mitoxantrone screened by the above method. The present invention can effectively measure the interaction of CK2α and Nopp140, and has the function of inhibiting the activity of CK2, a target protein of anticancer drug development, by using the measurement method can screen a substance that can be used as an anticancer drug, The mitoxanthone identified through screening proves to be a substance that can inhibit the activity of CK2, and can be used for development for another anticancer agent.

Kinase CK2, human nucleus phosphate protein (Nopp140), peptide, Nopp140 expression, protein interaction

Description

Inhibition Method of CK2α Activity Using Mitoxantrone}

The present invention relates to a method for measuring the interaction of CK2 with Nopp140, a method for screening the interaction modulator of CK2 and Nopp140 and a method for inhibiting CK2 activity using the material screened by the above method.

Phosphorylation enzyme CK2 is a protein composed of two active subunits (CK2α) and two regulatory subunits (CK2β) and can phosphorylate more than 300 proteins, including proteins responsible for the main functions of cells. have. CK2 is distributed in most cells and tissues and phosphorylates proteins necessary for cell growth and induces cell growth. CK2 also inhibits apoptosis by phosphorylating proteins involved in cell death. In particular, in many types of cancer cells, CK2 activity is expressed from 5 to 20 times higher than normal cells to induce the growth of cancer cells and to make them resistant to apoptosis inducers such as Fas or TNF-a. Inhibits cell death. Therefore, a substance that inhibits the activity of CK2 can be used as a new anticancer agent because it can inhibit the growth of cancer cells and induce apoptosis (Sarno, S., et al., 2002, Pharmacol. Ther. Vol. 93, pp 159-168). However, CK2 is also essential for normal growth of cells, so if CK2 activity is completely inhibited, it will inhibit normal cell growth. Therefore, proper regulation of CK2 activity is required to suppress anticancer effects without cytotoxicity.

Intracellular CK2 activity is not affected by signaling agents such as Ca ²⁺ , diacylglycerol, cAMP, and the like. However, the activity of intracellular CK2 is present in the state of 70-80% of the activity inhibited by a specific protein and high concentration of InsP ₆ (inositolhexakisphosphate) in which 6 phosphates are bound to inositol, a monosaccharide. It is known that the activity of the surface CK2 is increased by 3-4 times (Solyakov, L., et al., 2004, J. Biol. Chem. Vol. 279, pp 43403-43410).

Meanwhile, the inventors found that the human nucleolar phosphoprotein (hereinafter referred to as "Nopp140"), which is the human nucleus, binds to CK2 and inhibits CK2 activity by 3-4 times, and InsP ₆ inhibits the binding of CK2 and Nopp140. Observed. As a result, when the concentration of intracellular InsP ₆ is increased, CK2 is dissociated from Nopp140 and has high activity (Kim, YK et al., 2006, J. Biol. Chem. Vol. 281, pp 36752-36757). On the other hand, inositol polyphosphate such as InsP ₆ (inositol polyphosphate) is generated when the cells divide a lot, so the concentration is high, at this time the interaction of CK2 and Nopp140 is inhibited, resulting in increased CK2 activity. Inhibition of CK2α activity by Nopp140 and CK2α activation by InsP ₆ suggest a new model for CK2 activity regulation mechanism as shown in FIG. 1. As illustrated in FIG. 1, when CK2 and Nopp140 are present in cells, the two proteins bind with high affinity to each other and CK2 activity is inhibited. In particular, Nopp140 will inhibit the activity of CK2 overexpressed in cancer cells will inhibit cancer cell growth and reduce the resistance to apoptosis-inducing substances. When cells proliferate actively, the concentration of InsP _{6 in} the cells increases, and the increased concentration of InsP ₆ inhibits the binding of CK2α and Nopp140 to maintain high CK2α activity. The model of FIG. 1 infers that a substance capable of stabilizing the binding of Nopp140 and CK2α even in the presence of InsP ₆ has a function of inhibiting CK2 activity.

Accordingly, the present inventors have devised a method for measuring the binding of Nopp140 and CK2α in an effort to find a substance that can be used as an anticancer agent having a function of inhibiting the activity against CK2, and by using the same, Nopp140 and CK2α even in the presence of InsP ₆ The present invention was completed by screening a material for stabilizing binding of and confirming that the material identified by the screening method stabilizes the binding of Nopp140 and CK2α.

An object of the present invention is to provide a method for measuring CK2α and Nopp140 interaction, which is an active site of CK2, an anticancer drug target protein, and a method for screening a substance that inhibits CK2 activity by stabilizing the binding of CK2α and Nopp140.

Still another object of the present invention is to provide a method for inhibiting CK2 activity using a substance identified by the screening method.

In order to achieve the above object, the present invention comprises the steps of: i) fixing the GST-CK2α protein in a multi-well plate; Ii) treating the Nopp140 protein phosphorylated on the material; And iii) measuring the amount of Nopp140 bound to the GST-CK2α; and provides a method for measuring interaction between CK2α and Nopp140.

In addition, the present invention comprises the steps of reacting the CK2 subunit, CK2 and Nopp140 (human nucleolar phosphoprotein) interaction control candidate, and InsP ₆ (inositolhexakisphosphate); And ii) treating the reactants with Nopp140. The method provides a method for screening interaction modifiers of Nock140 with CK2.

The present invention also provides a method for inhibiting CK2 activity using mitoxantrone screened by the above method.

In addition, the present invention is a) GST-CK2α protein; b) phosphorylated Nopp140 protein; c) Screening kits of Nopp140-CK2α interaction modulators comprising InsP ₆ , His tag antibody and reaction quench solution.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: i) fixing the GST-CK2α protein to a multi-well plate; Ii) treating the Nopp140 protein phosphorylated on the material; And iii) measuring the amount of Nopp140 bound to the GST-CK2α; and provides a method for measuring interaction between CK2α and Nopp140.

In order to measure the interaction between CK2α and Nopp140, the present inventors measured binding affinity with GST-CK2α, a fusion protein containing an active site of His-Nopp140 and CK2 (see FIG. 2 ). In addition, InsP ₆ was measured to inhibit the binding between His-Nopp140 and GST-CK2α (see Figure 3 ). Through the above results, the interaction between phosphorylated His-Nopp140 and GST-CK2α can be measured (see FIG. 4 ).

Nopp140 inhibits the activity of CK2 by binding to CK2α, and InsP ₆ present in the cell activates CK2 by inhibiting this interaction. In order to quantitatively measure the interaction between Nopp140 and CK2α, the present inventors have introduced GST-CK2α protein, which is a fusion protein of GST (glutathione S-transferase) and CK2α, and Nopp140 (His- Nopp140) was expressed and purified in E. coli to examine the interaction of these proteins. GST-CK2α was immobilized on a glutathione-immobilized 96-well plate, and then bound by addition of His-Nopp140, and then the amount of bound His-Nopp140 was selected using His-tag antibody, which is a selective antibody to histidine tag. Detected. As a result, it was confirmed that His-Nopp140 selectively binds to GST-CK2α and has a dissociation constant K _D = 8 nM (see FIG. 2 ).

Meanwhile, InsP ₆ has an activity of inhibiting the interaction between Nopp140 and CK2α, and 25 μM or more of InsP ₆ inhibits the interaction of CK2α and phosphorylated Nopp140 by 50% or more (see FIG. 3 ). This means that CK2 is present in a non-binding state and CK2 activity is not inhibited at the time of proliferation of cells in which intracellular InsP ₆ concentration reaches several tens of μM or more. Therefore, substances that stabilize the binding of Nopp140 and CK2α in the presence of InsP ₆ inhibit the CK2 activity in cancer cells with active cell proliferation, thereby inhibiting cell proliferation and apoptosis of cancer cells by substances which induce apoptosis such as TNF-α. Will be able to promote.

In addition, the present invention comprises the steps of reacting the CK2 subunit, CK2 and Nopp140 (human nucleolar phosphoprotein) interaction control candidate, and InsP ₆ (inositolhexakisphosphate); And ii) treating the reactants with Nopp140. The method provides a method for screening interaction modifiers of Nock140 with CK2.

In the method for screening an interaction modulator of CK2 and Nopp140 of the present invention, the CK2 subunit is preferably CK2α, and the CK2α is a fusion protein of GST-CK2α to which GST (glutathione S-transferase) is bound. desirable.

In addition, in the screening method of the interaction modulator of CK2 and Nopp140 of the present invention, the Nopp140 is preferably phosphorylated or composed of His-Nopp140, His-Nopp140 is His- in order to facilitate the purification of Nopp140 and protein It is more preferable that it consists of a tag site | part.

In addition, in the method for screening the interaction modulator of CK2 and Nopp140 of the present invention, the candidate material is composed of an antibody, a ligand, a natural compound, an extract, a synthetic peptide, a new drug candidate, a peptide having a partial sequence of Nopp140, and a protein. It is preferred that the material is selected from the crowd.

In addition, in the screening method of the interaction modulators of CK2 and Nopp140 of the present invention, it is preferable that the screening method uses a detection method selected from the group consisting of chromophore, fluorescence, radioactivity and conjugated peptides using a labeled antibody. .

In addition, in the screening method of the interaction modulator of CK2 and Nopp140 of the present invention, the screening method comprises the steps of: i) immobilizing glutathione on the surface of the container, adsorbing GST-CK2α to the fixed glutathione; Ii) treating said substance with a candidate substance for controlling the interaction of CK2α with Nopp140; Iii) adding His-Nopp140 and InsP ₆ phosphorylated to the material; Iii) binding the labeled antibody conjugate to His-tag to the substance; Iii) developing the bound labeling antibody conjugate; And iii) comparing the degree of color development with the control.

The present invention provides a method for screening a substance that modulates the interaction of CK2 with a human nucleolar phosphoprotein (Nopp140), a human nucleolar phosphoprotein (Nopp140), which has a function of inhibiting the activity of the kinase CK2, which is one of anticancer drug target proteins. It is about. CK2, which is overexpressed in cancer cells, induces cell growth and inhibits apoptosis, is a target protein for anticancer drug development. Among these proteins interacting with CK2, Nopp140 was confirmed to bind to the active subunit of CK2 to inhibit the activity of CK2. Therefore, Nopp140, which has a function of binding to CK2 and inhibits activity, and a substance that stabilizes binding of Nopp140 and CK2, has a function of inhibiting CK2 activity and can be used as an anticancer agent. And the method of measuring the interaction between Nopp140 and CK2 can be used to discover a new concept of CK2 inhibitor.

Substances that stabilize the binding of Nopp140 and CK2α in the presence of InsP ₆ can be screened by measurement of the interaction between GST-CK2α and His-Nopp140 of the present invention (see FIG. 4 ), and as a representative material as a material excavated through Toxantrone (see FIG. 6 ) was excavated. Although this compound has been reported to have anticancer activity, anticancer activity has been reported to inhibit DNA topoisomerase or DNA replication by intercalation into DNA (Biophys. Chem. 2005, vol. 116, 199-205; Br. J. Clin. Pract. 1991, vol45, 208-211). However, the present invention found that mitozantron stabilizes binding of CK2α and Nopp140 even at a low concentration of 15 μM, so that CK2 maintains binding with Nopp140 even at a concentration of InsP ₆ of 50 μM or more (see FIG. 7 ). ). In addition, when the interaction between CK2α and Nopp140 was stabilized by mitozantron, it was confirmed that even in the presence of InsP ₆ , it was possible to inhibit the activity of CK2 which can phosphorylate α-casein (see FIG. 8 ). That is, a substance such as mitoxanthone having an activity of stabilizing the interaction of CK2α and Nopp140 identified by the screening method of the present invention has an anticancer activity that inhibits the function of CK2 by maintaining the activity of CK2 in a state inhibited by Nopp140. It can be used as a material having.

The present invention also provides a method for inhibiting CK2 activity using mitoxantrone screened by the above method.

In the method of inhibiting the CK2 activity, inhibition of the CK2 activity is preferably made by increasing the amount of His-Nopp140 binding to GST-CK2α under InsP ₆ exists.

In addition, the present invention is a) GST-CK2α protein; b) phosphorylated Nopp140 protein; c) Screening kits of Nopp140-CK2α interaction modulators comprising InsP ₆ , His tag antibody and reaction quench solution.

Hereinafter, the present invention will be described in detail in the above order.

(1) CK2α and Nopp140 interaction measurement method and CK2 activity modulator screening method

The interaction measurement method is a method of measuring the amount of Nopp140 binding to CK2α immobilized on the surface of a multi-well plate, and provides a method of screening a material to stabilize the binding of Nopp140 in the presence of InsP ₆ .

Hereinafter, the method for measuring the interaction of Nopp140 with CK2α of the present invention and the screening process of the CK2α-Nopp140 binding stabilizer using the same are composed of the following four steps.

1) preparing a GST-CK2α protein;

2) preparing phosphorylated Nopp140 protein;

3) immobilizing the GST-CK2α of step 1) on a multi-well plate and measuring the amount of Nopp140 bound to GST-CK2α by treating the phosphorylated hNopp140 protein of step 2); And

4) The degree of interaction of step 3) in the presence of the test compound and InsP ₆ was measured, and the test having the function of stabilizing the interaction between Nopp140 and CK2α in comparison with the degree of interaction of the control group in which there was no test compound and InsP ₆ was present. Screening the compound.

In addition, the screening method of such CK2α-Nopp140 binding stabilizer

a) GST-CK2α protein;

b) phosphorylated Nopp140 protein;

c) a screening kit of a Nopp140-CK2α interaction modulator comprising InsP ₆ , His tag antibody and reaction quench solution.

As described above, the screening method of the present invention can search for Nopp140-CK2α interaction modulators, and thus can be prepared a screening kit for analyzing the degree of Nopp140-CK2α interaction of various regulatory candidates. For example, the GST-CK2 α protein may be bound to a suitable container, treated with various candidates, treated with phosphorylated Nopp140 protein, InsP ₆ , His tag antibody, and finally the reaction stop solution may be Screening kits capable of measuring Nopp140-CK2α interactions can be prepared by appropriate detection means to process and measure color development. The detection means attaches a fluorescent material directly to His tag antibody or binds a coloring material to enable fluorescence or color development. Here, the fluorescent material may be Cy5 (red) and Cy3 (green). Specifically, the screening method of the candidate material using the screening kit

V) treating glutathione on the surface of the container and treating candidate interaction control material of CK2α with Nopp140 on the GST-CK2α protein adsorbed thereto;

Ii) reacting the phosphorylated His-Nopp140 with InsP ₆ in the mixture;

Iii) treating the mixture with a labeled antibody against His tag;

Iii) treating the mixture with a reaction stop solution to wash unreacted residues; And

Iii) most preferably including determining whether or not Nopp140 interacts with the CK2α of the candidate substance to the type of reaction or the degree of color development.

Hereinafter, the search method of the present invention will be described in detail.

The GST-CK2α protein of step 1) is preferably a GST-CK2α fusion protein consisting of a Glutathione S-Transferase (GST) protein and an active site of CK2 (CK2α). The site for facilitating purification of the protein is preferably GST (Glutathione-S-Transferase), but is not limited thereto.

The phosphorylated Nopp140 protein of step 2) is preferably a Nopp140 protein expressed in a human gene, and a form phosphorylated by CK2 is preferable. In addition, the site for facilitating protein purification is most preferably, but not limited to, six histidine tags at the N-terminus. Histidine tag introduced at the N-terminus of Nopp140 can be used to measure the amount of Nopp140 using an antibody. The Nopp140 is FITC, Cy3, Cy5, Alexa fluor or BODIPY to facilitate the search  It can be labeled by the conjugation of fluorescent materials, chromogenic materials, radioactive materials or synthetic peptides, and other markers known to those skilled in the art can be used in the present invention.

In the protein immobilization of step 3), the GST-CK2α of step 1) is preferably treated on a multi-well plate treated with glutathione. The phosphorylated Nopp140 protein prepared in step 2) can be treated with the immobilized GST-CK2α protein, and the amount of phosphorylated Nopp140 bound to GST-CK2α is reacted with an HRP-coupled anti-His tag antibody, followed by enzymatic immune reaction. It is preferable to use and measure. When the Nopp140 is labeled with a fluorescent material such as FITC, Cy3, Cy5, Alexa fluor or BODIPY to facilitate the search, the amount of bound Nopp140 may be measured by measuring fluorescence.

The test compound of step 4) may be an antibody, a ligand, a natural compound, an extract, a synthetic peptide, a drug candidate or a protein, but is not limited thereto. The test compound of step 4) can be reacted before, after or simultaneously with the treatment of phosphorylated Nopp140 with respect to the immobilized GST-CK2α of step 3). For example, the test compound may be pre-reacted with GST-CK2α protein and then treated with phosphorylated Nopp140 protein, and the test compound may be treated after first reacting the GST-CK2α protein with phosphorylated Nopp140. In addition, GST-CK2α protein, phosphorylated Nopp140, and the test compound can be treated simultaneously.

(2) CK2 inhibitory active substance And CK2 activity inhibition method using the same

The present invention provides a method for inhibiting CK2 activity using mitoxantrone, including mitoxantrone and derivatives thereof, as a substance having CK2 inhibitory activity discovered using a method for measuring the interaction between CK2α and Nopp140. .

The present invention relates to a method for screening a CK2 signaling modulator, a search kit, and a CK2 activity modulator. In the presence of a test compound, the binding of Nopp140 and CK2α is measured by measuring the interaction of purified GST-CK2α with His-Nopp140. Search for substances that stabilize and inhibit CK2 activity, and the excavated materials can be used as anticancer agents. Inhibitors for existing phosphatase including CK2 targeted the active site of phosphatase, but the substance discovered by the present invention has the characteristic of targeting the interaction between CK2α and Nopp140. The substance is expected to not bind to an active site of phosphatase other than CK2, and thus has an advantage of overcoming the selectivity problem that the inhibitory material that binds to the active site of the phosphatase may have in common. In addition, by using a purified protein and a high efficiency screening device, it can be usefully used for mass screening of compounds at low cost without the need for an expensive cell culture step.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Preparation of GST-CK2α Protein

To express and purify the GST-CK2α protein, first, the expression vector pGST-CK2α (Kim, YK et al., 2003, Protein Expression & Purification Vol. 31, pp 260-264) of GST-CK2α was expressed in E. coli DH5a (Invitrogen, USA) to prepare a transformant and then shaking culture at 37 ℃ using the LB culture medium containing the antibiotic ampicillin (ampicillin) at a concentration of 100 ㎍ / ㎖. When the absorbance of the culture at 600 nm reached 0.7, IPTG (Isopropyl-bD-Thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce the expression of the protein and further incubated at 18 ° C. for 16 hours, E. coli transformants were harvested by centrifugation (3,000 xg, 10 min). The E. coli transformant was suspended in a buffer solution (50 mM Na phosphate, pH 7.0), and passed through a French press at a pressure of 12,000 psi to break the cell wall and centrifuged (10,000 μg, 30 minutes). Insoluble materials such as cell walls were removed. The expressed protein was passed through a GST adsorption column and then extracted with a buffer containing 10 mM reduced glutathione. GST-CK2α protein expressed and purified by the above method has a purity of about 90%.

Example 2 Expression, Purification and Phosphorylation of Nopp140

PNopp140, an expression gene of His-Nopp140, was transformed into Escherichia coli BL21 (DE3) and shake-cultured at 37 ° C using an LB culture medium containing 0.05 mg / ml kanamycin (Kim, YK et al., 2003, Protein Expression & Purification Vol. 31, pp 260-264). When the culture solution reached an absorbance of 0.7 at 600 nm, 1 mM IPTG was added and further grown for 3 hours, followed by centrifugation to recover E. coli. The cells were lysed with a French Presser, and then the Ni-NTA column and the hydroxyapatite column were sequentially performed to purify the protein. Nopp140 has been reported to be phosphorylated to a very high degree by CK2 in cells (Pai et al., J. Cell. Sci ., 1995, 108, 1911-1920; Chen and Yeh, Biochem. Biophys. Res. Commun ., 1997, 230, 370-375). Therefore, the purified His-Nopp140 was phosphorylated using CK2. First, 1 mg of His-Nopp140 protein was added to 50 units of CK2 (New England Biolabs, USA) and 100 μl of reaction buffer solution (20 mM Tris-HCl, 50 mM KCl, 10 mM MgCl ₂ , 2.5 mM ATP, pH 7.5). After mixing in and reacted for 2 hours at 30 ℃ phosphorylated.

Example 3 Binding Measurement of GST-CK2α and Nopp140 by Enzyme Linked Immunosorbent Assay

To measure binding of GST-CK2α and His-Nopp140, 0.1 ml of GST-CK2α solution at a concentration of 5 μg / ml was placed in a glutathione-coated 96 well plate in a 96-well plate coated with Glutathione. The surface was adsorbed on the surface for 1 hour. After repeated washing with 0.2 ml wash buffer [20 mM Na phosphate, 150 mM Na chloride, pH 7.4] 4 times, buffer solution containing 0.3 ml of 5% skim milk powder (20 mM sodium phosphate, 150 mM sodium chloride, After standing at room temperature for 1 hour at pH 7.4), it was washed 6 times again with a washing buffer. 0.1 ml of 5 µg / ml phosphorylated His-Nopp140 (20 mM Na phosphate, 150 mM Na chloride, pH 7.4) was added to the surface to which GST-CK2α was attached and reacted at room temperature for 1 hour. After washing six times with washing buffer solution, 0.1 ml of His-Tag antibody conjugated with horseradish peroxidase (HRP) was conjugated with 1/1000 dilution in PBS solution (Note; BD biosciences, USA) at room temperature. After reacting for 1 hour, the mixture was washed 6 times. After washing, each plate was reacted for 3-5 minutes by adding 0.1 ml of peroxidase substrate aqueous solution containing 1 mg / ml of OPD (o-Phenylenediamine dihydrochloride) to stop the reaction by adding 0.1 ml of 2.5 M sulfuric acid. The absorbance of the aqueous solution was measured at 492 nm.

As a result, it was confirmed that phosphorylated His-Nopp140 binds concentration-dependently to CK2α ( FIG. 3 ), and InsP ₆ inhibited binding to phosphorylated His-Nopp140 and CK2α at a concentration of 25 μM or more ( FIG. 4 ). From the above results, it can be seen that Nopp140 selectively binds to CK2α with a very low binding dissociation constant (Kd = 8 nM), and InsP ₆ has an IC ₅₀ = 25 mM value for the interaction between Nopp140 and CK2α. It turns out that it suppresses.

Example 4 Screening Method of Interaction Stabilizing Material between CK2α and Nopp140

Searching for a material capable of stabilizing the interaction between CK2α and phosphorylated Nopp140 uses a method of measuring the interaction between GST-CK2α and Nopp140 as in <Example 3>.

Specifically, 0.1 ml of GST-CK2α solution at a concentration of 5 μg / ml was placed in a glutathione-coated 96-well plate and adsorbed onto the surface at 4 ° C. for 1 hour. After repeated washing with 0.2 ml wash buffer [20 mM Na phosphate, 150 mM Na chloride, pH 7.4] 4 times, buffer solution containing 20 ml skim milk powder (20 mM sodium phosphate, 150 mM sodium chloride) , pH 7.4) at room temperature for 1 hour, and wash it again with washing buffer 6 times. 50 μl of buffered test solution (0.1 to 50 mM) (20 mM Na phosphate, 150 mM Na chloride, pH 7.4) was added and reacted at room temperature for 10 minutes, followed by 50 μl of phosphorylated His-Nopp140 (5 ㎍ / ㎖, 20 mM Na phosphate, 50 μM InsP ₆ , 150 mM Na chloride, pH 7.4) is added and reacted at room temperature for 1 hour. After washing six times with washing buffer solution, 0.1 ml of His-Tag antibody conjugated with horseradish peroxidase (HRP) (1/1 000 dilution in aqueous PBS solution, Main; BD Biosciences) at room temperature for 1 hour. The reaction was washed six times. After washing, each plate was reacted for 3-5 minutes by adding 0.1 ml of an aqueous solution of peroxidase substrate containing 1 mg / ml of OPD (o-Phenylenediamine dihydrochloride), and then stopped by adding 0.1 ml of 2.5 M sulfuric acid. The absorbance of was measured at 492 nm.

Bioactive materials libraries were used to search for substances that could stabilize the interaction between CK2α and phosphorylated Nopp140. The search described above was performed by taking 1 μl of the test compound from a library (Microsource, NINDS collection) in which a total of 1040 bioactive substances were dissolved in DMSO (dimethylsulfoxide) at a concentration of 10 mM. As a result, when searching for 80 compounds containing mitoxanthrone, the search results as shown in FIG. 5 were obtained. It can be seen that in the presence of InsP6, mitozantron effectively stabilizes the interaction between CK2α and phosphorylated Nopp140 at the concentration used for screening.

Example 5 Inhibition of CK2α Activity Using Mitoxanthrone

100 units of CK2 (NEB, England), 100 μM InsP6, 100 μM ATP, 1 μM [γ- ³² P] on pH 7.5 reaction buffer consisting of 20 mM Tris-HCl, 50 mM KCl, 10 mM MgCl ₂ , final concentration ATP (100 cpm / pmol), 4 μg phosphorylated hNopp140, 10 μg casein, and mitoxanthrone were mixed at 1 to 50 μM, respectively, and reacted at 30 ° C. for 30 minutes. To the sample was added SDS gel sample buffer solution (final concentration 12 mM Tris-HCl pH 6.8, 5% Glycerol, 0.4% SDS, 2.8 mM 2-mercaptodthanol) and boiled for 5 minutes at 95 ℃ to stop the reaction and 12% SDS polyacrylic Electrophoresis was performed for 1 hour at 160 V using an amide gel (polyacrylamide gel) (Daniel et al., Protein methods, 1996, 108-126). After the electrophoresis, the gel was dried using a gel dryer (Bio-Rad, USA), and then imaged to the degree of phosphorylation using FUZIX BAS 2000 (Fuji, Japan).

As a result, mitoxantrone increased the amount of His-Nopp140 binding to GST-CK2α in the presence of InsP _{6 with} increasing concentration ( FIG. 7 ). In addition, the mitoxantrone stabilized the binding between Nopp140 and CK2 even in the presence of InsP ₆ to decrease the activity of CK2 ( FIG. 8 ). Specifically, the activity of CK2 was measured using α-casein and γ-P ³² ATP as substrate, and the result of measuring the amount of α-casein with P ³² attached to the isotope by autoradiography. Although, mitoxanthrone maintains the activity of CK2 inhibited by Nopp140 in the presence of InsP ₆ ( Fig. 8 (a) ), mitoxanthrone alone affects the activity of CK2 itself. Was not reached ( FIG. 8 (b) ).

1 is a schematic diagram of the mechanism of regulating CK2 activity by Nopp140, wherein the state of activated CK2 and the interaction of Nopp140 with CK2 activity is inhibited, and the process of inhibiting the interaction of Nopp140 and CK2 by InsP ₆ It is a schematic that simulates

2 is a graph measuring binding affinity between His-Nopp140 and GST-CK2α, a fusion protein containing an active site of CK2,

3 is a graph measuring the activity of InsP ₆ inhibiting binding between His-Nopp140 and GST-CK2α,

4 is a schematic diagram of a method for measuring the interaction of phosphorylated His-Nopp140 and GST-CK2α,

5 is a graph of searching for a bioactive material library using a system for measuring interaction between His-Nopp140 and GST-CK2α.

FIG. 6 is a structural formula of mitoxantrone, a compound that is active by stabilizing the interaction between His-Nopp140 and GST-CK2α as a compound detected by the interaction measuring system of His-Nopp140 and GST-CK2α,

FIG. 7 shows the increase in the amount of His-Nopp140 binding to GST-CK2α in the presence of InsP _{6 with} increasing concentrations of mitoxantrone, a compound detected by His-Nopp140 and GST-CK2α interaction measurement system. , Is a graph measuring the activity of stabilizing the binding between Nopp140 and CK2α,

Figure 8 (a) and 8 (b) is an electrophoresis photograph showing the degree activity which reduces the CK2 activity to stabilize the bond between Nopp140 and CK2 of my Tozan anthrone (mitoxantrone) the searched compound in the presence of InsP ₆ , wherein FIG. 8 (a) is My Tozan torch is in the presence of a CK2 a by Nopp140 activity is inhibited InsP ₆ Fig electrophoresis image showing the maintaining in the activity is inhibited state, 8 (b) also is My Tozan Tron Is an electrophoretic photograph showing no effect on the activity of CK2 itself (in this case, the activity of CK2 was measured using α-casein and γ-P ³² ATP as substrates, and isotope P ³² was attached. The amount of a-casein added was measured by autoradiography).

Claims (12)

delete delete delete delete delete delete delete delete delete delete Iii) reacting CK2α, CK2α with Nopp140 (human nucleolar phosphoprotein), and a candidate substance for controlling the interaction, and Insitol ₆ (inositolhexakisphosphate); And Ii) treating the reactants with Nopp140; and enhancing the inhibitory activity of CK2α by Nopp140 using a screened mitoxantrone. 12. The method of claim 11, a method of enhancing the CK2α inhibitory activity of which comprises, by increasing amounts of His-Nopp140 binding to the inhibitory activity of the GST-CK2α is CK2α under InsP ₆ it exists.

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