WO2014142619A1 - Method for screening therapeutic agent for cancers by using aimp2-dx2 and pl4/arf interaction - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for cancers by using AIMP2-DX2 and pl4/ARF interaction, more specifically, relates to a method for screening an anticancer drug that suppresses the AIMP2-DX2 and pl4/ARF interaction, which includes the steps of: (a) bringing a test substance into contact with a separated AIMP2-DX2 protein or a fragment thereof and a separated pl4/ARF protein or a fragment thereof; and (b) for a measurement to check whether or not the test substance suppresses the interaction of an AIMP2-DX2 protein or a fragment thereof and a pl4/ARF protein or a fragment thereof, selecting the test substance as a first candidate when the substance provides a decrease in the AIMP2-DX2 and pl4/ARF interaction due to a contact with the test substance as compared to a control group which has not been brought into contact with the test substance, and to a pharmaceutical composition for treating cancers which includes as an active ingredient a substance that has been screened by the screening method. The method is for screening an anticancer drug that suppresses the AIMP2-DX2 protein and pw/ARF protein interaction and is effective in manufacturing an anticancer agent by screening anticancer substances.

Description

 【Specification】

 [Name of invention]

 Screening method for cancer drug using the interaction of AIMP2-DX2 and pl4 / ARF

 This application claims the priority of Korean Patent Application No. 10-2013-0027421 (Application No.) filed on March 14, 2013, the entirety of which is a reference to the present application. The present invention relates to a method for screening a cancer therapeutic agent using the interaction of AIMP2-DX2 with pl4 / ARF, and more particularly, (a) isolated AIMP2-DX2 protein or fragment thereof and isolated P14 / ARF protein or its Contacting the fragment with the test substance; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, and the method for screening an anticancer agent that inhibits the interaction between the two proteins. It relates to a pharmaceutical composition for treating cancer comprising the substance screened by the screening method as an active ingredient.

[Technique to become background of invention]

Development of cancer specific markers is required not only for the diagnosis of cancer but also for cancer specific treatment. New treatment (cytotoxic therapies) ever between the written extensively been written in cancer therapy for 5 years since yongdoen four in the first anti-cancer drug shows a strong toxicity to non-specific effects on the ^'wateuna addition to the relatively fast another division rate tumor organ cells, serious There is a problem that causes side effects. In order to overcome the side effects and resistance of the existing anticancer drugs, studies have been conducted to develop therapeutic agents that specifically act on tumor cells using cancer specific markers that appear in the process of cancer cell normalization. The key to cancer targeted therapy, which has emerged as a way to minimize the toxicity of anticancer drugs, is to identify cancer cell specific genes.

 ARF, known as P14 / ARF in humans and pl9ARF in mice, is a tumor suppressor protein. ARF proteins bind to E2F-1, -2 and -3 transcriptional activators to inhibit their transcriptional activity and degrade them via the 26S proteasome pathway. This binding results in a p53-independent ARF activity mechanism that is seen in cell cycle progression and tumor sensitivity.

AIMP2 (ARS-interacting multi-functional protein 2) is the aminoacyl-tRNA sum One of the proteins involved in the formation of aminoacyl-tRNA synthetase (ARSs), also called p38 / JTV-l or p38. The present inventors have shown that AIMP2 is a novel cancer suppressor that enhances the signaling of TGF-β through direct interaction with Smad2 / 3, cancer cell lines and tissues. It was confirmed that AIMP2-DX2, a variant of AIMP2 lacking in AMP2, was specifically expressed in. In addition, the present inventors confirmed that AIMP2 levels are dramatically reduced in AIMP2-DX2-type transfected cells irrespective of TGF-β, resulting in loss of AIMP2 activity in AIMP2-DX2 production (Patent 10-0762995). .

 Although the AIMP2-DX2-knock down model has excellent anticancer effects in benzopyrine-induced lung cancer, the role of AIMP2-DX2 in lung cancer progression is not yet clear. Since AIMP2-DX2 alone does not reliably induce lung cancer, the tumorigenic properties of AIMP2-DX2 may be dependent on other genetic factors.

[Content of invention]

 Problem to be solved

Therefore, while searching for other genetic factors affecting the carcinogenicity of AIMP2-DX2, the present inventors found that pW / ARF is related to AIMP2-DX2 and completed the present invention. Accordingly, an object of the present invention is the step of (a) contacting a test substance with an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, as compared to the control group to which the test substance was not contacted. When the interaction of AIMP2-DX2 with pl4 / ARF is reduced in contact with the test substance, inhibiting the interaction of AIMP2-DX2 with pl4 / ARF, including selecting the test substance as a primary candidate. It is to provide an anticancer screening method. Another object of the present invention is to contact a test substance with a cell or tissue expressing an isolated AIMP2-DX2 protein or fragment thereof and an isolated P14 / ARF protein or fragment thereof; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in the cell or tissue to which the test substance is contacted, wherein the test substance is not contacted. Is an interaction between AIMP2-DX2 and P14 / ARF, comprising the step of selecting the test substance as a primary candidate when the expression of AIMP2—DX2 is reduced in contact with the test substance as compared to the control. It is to provide an anticancer screening method that inhibits. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises a substance screened by the screening method as an active ingredient.

[Measures of problem]

In order to achieve the above object of the present invention, the present invention comprises the steps of: (a) contacting the test substance with the isolated AIMP2-DX2 protein or fragment thereof and the isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2—DX2 protein or fragment thereof and the p / ARF protein or fragment thereof, the test as compared to the control group not contacted with the test substance. An anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF when the interaction of AIMP2-DX2 with pl4 / ARF is reduced in contact with the substance; Provides a screening method. The present invention comprises (a) testing the AIMP2-DX2 separating material protein or of the fragment mat separate pl4 / ARF protein ^or, cells to a fragment thereof string or tissue and on contact with the key in order to achieve all-object of the present invention Is a step; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in the cells or tissues to which the test substance is contacted, wherein the AIMP2- is in contact with the test substance when compared to the control group to which the test substance is not contacted. When the expression of DX2 is reduced, the present invention provides a method for screening an anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF, including selecting the test substance as a primary candidate. In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a substance screened by the screening method as an active ingredient. Hereinafter, the present invention will be described in detail. The present invention comprises the steps of (a) contacting a test substance with an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, when compared with the control group to which the test substance is not contacted. If the interaction of AIMP2-DX2 with pl4 / ARF in contact with the test substance is reduced, selecting the test substance as a primary candidate, inhibiting the interaction of AIMP2-DX2 with P14 / ARF An anticancer drug screening method is provided. The AIMP2-DX2 protein and pl4 / ARF protein are characterized in that it is provided in the form of cells or tissues expressing it.

Step (b) is a two-hybrid method, a co-immunoprecipitation method (c immunoprecipitat ion assay), a co-localization assay, a scintillation proximity assay (SPA), UV or chemical crosslinking. Binding methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance, fluorescence polarization assays (FPA), and in vitro pull-down assays It is characterized in that it is carried out by any one method selected from the group consisting of in vitro pull-down assay. ^In addition, the present invention comprises the steps of (a) contacting a test substance with a cell or tissue expressing the isolated AIMP2-DX2 protein or fragment thereof and the isolated pM / ARF protein or fragment thereof; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in cells or tissues to which the test substance is contacted, wherein the test substance is in contact with the test substance as compared to the control group not contacted with the test substance. When the expression of AIMP2-DX2 is reduced, the present invention provides a method for screening an anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF, including selecting the test substance as a primary candidate. Step (b) comprises co-immunoprecipitation, enzyme-linked unosor bent assay, radioimmunoassay (RI A), immunohistochemistry, Westton blotting ( Western Blotting) and flow cytometry (FACS) is characterized in that it is measured by a method selected from the group consisting of. The AIMP2-DX2 is a variant in which the region of exon 2 is deleted in the AIMP2 protein sequence (312aa version: AAC50391.1 or GI: 1215669; 320aa version: AAH13630.1, GI: 15489023, BC013630.1), and AIMP2 equivalent (amino acid Functional derivatives having a substantially equivalent activity to AIMP2, or a derivative having a substantially equivalent activity to AIMP2 but having a modification that increases or decreases physicochemical properties with a modification by substitution, deletion, insertion, or combination thereof in the sequence) Contains a protein from which the region of axon 2 is deleted. In the present invention, "deleting the axon 2 region" in the AIMP2 protein sequence means that the variant resulting from the partial or total loss of the amino acid sequence of the axon 2 region in the AIMP2 protein forms an AIMP2 protein and a heterodimer, thereby reducing the normal function of AIMP2. It means to disturb. Thus, the AIMP2-DX2 protein may have deleted all of the amino acid sequences of axon 2 of the AIMP2 protein or the amino acid sequence of this region, including some of these regions in exon 1, exon 3, axon 4 or both of these regions, Only part of the amino acid sequence includes the deleted protein.

AIMP2-DX2 proteins and pl4 / ARF proteins of the present invention are also within the scope of the present invention, as well as proteins having their native amino acid sequences, as well as amino acid sequence variants thereof. Variant means a protein in which the natural amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / GIy. In some cases, it is modified by phosphorylation, sulfation, acrylation, glycosylat ion, methylat ion and pansylation (^ 1 ^" 1 3 131 ^ 011). May be.

The AIMP2-DX2 protein or pl4 / ARF protein and variants thereof may be extracted or synthesized in nature (Merrifleld, J. Araer. Chem. Soc .. 85: 2149-2156, 1963) or recombinant based on DNA sequences. It may be prepared by the method (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989). In the case of gene recombination technology, the nucleic acid encoding the protein is inserted into an appropriate expression vector, the host cell is cultured to express the protein in a transformant transformed with the recombinant expression vector, and then Recovering the proteins It can be obtained by the process.

 Preferably, the AIMP2-DX2 protein of the present invention may be represented by the amino acid sequence of SEQ ID NO: 4 or 5, more preferably AIMP2-DX2 may be represented by the amino acid sequence of SEQ ID NO: 4, pl4 / ARF is It may be represented by the amino acid sequence of SEQ ID NO. In addition, the AIMP2-DX2 protein fragment of the present invention may be composed of at least 5 to 100 amino acid sequences including exon 1-3 junction of AIMP2 protein.

 In addition, fragments of the pM / A F protein of the present invention may be represented by the 1st to 29th or 2nd to 29th amino acid sequence of SEQ ID NO: 6. The anticancer screening method comprising the steps (a) to (b) described above,

 (c) contacting the primary candidate selected in step (b) with a protein other than pl4 / ARF and AIMP2-DX2 binding thereto

 (d) the step (c) may further comprise the step of selecting a primary candidate material that does not affect the interaction of the protein other than pl4 / ARF and AIMP2-DX2 binding thereto as a secondary candidate material.

The proteins other than the AIMP2-DX2 are, but are not limited to, p53, MDM2 (Cell, Vol . 92, 713723, March 20, 1998), E2F1 (Oncogene (2001) 20, 1033土1041), • prOgerin (Ceir Cycle 12: 14, 22772290; July 15, 2013), and may preferably be p53. Step (c) is a two-hybrid method, a coimmunoprecipitation method, a co-localizatkm assay, a scintillation proximity assay (SPA). , UV or chemical cross-linking methods, bimolecular interaction analysis (BIA), mass spectrometry ^' -MS, nuclear magnetic resonance (NMR), fluorescence polarization assays (FPA) and in vitro It is characterized in that it is carried out by any one method selected from the group consisting of a pull-down assay (in vitro pull-down assay). As used herein, the term "interaction" means that AIMP2-DX2 and pl4 / ARF proteins directly bind AIMP2—DX2 to inhibit the function of pl4 / ARF. All.

 The term "anti-cancer agent that inhibits interaction" used in the present invention means treating and preventing cancer by inhibiting AIMP2-DX2 from inhibiting the function of pl4 / ARF.

 As used herein, the term "inhibit interaction" includes both inhibiting, alleviating and eliminating the interaction between AIMP2-DX2 and pl4 / ARF. Inhibition of interaction may inhibit the interaction between AIMP2-DX2 and pl4 / ARF itself, but also inhibits the binding of each other by reducing the activity of AIMP2-DX2.

As used herein, the term "anticancer" means inhibiting or preventing the growth of cancer. "Inhibiting or preventing cancer growth" is a concept that includes reducing cancer growth and cancer metastasis as compared to when not treated or treated. "Metastasis" means that tumor cells It refers to a process that spreads to distant parts of the body, as used herein, the term also encompasses cancer that occurs through metastasis.

As used herein, the term “recombinant vector” refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell and comprises an essential regulatory element operably linked to express the gene insert. According to the previous release, P14 / ARF is present in the nucleus and is involved in cell cycle resting and apoptosis. Moreover, inactivation of pl4 / ARF can promote metastatic phenotype of lung cancer. Because the carcinogenic properties of AIMP2-DX2 were similar to those of pl4 / ARF inactivated cancers, we tested the functional association between AIMP2-DX2 and pl4 / ARF. First, the effect of AIMP2-DX2 on pl4 / ARF expression was observed. Transfection of AIMP2-DX2 reduced P14 / ARF expression. However, since the negative control RKIP was not affected by AIMP2-DX2 transfection, it can be seen that AIMP2-DX2 selectively inhibits pl4 / ARF expression (see FIG. 1). Subsequently, the protein half-life was measured after each of the two proteins was removed using si-RNA acting on each protein. Removal of AIMP2-DX2 prolonged the half-life of pl4 / ARF and similarly removal of pl4 / ARF prolonged expression of DX-2 (see FIG. 2). Si-AIMP2-DX2 also promoted intranuclear expression of pl4 / ARF. (See Figure 3).

Based on the presence of AIMP2-DX2 and pl4 / ARF, we examine the binding of the two proteins. Bought. IP analysis using cell lysates and recombinant AIMP2-DX2 revealed an association between pl4 / ARF and His-AIMP2-DX2. However, under the same conditions, GFP-pRb or nucleolin was not associated with AIMP2-DX2 (see Figure 4). In addition, it was confirmed by IF staining that AIMP2-DX2 and P14 / ARF existed together (see FIG. 5). Direct interaction of the two proteins was confirmed by IP assay using recombinant protein. As a result, AIMP2-DX2 was directly bound to pl4 / ARF. (See Figure 6). To identify the binding domains of the two proteins, pull-down was performed using the N-terminal fragments of AIMP2-DX and pl4 / ARF. As a result, the N-terminal fragment of pl4 / ARF alone binds to AIMP2-DX. (See FIGS. 16 and 17).

 To confirm that AIMP2-DX2-pl4 / ARF binding was direct and selective, Glutathione S-transferase (GST) -pul 1 down was performed. In the GSP-pull down assay using p53 ol, the interaction with AIMP2 as well as AIMP2-DX2 was observed. This result is in agreement with previous findings that AIMP2 and AIMP2-DX2 bind directly to p53. In contrast, GST-pl4 / ARF shows selective interaction with AIMP2—DX2 (see FIG. 7).

pRb deficiency is essential for small cell lung cancer (SCLC) progression, and pRb is known to be involved in cell differentiation and proliferation. Therefore, we examined how AIMP2-DX2 affects pRb function. When AIMP2-DX2 was transfected, the nuclear pRb was released into the cytoplasm. (See Figure ^' 8)-. And this, again, check the connection through the cell ^fractions', AIMP2 DX2 has confirmed that interferes with pRb expression in the nucleus. (See FIG. 9)

As a result of observing whether the result was due to the binding between AIMP2-DX2 and pRb, pRb interacted with pl4 / ARF rather than AIMP2-DX2 (see FIG. 10), and _P 14 / ARF by GST-pull down assay. Was found to be selectively associated with wild type pRb rather than mutant (see FIG. 11). In addition, the intracellular location of the mutant pRb was unaffected by AIMP2-DX2 (see FIG. 12), and the results show that pl4 / ARF is required for pRb's intracellular placement.

To examine the molecular correlation between AIMP2-DX2 and pRb-pl4 / ARF, we observed the effect of AIMP2-DX2 on the binding of pRb and P14 / ARF. In this experiment, it was confirmed that AIMP2-DX2 prevented the interaction between P14 / ARF and pRb (see FIG. 13), and to confirm this, after removing AIMP2-DX2 from the A549-pl4 / ARF transfectant, expression of pRb was suppressed. As a result, expression of P14 / ARF and pRb increased (see FIG. 14). These results show that AIMP2-DX2 inhibits pRb function through pl4 / ARF blockade. Therefore, it was found that the inhibitors of AIMP2-DX2 and pl4 / ARF binding have anticancer effects.

ELISA-based screening for the selection of inhibitors specific for P14 / ARF-DX2 binding resulted in the selection of primary candidates that inhibit more than 70% of pl4 / ARF-DX2 binding (see 18-20). ). P14 / ARF-DX2 specific binding inhibitor that does not affect p53-pl4 / ARF binding by confirming by GST pull down assay whether the primary candidate inhibits p53-pl4 / ARF pair binding in addition to DX2-pl4 / ARF Could be screened (FIGS. 21 and 22). In addition, the GST-pull down assay confirmed that the candidate selected as a pl4 / ARF-DX2 specific binding inhibitor inhibited the _P 14 / ARF-DX2 binding, and actually inhibited the binding of pl4 / ARF-DX2. It could be confirmed (see FIG. 23).

 Therefore, it can be seen that the screening method of the present invention can effectively select pl4 / ARF-DX2 specific binding inhibitors.

 It provides a pharmaceutical composition for preventing or treating cancer, comprising the substance screened by the screening method of the present invention as an active ingredient.

 The composition of the present invention comprises an effective amount of the substance screened by the screening method of the present invention or a pharmaceutically acceptable carrier of the substance. As used herein, the term “effective amount” means an amount sufficient to exert the therapeutic efficacy of cancer.

- ^i, a pharmaceutically pharmaceutical less contained in eu compositions of the balttang - as carrier duty is commonly used at the time ^o preparation ^■, carbohydrate-type compound (e.g., lactose, amylose, dextrose, sucrose, sorbitan Mold, mantle, starch, cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, salt solution, alcohol , Gum arabic, vegetable oils (e.g. corn oil, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, stearic acid Nesium, mineral oil, and the like, but is not limited thereto.

 In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like. Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response. Ordinarily, the skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the invention, a suitable daily dosage is 0.0001-100 mg / kg body weight. Administration may be once a day or may be divided several times.

 The pharmaceutical composition of the present invention may be formulated using a pharmaceutically acceptable carrier and / or excipient, in unit dosage form, according to a method which can be easily carried out by those skilled in the art. It can be made or prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or an aqueous medium, or may be in the form of axes, powders, granules, tablets, or tablets, and may further include a dispersant or stabilizer.

 In the pharmaceutical composition of the present invention, AIMP2-DX2 and

Inhibitors of interaction of P14 / ARF can be a variety of substances. For example, the active ingredient includes a single compound, a combination of compounds (eg, a natural extract or a cell or tissue culture), an antibody or a peptide.

【Effects of the Invention】

The eu -ΑΊΜΡ2- DX2 protein and pl4 / ARF danbek query ^"method for cleaning a disc anti-cancer agent for inhibiting the interaction, it is effective in the manufacture of a cancer by screening for anti-cancer substances.

[Brief Description of Drawings]

 FIG. 1 shows the expression level of pl4 / ARF following AIMP2-DX2 transfection. (EV: Vector not including AIMP2-DX2 gene / Myc-AIMP2-DX2: Contains AIMP2-DX2 gene tagged with Myc) Vector)

 Figure 2 is a result of measuring how the removal of AIMP2-DX2, pl4 / ARF affect each other half-life. (si-Count: control group, si-AIMP2-DX2: experimental group inhibited AIMP2-DX2 expression, si-pl4 / ARF: experimental group inhibited pl4 / ARF expression, CHX: chlorhexidine,-, 2,7 (hr): CHX Time processed)

 Figure 3 looks at the positional shift of pl4 / ARF to the nucleus following AIMP2-DX2 removal. (Green: pl4 / ARF, blue: nucleus, 1.5, 2.5: concentration treated with si-AIMP2-DX2)

4 shows selective binding between AIMP2-DX2 and pl4 / ARF with AIMP2-DX2 immunoprecipitation. (1 ?: 3- ^ 2-02: group in which AIMP2-DX2 was precipitated, Sup: group in which supernatant liquid was separated (group not combined with AIMP2-DX2), GFP-EV, GFP-pl4) , GFP-pRb, GFP-nucleolin, pRb + l4, Nucleol in + pl4: each protein expressed in HEK293 cells)

 Figure 5 shows the presence of AIMP2-DX2 and pl4 / ARF in the nucleus by immunofluorescence staining.

 Figure 6 is the result of observing the interaction between proteins made AIMP2-DX2 pl4 / ARF directly recombinant protein. (Sup: Group separating sediment supernatant (Group not associated with AIMP2-DX2), IP: GST: Group precipitated with GST antibody)

 7 is a result confirming the specific interaction of AIMP2-DX2 and pl4 / ARF through GST-pull down.

 8 shows the results of observing with immunofluorescence that AIMP2—DX2 influences the migration of pRb. (EV: HCT116 cells transfected with vector containing no AIMP2-DX2 gene, AIMP2-DX2: HCT116 cells transfected with vector containing AIMP2-DX2 gene) FIG. 9 shows Western pRb expression by cell fraction. This is the result confirmed by the blot. (pRb (S.E.): Short Expose / pRb (L.E.): Long Expose)

 Fig. 10 shows the result of confirming that pl4 / ARF and pRb bind to each other by Western blot. (Input: cell lysate)

11 is a result of confirming that ^", that interacts with ^pRb, P14 / ARF is the wild type.

 12 is a result of confirming that two mutant pRb is present in the cytoplasm irrespective of whether AIMP2-DX2 is expressed by immunofluorescence.

 13 shows the results demonstrating that AIMP2-DX2 inhibits the interaction of pl4 / ARF with pRb.

 14 is a result showing that reduction of AIMP2-DX2 increases the expression of pl4 / ARF and pRb.

 15 shows that pRb and pl4 / ARF migrate to the nucleus without AIMP2-DX2.

 16 shows the results of confirming the specific interaction of AIMP2-DX2 with pl4 / ARF through His-DX2 Co-immunoprecipitation. (pl4-F: pl4 / ARF full length, pl4-N: P14 / ARF N-terminal part)

It is a schematic diagram which shows that AIMP2-DX2 binds to the N-terminal site of pl4 / ARF. 18 is a schematic diagram showing a pl4 / ARF-DX2 specific inhibitor screening method. 19 shows a method for finding a test substance that inhibits pl4 / ARF-DX2 binding using ELISA.

 20 shows the results of ELISA-based screening of test substances.

 FIG. 21 is a GST pull down result confirming pl4 / ARF-DX2 binding inhibition of primary candidates selected by ELISA-based screening.

 FIG. 22 shows specificity test results confirming that primary candidates selected through ELISA-based screening inhibit protein binding other than P14 / ARF—DX2.

23 is a GST pull down result confirming the inhibition of _P 14 / ARF_DX2 binding of the candidate material selected by the specificity test.

[Specific contents to carry out invention]

 Hereinafter, the present invention will be described in detail.

 However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments.

Experimental Method

1. Cells and reagents

 Cells HEK293 and HCT116 were purchased from American Type Culture Collection (ATCC, Manassas, VA). NCI-H23 cells were obtained from Prof. Kim Sung-hoon of Seoul National University. The medium contains RPMI— 1640 or DMEM containing 10% FBS and antibiotics. Used. Common reagents were purchased from Sigma (St Louis, MO, USA) and chemical inhibitors were purchased from Calbiochem (San Die, CA, USA).

2. Western blot analysis

Protein extraction from cells was performed using a radioimmunoprecipitation assay (RIPA; 150 mM NaCl, 25 mM Tris-Cl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture). After cell lysate in the buffer solution, it was stored at 95 ^° C for 7 minutes. Samples were subjected to Western blot after SDS-PAGE. The membrane to which the protein was transferred was incubated with the primary antibodies. The antibodies used are shown in Table 1 below. The membrane was washed for 5 minutes and then incubated with HRP-conjugated 2nd antibody for 1 hour. stomach The positive membrane was washed three times for 5 minutes.

 [Table 1]

Antibodies used.

3. Recombinant protein, immunoprecipitation, GST pull-down

 The pl4 / A F fragment (Full-length) was ligated to the Hind II site of the pGEX-TEV vector prepared by adding the TEV protease cleavage site to the pGEX-4TL site. Recombinant protein is expressed in GST binding form in E. coli strain BL2KDE3). Proteins are separated by glutathione affinity chromatography. The amino acid sequence of the isolated pl4 / ARF is shown by SEQ ID NO: 6.

To confirm direct eye binding of the two proteins, place them in agarose-bead conjugated GST (negative control) or GST-proteinol RIPA buffer at 4 ^° C for 1 hour. Immunoprecipitation (111111111: 1013 ^ (^ 1) 3 011, IP) analysis was performed with cell lysates or recombinant proteins in RIPA buffer. All cell lysates were incubated with suitable antibodies for 2 hours at 4 ^° C and then mixed with A / G agarose beads ᅳ conjugaed ^' secondary antibody (Invitrogen, Carlsbad, GA, USA) for 2 hours. After incubation, the mixture was washed twice with RIPA buffer, and the precipitated protein was confirmed by Western blot analysis.

4. Transfection and si-RNA of Mammalian Expression Plasmids

 Mammalian expression plasmid encoding GFP-pl4 / ARF (attach GFP to the N terminus of pl4 / ARF) or GFP-pl4 / ARF-N (N-terminal region) (Cancer Research UK, London Research Institute), His-AIMP2 , His ᅳ AIMP2-DX2 vector (Seonghoon Kim, Seoul National University) was prepared. When the GFP-pl4 / ARF or GFP-pl4 / ARF-N were expressed, pl4 / ARF protein was expressed from the second amino acid sequence using the start codon of the GFP protein.

 Wild type and two variant pRb (567L, 661W; point mutant; Sellers, 1998) were purchased from Addgene (Cambridge, USA).

Si-RNAs that interfere with each gene are listed in Table 2 below. Transfection was performed for 24 hours using Jet-pei (Polypi us Trans feet ion, New York, NY) reagent It became. In short, the cells sown a day ago were washed with PBS and incubated with DNA / Jet-pei mixture for 4 hours under serum-free conditions. The control is a sequence without a target.

 Table 2

si-RNA sequence

 5. Immunofluorescence Staining

In the coverslip, cells are fixed with 100% MeoH at -20 ^° C for 1 hour. After washing with PBS, nonspecific reaction was blocked with PBS buffer (non-related Goat antibody (1: 500)). Cells were then incubated with primary antibodies (1: 50-1: 200; overnight at 4 ^° C) and Rhodamine-conjugated secondary ant i bodies (room temperature, 1 to 12 hours). DAPI, 6—diamidino-2-phenylindole) was used for nuclear staining. After washing with PBS, mounting solut ion (Vector Laboratories (Bur 1 ingame, CA), H. 5501) was dropped on the coverslip and subjected to fluorescence microscopy (Zeizz).

6. Screening pl4 / ARF-DX2 Specific Inhibitors

<6-1> chemical library

 Test materials used for screening are described in Synthetic Compounds and Natural Products Libraries (Lee, SH et al., Oncogene 29, 4576-4587, 2010; Lee, SH et al., P53, Oncogene 28, 2005-2014, 2009). The 8000 chemicals provided by the Korean Chemical Society were used.

<6-2> enzyme immunoassay

To find inhibitors that inhibit DX2 binding of P14 / ARF, an enzyme immunoassay-based screening system was made. His-DX2 recombinant protein was immobilized with 1% paraformaldehyde in a 96-well plate. After the plates were dried and washed, the final concentration of the O.lmM chemicals and the GST-pl4 / ARF protein were incubated. After 1 hour, the plate was washed with Tris-buf fered saline-Tween20 and incubated with anti-GST antibody (1: 10,000) for 30 minutes and anti-mouse IgG-HRP (1: 50,000) for 1 hour. After washing twice, the plate 3,3 ^{', 5,5'} - Incubated with tetramethylbenzidine solution (Calbiochem) and stop solution (IN H ₂ S0 ₄ ). Thereafter, absorbance at 450 nm was measured using an ELISA reader.

Based on ELISA results, chemicals that inhibit at least 7 pl4 / ARF-DX2 binding are identified _. P14 / ARF-DX2 inhibitors were selected as primary candidates. Among them, other known chemicals were excluded by screening. Among the primary candidates, pl4 / ARF-DX2 specific binding inhibitors were selected by excluding non-specific inhibitors that inhibit the binding of p53-pl4 / ARF through the GST pull down assay.

<Experiment Result>

1. AIMP2-DX2 regulates the expression of pl4 / ARF.

After dividing AIMP2-DX2 into the group transfected and the group not transfected, _P 14 / ARF expression was measured by Western blot, and the results are shown in FIG. 1. In FIG. 1, it can be seen that transfection of AIMP2-DX2 reduced pl4 / ARF expression. On the other hand, RKIKRaf kinase inhibitory protein / cancer inhibitory function used as a negative control was expressed regardless of AIMP2-DX2 type vaginal injection. Therefore, AIMP2-DX2 appears to selectively inhibit pl4 / ARF.

 In addition, si- 腿, which acts on AIMP2 3X2 and pl4 / ARF, respectively, inhibited the expression of proteins, and then treated with CHX (Cycloheximide) every 0, 2 and 7 hours to observe the half-life of the expression level of the remaining proteins. The result is shown in FIG. Removal of AIMP2-DX2 extended the half-life of pl4 / ARF, and pl4 / ARF removal extended the half-life of AIMP2-DX2.

In addition, it was observed by immunofluorescence staining that AIMP2-DX2 removal promotes the expression of _P 14 / ARF in the nucleus, which can be seen in FIG. 3.

2. AIMP2—DX2 and pl4 / ARF combine directly.

We examined whether AIMP2-DX2 and pl4 / ARF directly bind and interact. HEK293 cell lysates expressed by GFP-pl4 / ARF were incubated with Histidine-labeled AIMP2-DX2 (His-AIMP2-DX2). After the culture was immunoprecipitated with Histidine antibody, it was confirmed by GFP antibody whether the precipitated protein contained pl4, pRb (cancer inhibitor protein), nucleoline (cancer inhibitor inhibitor protein). As a result, only pl4 was found in the precipitated protein. All. On the other hand, after mixing with AIMP2-DX2, all of pl4, pRb and nucleolin were found in the supernatant except the precipitate (see Fig. 4). Thus it can be seen that AIMP2-DX2 binds to pl4.

 It was confirmed by immunofluorescent staining (GFP-pl4 / ARF: green / recombinant protein Myc-AIMP2-DX2: red of HEK293 cells) that AIMP2-DX2 and pl4 / ARF were present in the same place. The results are shown in [FIG. 5].

 Two proteins were prepared as recombinant proteins to see if AIMP2-DX2 and pl4 / ARF bind. AIMP2-DX2 was labeled with His (His-AIMP2-DX2) and pl4 / ARF was labeled with GST (GST-pW). As a result of immunoprecipitation of GST antibody, His-AIMP2-DX2 was not detected when added alone, but when GST-pl4 was added. Thus it can be seen that AIMP2-DX2 binds to pl4 / ARF (see FIG. 6).

 In addition, to confirm that the AIMP2-DX2-pl4 / ARF binding is direct and selective, GST pulldown was performed using p53. p53 has been shown to interact with AIMP2-DX2, AIMP2. In the results of GST-p53, protein was detected after pull-down and His immunoprecipitation, regardless of AIMP2-DX2 and AIMP2. However, in GST-pl4 only when His-AIMP2-DX2 was added, His immunoprecipitation was performed. Protein was detected.

To identify the binding domains of P14 / ARF and AIMP2-DX2, cell lysates transfected with pl4 / ARF full-length or N-terminal sites were analyzed by His DX2 C0-i ^' mm ¾) pfecipitatiori. As a result, it was confirmed that AIMP2-DX2 binds to the N-terminus of pl4 / ARF, and AIMP2 does not bind (see FIG. 16). AIMP2-DX2 is the deletion of the exon 2 region of AIMP2, the AIMP2-DX2 domain corresponding to the site where the axon 1 and 3 regions of AIMP2 are linked to the pl4 / ARF N-terminal region (see Figure 17).

3. pRb is inhibited by AIMP2-DX2 via pl4 / ARF.

 pRb deficiency is essential for small cell lung cancer (SCLC) progression, and pRb is known to be involved in cell differentiation and proliferation. Therefore, we examined how AIMP2-DX2 affects pRb function. AIMP2-DX2 and pRb vectors were transfected into HCT116 cells for 24 hours. After fixation, the cells were anti-pRb (green) and DAPI stained (blue). As a result, the position of pRb moved from the nucleus to the cytoplasm by AIMP2-DX2 (see FIG. 8).

The transfected HCT116 cells were fractionated and subjected to Western blot in each fraction. As a result, AIMP2-DX2 reduced the expression of pRb in the nucleus (see FIG. 9). In order to examine the binding of AIMP2-DX2 and pRb, pRb and pl4 overexpressed HEK293 cell lysates were incubated with His labeled AIMP2-DX2 recombinant protein. Thereafter, after co-immunoprecipitation with anti-pRb antibody, Western blot was performed. As a result, pRb did not bind to AIMP2-DX2 but directly to pl4 / ARF (see FIG. 10).

 Whether the pRb to which P14 / ARF binds was wild-type or mutant, GST-pull down assay revealed. Agarose bean conjugated GST-pl4 / ARF was incubated with HEK293 cell lysates overexpressed with wild type and mutant pRb (567L, 661W), respectively, and binding was tested by Western blot. GFP-pRb was used as a positive control. It was confirmed that P14 / ARF interacts with wild type pRb but not with variant (see FIG. 11). In addition, as a result of immunofluorescence staining by transfection of the two variant vectors into HCT116, two variant types were present in the cytoplasm regardless of AIMP2-DX2 overexpression (see FIG. 12).

 To determine whether AIMP2-DX2 inhibited the binding of pl4 / ARF and pRb, immunoprecipitation analysis was performed on HEK293 cell lysates expressing His-AIMP2-DX2 and Myc-pl4 / ARF + pRb. As a result, it was confirmed that AIMP2-DX2 inhibited the interaction between pl4 and pRb (see FIG. 13). After transfection of A549 cells with pl4 / ARF, treatment of si_AIMP2-DX2 to remove endogenous AIMP2-DX2, immunoprecipitation analysis showed that the reduction of AIMP2-DX2 increased pl4 and pRb ( See FIG. 14). Furthermore, GFP-pW / ARF was transfected into NCI-H23 cells (human lung cancer cell line expressing endogenous AIMP2-DX2) and treated with si-AIMP2-DX2, followed by IF. It was confirmed that the deficiency promoted the migration of pRb and pl4 / ARF into the nucleus (see FIG. 15).

4. pl4 / ARF-DX2 binding specific inhibitors were selected.

ELISA-based screening was performed to select inhibitors specific for P14 / ARF-DX2 binding. After screening chemicals that inhibited more than 70¾> of pl4 / ARF-DX2 binding by 9200 ELISAs, the screened material was removed from the candidates (see 18-20). Fourteen candidates obtained through the above process were confirmed to inhibit _P 53-pl4 / ARF pair binding or DX2-pl4 / ARF pair binding by GST pull down assay. As a result, it was confirmed that only five candidates (D12, F6, F9, 889-A4, SLC36) are pl4 / ARF-DX2 specific binding inhibitors that do not affect p53-pl4 / ARF binding (FIG. 21 and 22). In addition, GST-pull down assay confirmed that SLC36, which was selected as a pl4 / ARF-DX2 specific binding inhibitor, inhibited pl4 / ARF-DX2 binding. It was confirmed that the inhibition of the binding (see Fig. 23).

Industrial Applicability

 By screening an anticancer agent that inhibits the interaction of AIMP2-DX2 protein and pl4 / ARF protein, there is an industrial applicability since an anticancer agent can be selected to produce an anticancer agent.

Claims

[Patent Claims]  [Claim 1]  (a) contacting a test substance with an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And  (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, the test substance when compared to the control group to which the test substance is not contacted. Screening anticancer agents that inhibit the interaction of AIMP2-DX2 with pl4 / ARF, comprising the step of selecting the test substance as a primary candidate when the interaction of AIMP2-DX2 with P14 / ARF is reduced in contact with Way. [Claim 2]  The method of claim 1, wherein the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof are provided in the form of cells or tissues expressing the same. [Claim 3]  According to claim 1, wherein the AIMP2-DX2 protein is represented by the amino acid sequence of SEQ ID NO: 4 or 5--, the P14 / ARF protein is characterized in that intended to be represented by the amino acid sequence of SEQ ID NO-6 Anticancer Screening Method. [Claim 4] The method of claim 1, wherein step (b) comprises: a two-hybrid method, a co-immunopreci itat ion assay, a co-localizat ion assay, a scintillation proximity assay: SPA), UV or chemical cross-linking methods, bimolecular interaction analysis (BIA), mass spectrometry ^' -MS, nuclear magnetic resonance (NMR), fluorescence polarization assays' FPA And in vitro pull-down assays. [Claim 5] (a) contacting a test substance with a cell or tissue expressing an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And  (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in cells or tissues to which the test substance is contacted, wherein the AIMP2- is in contact with the test substance when compared to the control group to which the test substance is not contacted. If the expression of DX2 is reduced, the screening method for screening the anticancer agent comprising the step of screening the test candidate as a primary candidate, inhibiting the interaction of AIMP2-DX2 and pl4 / ARF. [Claim 6]  The method of claim 5, wherein the AIMP2-DX2 protein is represented by the amino acid sequence of SEQ ID NO: 4 or 5 and the pl4 / ARF protein is represented by the amino acid sequence of SEQ ID NO: 6. [Claim 7]  6. The method of claim 5, wherein step (b) comprises: co-immunoprecipitat ion, enzyme-1 inked immunosorbent assay, radioimmunoassay (RIA), immunohistochemistry, western blot. The method characterized in that the measurement by a method selected from the group consisting of Western Blotting and flow cytometry (FACS) [Claim 8]  A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a substance screened by the screening method according to any one of claims 1 to 7. [Claim 9]  According to claim 8, wherein the cancer is colon cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, chorionic cancer, ovarian cancer breast cancer, thyroid cancer , Brain cancer, head and neck cancer, malignant melanoma, lymphoma, aplastic anemia and hematologic cancer, characterized in that the pharmaceutical composition is selected from the group consisting of. [Claim 10] The method of claim 1, wherein the fragment of the pW / ARF protein is 1 to 29 of SEQ ID NO: 6 Anticancer drug screening method characterized in that the first amino acid sequence. [Claim 111]  The method of claim 1, wherein the fragment of the P14 / ARF protein is anticancer drug screening method, characterized in that represented by the second to 29th amino acid sequence of SEQ ID NO: 6. [Claim 12]  The method of claim 1 or 5, wherein the screening method  (c) contacting the primary candidate selected in step (b) with a protein other than pM / ARF and AIMP2-DX2 binding thereto; And  (d) selecting a primary candidate as a secondary candidate that does not affect the interaction of proteins other than pl4 / ARF and AIMP2-DX2 bound thereto in step (c) An anticancer screening method that inhibits the interaction of AIMP2-DX2 with pl4 / ARF. [Claim 13]  The method of claim 12, wherein the protein other than AIMP2-DX2 is one selected from the group consisting of p53, MDM2 (Mouse double minute 2), E2FKE2F transcription factor 1), and progerin. [Claim 14]  13. The method of claim 12, wherein step (c) comprises a two-hybrid method, a co-immunoprecipitation method, a co_localization assay, a scintillation proximity assay. : SPA), UV or chemical cross-linking methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), NMRCnuclear magnetic resonance, fluorescence polarization assays (FPA) and testing A method characterized in that it is carried out by any one method selected from the group consisting of an in vitro pull-down assay.