WO2016013909A1 - Method for screening therapeutic agent for cancer using interaction between ikba and aurkc - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for cancer using interaction between IkBa and AURKC and, more specifically, to an IkBa and AURKC binding inhibitor compound, and a composition for the prevention and treatment of cancer comprising the same as an active ingredient. The present invention is effective in that a therapeutic agent for cancer can be screened at a molecular level and the action of the therapeutic agent can be identified through molecular interaction.

Description

 【Specification】

 [Name of invention]

 Cancer drug screening method through interaction of IkBa and AURKC

 <ι> This application claims the priority of Korean Patent Application No. 10-2014—0094260, filed July 24, 2014, the entirety of which is a reference of the present application,

 <2>

 The present invention relates to a method for screening cancer therapeutic agents through the interaction of IkBa and AURKC. More particularly, the present invention relates to a binding inhibitor compound of ikBa and AURKC and a composition for preventing and treating cancer comprising the same as an active ingredient. .

 <4>

 Background Art

 <5> IkBa (nuc lear factor of kappa light polypeptide gene enhancer in B eel I s inhibitor, alpha) protein is a type of control subunit that inhibits NF- κ Β activity. IkBa binds to the nuclear position signal (NLS) site of NF-kB to mask the nuclear position signal so that NF-kB remains inactivated in the cytoplasm. IkBa also prevents the NF—kB translation factor from binding to the DNA needed for NF-kB to function. IkBa protein has been found to have IkBa protein mutations in some Hawkins lymphoma cells. This mutation inactivates IkBa, resulting in chronic activation of NF-kB. Lymphoma tumors have been reported to be directly associated with the development of malignant tumors (Cabannes E et al, Oncogene 18 (20): 3063770).

 <6>

 SerURe / threonine-protein kinase 13 (AURKC) protein is an enzyme encoded by the AUPKC gene (Bernard M et al., Genomi cs 53 (3): 40679). This gene encodes the aurora subfami ly of serine and threonine protein kinase. The encoded protein is a chromosomal transport protein that complexes with Aurora B and the internal centrosome protein and can play a role in tissue microtubules with respect to centrosome and spindle function during mitosis. This gene is overexpressed in some cancer cell lines, indicating that it is involved in tumor signal transduction.

 <8>

On the other hand, the growth, differentiation, and death of cells can be attributed to protein-protein, protein-nucleic acid. It is made by the interaction between high molecular materials. Out-of-cell signals pass through receptors on the cell membrane and pass through the cell's nucleus through several biochemical reactions in the cytoplasm, where they express specific genes. This intracellular delivery of these external signals is achieved by interactions between proteins at different levels. For example, growth factors or cytokines bind to the corresponding receptors on the cell surface, which induces clustering of the receptors. Ligand aggregation by these receptors causes the intracellular domains of the receptors to aggregate together, leading to interactions with various proteins involved in intracellular signaling. This signaling system creates intermediate proteins that can deliver multiple levels of signal through protein kinase phosphorylation, protein phosphatase dephosphorylation, and so on, and eventually the signal is transferred to transcriptional activators. (Helden, CH, Cell 80, 213-223 (1995)). Activated transcriptional promoters bind to DNA and interact with basic transcriptional apparatus such as RNA polymerase that synthesizes mRNA to activate specific genes. Therefore, these interactions control specific transcription, specifically in response to specific tissues, developmental processes and external stimuli. At this time, it can be said that the cause of the disorder is that abnormal interactions such as alteration, inhibition, and promotion between specific proteins are caused by invasion of foreign substances or genetic variation of internally active proteins. Therefore, the research that has been continued because the substance that can control this interaction can provide a method of treatment for the disease caused by it.

<ιο> Cross-linking, affinity chromatography, immunoprecipitation (i mmunoprec ipit at i on, Traditional in vitro methods such as IP) are used, but these methods require the production and separation / purification of proteins, and in vivo, due to problems such as in vitro lye solution and secondary modification of extracted proteins. The disadvantage is that it can provide different information than the actual interaction that takes place.

 <ιι> To complement the shortcomings of these in vitro methods, yeast two hybrids (Y2H), fluorescence resonance energy transfer (FRET), and recombination of split proteins (biniolecular fluorescence com) lementation (Bi-FC) methods have been developed and used.

<12> <13> The researchers performed intracellular intracellular preparations of protein kinase C carriers, first and second markers and markers. The company developed a system that can express whether the expressed target substance and the target substance interact with each other and received patent registration (equivalent number: 10-0948767). This system presents a useful experimental method in that it can confirm the interaction between proteins. However, no previous studies have screened for drug candidates through this system.

 <14>

 [Detailed Description of the Invention]

 [Technical problem]

 Thus, the inventors of the present invention have completed a cancer drug screening method using IkBa and AURKC based on the system for confirming the protein-protein interaction.

Accordingly, an object of the present invention is to provide a method for screening a cancer drug through IkBa and AURKC.

 Another object of the present invention is to provide a compound screened through the cancer drug screening method.

Still another object of the present invention is to provide a composition for preventing and treating cancer, which comprises the screened compound as an active ingredient.

 Another object of the present invention is to provide a use of the screened compound for the manufacture of a prophylactic and therapeutic agent for cancer.

Still another object of the present invention is to provide a method for preventing and treating cancer, wherein the screened compound is administered to an individual in need thereof in an effective amount.

<21>

 Technical Solution

 In order to achieve the above object, the present invention provides a method for screening a cancer drug through IkBa and AURKC.

 In order to achieve another object of the present invention, the present invention provides a compound screened through the cancer drug screening method.

 In order to achieve another object of the present invention, the present invention provides a composition for preventing and treating cancer comprising the screened compound as an active ingredient.

 <25>

Hereinafter, the present invention will be described in detail. <27>

 <28> The present invention

 (A) (i) expressing a fusion protein in which sequentially bound fusion proteins, a first marker, and IkBa, and a fusion protein in which a second marker, AURKC are sequentially coupled, Preparing a cell expressing a fusion protein to which a label, IkBa is sequentially coupled, mobile mothers, second markers, and AURKC are sequentially coupled to each other;

(B) adding a cancer drug candidate;

 (C) allowing the cancer drug candidate to interact with the fusion protein of step (a);

 (D) processing the signal material; And

 (E) confirming the interaction through the distribution of the candidate drug for cancer therapy and the intracellular fusion protein,

 The present invention provides a method for screening a cancer drug, wherein the cancer drug candidate is identified as a cancer drug when it inhibits binding of IkBa protein and AURKC protein.

 <35>

 In order to analyze the interaction between IkBa and AURKC directly and in real time, the present invention used intracellular localization of proteins generated by external stimulation or intrinsic signaling mechanisms. In other words, a mobile parent whose position is moved by an external stimulus or an intrinsic signal transduction operation, a constituent in which the labeling material and the IkBa or AURKC which can be tracked are sequentially designed, and the labeling material and the AURKC or IkBa are A second construct was sequentially designed. The first label and the second label should each contain one different IkBa and one AURKC. For example, if the first component is a carrier, the first marker, and IkBa, the second component should be composed of the second marker, AURKC, and if the first component is the carrier, the first marker, and the AURKC, the second component is: It should consist of two markers, IkBa.

 <37>

 According to the present invention, whether the first component and the second component bind to each other, and whether the first component and the second component interfere with the binding of the first component and the second component to the cancer therapeutic agent. If inhibition of the binding of the first component and the second component is determined as a cancer treatment.

<39>

Mobile hair in the present invention is a site that functions to move the crab 1 to a specific area in the cell. In this case, movement to a specific region within a cell is caused by an external signal. It is possible to migrate haphazardly or intrinsically, and certain regions within the cell represent separate, discrete and identifiable elements present in the cell as intracellular structures. The intracellular specific region may preferably be a membrane structure such as a cell membrane or a nuclear membrane or intracellular organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes, and other intracellular specific regions.

<4i> The "mobile mothers" of the present invention is a protein kinase C (protein kinase C,

PC), cPKC (classic PKC; PKC-alpha, P C-beta, PKC— ga 隱 a), nPKC (novel PKC; PKC-delta, PKC-epsilon, PKC-eta, PKOtheta) It is done.

 They all have a common part called the C1 domain, and their binding to the cell membrane is induced by binding to DAG (diacyl glycerol) or TPA (phorbol ester (PMA)) in the C1 domain. Preferably, the carriers of the present invention may use a variant of PKC, and the variant is more preferably a variant in which the intrinsic phosphorylation activity of PKC is eliminated in order to minimize disturbance caused by the intrinsic signaling mechanism.

 The "first label" of the present invention is GFP (Green Fluorescent Protein),

Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), Monomeric Red Fluorescent Protein (mRFP), Discosoma sp. Red fluorescent protein (DsRed), Cyan Fluorescent Protein (CFP), Cyan Green Fluorescent Protein (CGFP), YFP (Yel low Fluorescent Protein), AzG (Azami Green), HcR (HcRed, Heteractis cr ispa red fluorescent protein) and BFP (Blue Fluorescent Protein).

 The "second label material" of the present invention is different from the one label material of the crab and GFP (Green

Fluorescent Protein (EGFP), Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), Monomeric Red Fluorescent Protein (mRFP), Discosoma sp. Red fluorescent protein (DsRed), Cyan Fluorescent Protein (CFP), Cyan Green Fluorescent (CGFP) Protein), YFP (Yel low Fluorescent Protein), AzGCAzami Green (HFP), HcRCHcRed, Heteractis cr ispa red fluorescent protein) and BFP (Blue Fluorescent Protein).

<45>

As used herein, the term “fusion protein” refers to a protein or polypeptide having an amino acid sequence derived from two or more proteins. It may also comprise a linkage region of amino acids between amino acid moieties derived from. The cell may be a cell of an animal, a plant, a yeast and a bacterium. Preferably, the cell may easily receive a first construct and a second construct, which are introduced from outside of the bacterium, and may be used for intracellular organelles such as cytoplasm and nucleus. Cells with distinct boundaries are preferred. More preferably CHO-kl (ATCC CCL-61, Cricetulus griseus, hamster, Chinese), HEK293 (ATCCCRL-1573, Homo sapiens, human), HeLa (ATCC CCL-2, Homo sapiens, human), SH-SY5Y ( ATCC CRL-2266, Homo sapiens, human), Swiss 3T3 (ATCC CCL-92, Mus musculus, mouse), 3T3-L1CATCC CL-173, Mus musculus, mouse), NIH / 3T3CATCC CRL-1658, Mus musculus, mouse) , L-929 (ATCC CCL-1, Mus musculus, mouse), Rat2 (ATCCCRL-1764, Rat t us norvegicus, rat), RBL-2H3 (ATCC CRL-2256, Rat t us norvegicus, rat), MDCK (ATCC CCL-34, Can is familiar is). In addition, it may be other stem cells, cells extracted from various tissues, and artificially made similar membrane structures. In the present invention, the cells comprising the crab first construct and the second construct may be prepared by molecular biological methods known in the art. Although not limited thereto, the cells are transformed into individual expression vectors capable of expressing the first component and the second component or expression vectors capable of expressing both the first component and the second component, and then the system is expressed in the expression vector. Preferred is a method by which the first and second constructs are expressed. The transformation of each expression vector into cells can be carried out using transformation methods known in the art, such as calcium phosphate, calcium chloride, rubidium chloride, microprojectile bombardment, electroporation, Particle gun bombardment, silicon carbide whiskers, sonication, PEG-mediated fusion, microinjection, liposome mediation (1 iposome-mediated method), magnetic nanoparticle mediated method, and the like. Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory). Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Si lhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and by Ausubel, FM et al. , Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wi ley-lnterscience (1987)).

 <55>

 As used herein, the term “interaction” refers to the proximal state between a particular ligand or compound, or a portion or fragment thereof, and a portion of the second molecule of interest. It may be non-covalent or covalent as a result of hydrogen-bonding, van der Waals interactions, electrostatic or hydrophobic interactions. 12-myri state 13-acetate,

Phorbol ester), TPA (12-ot et r adecanoy lphor bo 1 -13-acetate), PDBu (phorbol 12, 13-dibutyrate), Adenosine triphosphate (ATP), tr idecanoic ac id, arachidonic acid, linoleic acid, DiC8 It is characterized in that selected from the group consisting of 130C937.

 <58>

 The "treatment of signal substance" of the present invention is characterized by treating PM Phorbol 12-myri state 13-acetate, Phorbol ester) at a concentration of 50nM to 5υΜ. More preferably luM. When the concentration of the PMA is less than 50 nM, movement of the mobile mothers using the PKC is not difficult, and when the concentration of the PMA is greater than 5 uM, abnormal phenomena (eg, cell death signal disturbances, etc.) occur due to overtreatment of the chemical.

 <60>

 The "confirming the interaction" is to detect the distribution of the first label and the second label, for example, coverslips containing the cells to be identified.

It may be a method of fixing the coverslip to a perfusion chamber and mounting it on a material stage of a confocal laser fluorescence microscope (Cal Zeiss LSM710) and obtaining images of the component vectors before and after external stimulation.

 <62>

 The present invention provides a compound screened through the cancer drug screening method.

The compound of the present invention may be represented by the following formula (1). [Formula 1]

 The compound of the present invention is characterized by inhibiting IkBa protein and AURKC protein binding.

 The compound of the present invention is characterized by inhibiting the binding of the IkBa protein and AURKC protein to show cancer prevention and treatment effects.

 The "cancer" of the present invention is gastric cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosis, uterine cancer, cervical cancer head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney Cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, hematologic cancer, lymphoma, psoriasis or fibroadenoma. The present invention provides a composition for preventing and treating cancer, comprising an inhibitor of interaction between IkBa and AURKC as an active ingredient. The inhibitor may be represented by the following formula (1).

 [Formula 1]

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceut i Cal Science, recently, Mack Publ i Shing Company, Easton PA. Carrier excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, manny, xylly, erythritol, maltitol, starch, acacia rubber, alginine. , Gelatin, Calcium Phosphate Shampoo, Cellulose, Methyl Cellulose, Microcrystalline Salose Polyvinyl Pyridone, Water, Methylhydroxy Benzoate, Propylhydroxyxbenzoate, Talc, Magnesium Stearate and Mineral oil and the like. When formulating the composition, it is prepared using diluents or excipients such as layering agents, extenders, binders, wetting agents, disintegrating agents, and surfactants which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witemsol, macrogol, twenty-one, cacao butter, laurin butter, glycerogelatin and the like can be used.

 The term “treatment” of the present invention refers to amelioration of a particular disease or disorder symptom, which may include treating such disorder, substantially preventing the development of the disorder, or improving the condition of the subject. . As used herein, the term “treatment” refers to the full spectrum of treatment for a given disorder that afflicts a patient, which alleviates one or most of the symptoms resulting from that disorder, Treating certain disorders or preventing the development of a disorder.

<78>

 Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with a known compound having an effect of preventing and treating cancer or a known compound having an effect of inhibiting cancer metastasis.

 <80>

 <8i> The present invention provides a use of the inhibitor of the interaction between IkBa and AURKC for the manufacture of a prophylactic and therapeutic agent for cancer.

 The present invention provides a method for preventing and treating cancer, comprising administering an effective inhibitor of the interaction between IkBa and AURKC to a subject in need thereof.

<83> The inhibitor may be represented by the following formula (1).

 [Formula 1]

H

Inhibitors of interaction between IkBa and AURKC of the present invention can be administered in an effective amount via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount that exhibits a therapeutic and prophylactic effect or a cancer metastasis inhibiting effect when administered to a patient, and the term 'subject' includes an animal, preferably a mammal, particularly a human. It may be an animal, cells, tissues, organs and the like derived from the animal. The subject may be a patient (pat i ent) in need of treatment. The interaction inhibitor between IkBa and AURKC of the present invention may be administered by itself or prepared and prepared in various formulations as described above, and is preferably administered until a desired effect, i.e., cancer prevention and treatment effect, is obtained. Can be. The compounds of the present invention and their pharmaceutically acceptable salts can be administered by various routes according to methods known in the art. That is, orally or parenterally, such as oral, intramuscular, intravenous, intradermal, intraarterial, intramedullary, intradural, intramural, intranasal, intravaginal, intra rectal sublingual or subcutaneous, gastrointestinal tract, mucosa or respiratory tract. May be administered. Preferably it can be applied directly to the skin. In addition, the compounds of the present invention and pharmaceutically acceptable salts thereof may be administered in a form that is bound to or encapsulated within a molecule that induces high affinity binding to the target cell or tissue. Inhibitors of interactions between IkBa and AURKC of the present invention may be prepared using techniques known in the art, such as sterols (e.g. cholesterol), lipids (e.g. cationic lipids, bivalent or liposomes) or target cell specific binding agents (e.g. Ligands recognized by target cell specific receptors). Suitable coupling or crosslinking agents can include, for example, Protein A, carbodiimide, N-succinimidyl-3- (2-pyridyldithio) propiotate (SPDP) and the like. These formulations are described in Remington's Pharmaceut i cal Science, 15th Edition, 1975, Mack Publ i Shing Company, East on, Pennsyl vani a).

 <93>

 Advantageous Effects

 Accordingly, the present invention provides a method for screening a cancer drug through the interaction of IkBa and AURKC. More specifically, the present invention provides a binding inhibitor compound of IkBa and AURKC and a composition for preventing and treating cancer comprising the same as an active ingredient. The screening method of the present invention can be usefully used in that the cancer therapeutic agent can be easily screened depending on the inhibition of binding of IkBa and AURKC.

 <95>

 [Brief Description of Drawings]

 FIG. 1 is an overexpression of TMD-mRFP-IkBa (first construct) and EGFP-AURKC (second construct) in HEK293T cell line and analyzed by a method using cell imaging (Registration No. 10-0948767) (left) Panel; first component, middle panel; second component, right panel; first component + second component).

 Figure 2 shows pTMD-mRFP-C3 p i asm i d (Crab 1 construct) and EGFP-AURKC (near 12 constructs)

Results of overexpression in HEK293T cell line followed by treatment with 1 M PMA (external stimulus) for 3 minutes (left panel; first construct, middle panel; second construct right panel; first construct + second construct).

 3-98. TMD-niRFP-AURKC (Crab 1 constituent) and EGFP-IkBa (Second constituent) were overexpressed in HEK293T cells and treated with 1 μΜ Ρ Α (external stimulus) for 3 minutes. Results of real-time analysis of protein binding (left panel; first component, middle panel; second component, right panel; first component + second component). 3-I overexpresses pTMD-mRFP—C3 pl asm id (first construct) and EGFP-IkBa (crab construct) to HEK293T cells, followed by treatment of 1 μM PMA (external stimulus) for 3 minutes. Results of real-time analysis of binding (left panel; first component, middle panel; second component, right panel; first component + second component).

 4 shows that TMD-mRFP-IkBa (first construct) and EGFP-AURKC (second construct) were overexpressed in the CH0-K1 cell line, followed by treatment of 1 μΜ ΡΜΑ (external stimulus) for 3 minutes. This is a photo showing the binding of proteins in real time (left panel: first component, middle panel: second component, right panel: first component + second component).

5A to 5D show gastric cancer cell lines AGS (A) and SNU638 b) and colorectal cancer cell lines.

HCT116 (C) and SW620 (D), uterine cancer cell line He la (E), breast cancer cell line MDA-MB-23U), Cytotoxicity was measured in lung cancer cell line A549 (A) and liver cancer cell line HepG2 (H).

 FIG. 6 shows that TMD-mRFP-IkBa (first construct) and EGFP-AURKC (second construct) are overexpressed in HEK293T cells (Panel A) and CH0-K1 cell lines (Panel B) in the same manner and Compound 50 μM was treated for 3 hours. After that, 1 μΜ ΡΜΑ (external stimulus) was treated for 3 minutes to analyze the inhibition of binding of the two proteins in real time (left panel: 1st component, middle panel: 2nd component ᅳ right panel: 1st component + 1st) 2 components).

 FIG. 7 shows a wound healing assay (A), a Transwell migration assay (B) and a soft agar assay by culturing breast cancer cell line MDA-MB-231 cells. As a result of (c), the effect of compounds inhibiting the interaction of IkBa and AURKC on cancer cell proliferation was confirmed.

 <103>

 [Form for implementation of invention]

Hereinafter, the present invention will be described in detail.

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

 <106>

 <Example 1>

 <108> Animal Cell Lines and Transformation

 <109>

 <ιιο> <1-1> Animal cell lines and their culture

 <ιιι> Experiments in the present invention are CHOKKATCC CCL-61, Cricetulus griseus, hamster,

Chinese), HEK293 (ATCC CRL-1573, Homo sapiens, human), MDA-MB-231 (ATCC HTB-26, Homo sapiens, human), A549 (ATCC CC 一 185 Homo sapiens, human), HepG2 (ATCC HB -8065, HepG2, Homo sapiens, human) cell line was used. Culture conditions of animal cell lines used in the present invention was used for cell line culture method of ATCC (American Type Culture Collection), which is the organ of each cell line. However, CH0-K1 was used for F-12 culture, HEK293 and MDA-MB-231 cell lines were used for DMEM culture, A549 and HepG2 cells were used for RPMI 1640, and other culture conditions were used in the same manner. On the other hand, the common culture method of each cell is as follows (detailed culture conditions may vary depending on the purpose of the skilled person). Incubation at pH 7.4 with 25 niM HEPES, 10% FBS (fetal bovine serum, v / v), 100 units / ml penicillin, 100 ug / ml straptomycin In cell (F-12 and DMEM), each cell line was incubated in an incubator maintained at a temperature of 37 ^° C., CO ₂ partial pressure of 5%.

 <112>

 <! 13> <1-2> Transformation of Cell Line

 Intracellular gene introduction method used in the embodiment of the present invention used Fugene HD (Promega), one of the liposome-based methods commonly used, all conditions for gene introduction, such as the concentration of the gene is a manufacturer Follow the instructions. More specifically, the coverslips were placed in 6-plates, and the cells were cultured for one day, and then exchanged with 2 ml of fresh culture without penicillin, streptomycin, or FBS. About one transgenic white sample was added to the culture medium without 0.1 ml of penicillin and streptomycin FBS, followed by complete mixing and addition of 3 Fugene HD reagents. The solution was allowed to stand at room temperature for 15 minutes, then added to each well of a 6-plate containing the coverslips in which the cells were grown, incubated for 4 hours, and 200 μl of FBS was added for transformation for 18 hours. It was.

 <] 15>

 <Example 2>

 <ιΐ7> Design and manufacture of the first and second components

 <118>

 <1! 9> <2-1> Design and Fabrication of First Component

 In the present invention, the first constituent includes a module (mobile mothers) that can move to the cell membrane when the protein evenly expressed in the cytoplasm has been treated with the signal amorphous substance, and can be analyzed using these microscopes. The protein is labeled and bound, and finally consists of a fusion sequence to which the target substance can be bound. The first construct was constructed using restriction enzymes EcoRI / Kpnl (IkBa) and EcoRI / Xinal (AURKC) with IkBa and AURKC in a TMD-mRFP-empty vector. TMD—mRFP-empty vector is a vector comprising a Protein Kinase C mutant (TMD) represented by SEQ ID NO: 5 and mRFP represented by SEQ ID NO: 7.

 <i2i> IkBa is the template for pcDNA3.1-IkBa vector (NCBI Reference Sequence:

NM_020529.2; purchased from Mediscov Inc), and PCR was performed using primers of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, AURKC performed PCR using a PPTB7-AURKC vector (GenBank: BC002363) as a template and primers of SEQ ID NO: 3 and SEQ ID NO: 4. Obtained after PCR amplification using primers Water was prepared by inserting into the EcoRI / Kpnl (IkBa) and EcoRI / Xmal (AURKC) positions of the pTMD-mRFP-C3 vector.

 <122>

 <123> <2-2> Design and Fabrication of Second Component

 The second construct includes a label for analyzing the movement of a target substance having intrinsic properties of binding to a target substance bound to the first component. Crab bicomponent uses green fluorescent protein (EGFP), a fluorescence that is different from the label contained in the first construct. The second construct was constructed using restriction enzymes EcoRI / Kpnl (IkBa) and EcoRI / Xmal (AURKC) from IkBa and AURKC in an EFRP-C3 vector (clontech) of a previously registered patent (e.g. 10-1217718). . <K> IkBa is the template for the pcDNA3.1—IkBa vector (NCBI Reference Sequence:

 M_020529.2; purchased from Mediscov Inc), and PCR was performed using primers of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, AURKC was performed using a PPTB7-AUR C vector (GenBank: BC002363) as a template and using primers of SEQ ID NO: 3 and SEQ ID NO: 4. After PCR amplification using the primers, the obtained product was prepared by inserting into the EcoRI / Kpnl (IkBa) and EcoRI / Xnial (AURKC) positions of the pTMD-mRFP-C3 vector.

 <126>

 <127> <Example 3>

 Intracellular migration of the first and second constructs

 <129> Coverslips in which cells in which the first and second construct vectors are introduced grow

 (coverslip) mounted in a perfusion chamber, mounted on the product of the confocal laser fluorescence microscope (Cal Zeiss LSM710) and image of the composition vectors before and after external stimulation (ΙμΜ Phorbol ester treatment) Obtained.

The confocal laser microscope uses a 488 nni Argon laser (EGFP) and a 543 nm HeNe laser (niRFP) to excite the fluorescent label, and the fluorescent signal generated from each fluorescent label is a band path filter BP505. -530 (EGFP), Long path filter LP560 or BP560-630 (niRFP) were used, and images were obtained after the interference between each fluorescence was completely removed.

As a result, as shown in FIGS. 1, 3, and 4, the red fluorescence caused by the first component vector including the moving mothers (TMD) after the external stimulation using the confocal laser fluorescence microscope is transferred to the cell membrane. However, the green fluorescence by the second material vector for the target material without the mobile hairs is distributed evenly in the cytoplasm as in stimulation. Thus, a 12-element vector is an external stimulus Inevitably, the migration of the second constituent to the cell membrane is necessarily based on the binding of the target substance to the target substance.

 <132>

 <133> <embodiment Γ 4>

 Intracellular Binding Analysis of IkBa and AURKC Using First and Second Constructs

 The interaction of IkBa proteins regulated by TNF-a pha is known to be involved in various cellular signaling mechanisms in cells. Of particular interest among the various mechanisms is that they have a close relationship with provoking inflammation in response to various risk signals occurring in vivo. The binding of AURKC protein interacting with IkBa was performed in real time using living cells.

 The first and second constructs prepared above were transformed into HEK293T and CH0-K1 cell lines, and their fluorescence was confirmed at 0 and 3 minutes.

 As a result, as shown in Fig. 1, the first construct (TMD-mRFP-IkBa) was evenly distributed in the cell at 0 minutes, and then moved to the cell membrane after 3 minutes (left panel in Fig. 1). EGFP-AURKC) was also distributed evenly in the cell at 0 min and then moved to the cell membrane with the first construct after 3 min (panel in FIG. 1). This means that the first component and the second component are combined.

 <138>

 In contrast, the first component is replaced by TMD-mFRP-AU KC and the second component is replaced by EGRP—IkBa.

 HEK2953T and CH0-K1 cell lines were transformed and fluorescence was detected at 0 and 3 minutes.

 As a result, as shown in Fig. 3A, the first construct (TMD-mRFP-AURKC) was evenly distributed in the cells at 0 minutes, and then moved to the cell membrane after 3 minutes (Fig. 3 is the left panel). The two constructs (EGFP-IkBa) were also distributed evenly within the cell at 0 min and then migrated to the cell membrane with the first construct after 3 min (Figure 3-middle panel). This means that the first component and the second component are combined.

 <141>

 <142> <Release> 5>

 <143> Screening Compounds That Inhibit the Binding of IkBa and AURKC

 Using the screening method, the first and second compositions prepared above are prepared.

The cells were transformed into coverslips in which HEK293T and CH0-K1 cells grew. Afterwards, IkBa protein and AURKC protein binding inhibitor candidates were Treated during. The coverslip is secured to the perfusion chamber, mounted on the stage of the confocal laser fluorescence microscope (Cal Zeiss LSM710), for component vectors before and after external stimulation (ΙμΜ Phorbol ester). Image was acquired.

 As a result, the compounds represented by the following formula (1) inhibiting the interaction of IkBa protein and AURKC protein were screened.

 [Formula 1]

 Figure 6 is an analysis of the binding inhibitory effect of the IkBa protein and AURKC protein of the compound TMD-mRFP-IkBa (first component) and EGFP-AURKC (component 2) in the same manner in HEK293T cells and CH0-K1 cell line Overexpressed and treated 50 μM of the compound for 3 hours. Thereafter, 1 μΜ of ΡΜΑ (external stimulus) was treated for 3 minutes to analyze the inhibition of binding of both proteins in real time. It was confirmed that the above compound did not bind the IkBa protein and the AURKC protein, and the red fluorescence reflecting the moving mothers and IkBa moved to the cell membrane, but the green fluorescence reflecting the AURKC protein was located in the cytoplasm. Therefore, it can be seen that the compound represented by Chemical Formula 1 can be used as an inhibitor that inhibits the binding of IkBa and AURKC.

<Example 6>

 Cytotoxicity Measurements of Screened Compounds

 Cytotoxicity of the compounds used in the examples of the invention was used a commonly used CCK8 kit (Dojindo Molecular Technologies) and all conditions were performed according to the manufacturer's instructions. More specifically, cells were cultured in 96-plate for one day, 10, 50, and 100 μΜ were treated with the compound and incubated for 24 and 48 hours, followed by 10 μl of CCK8 solution, and the absorbance was measured at 450 nm for one hour.

As a result, gastric cancer cell lines AGS and SNU638 (FIG. 5, B) and colon cancer cell lines HCT116 and SW620 (C), uterine cancer cell line Hela (MA) ᅳ breast cancer cell line MDA—MB-231 (F), as shown in FIG. Cytotoxicity was measured in lung cancer cell line A549 and HepG2 cell line, and these cancer cells were inhibited by compounds that inhibit the interaction of IkBa and AURKC. Death increased.

 <154>

 <155> <Example 7>

 Inhibition of Cancer Cell Proliferation of Screened Compounds

 <157>

 <158> <7-1> Analysis method of cancer cell migration ability

The cancer cell migration assay was performed using a breast cancer cell line MDA—MB-231 cultured in DMEM containing 10% FBS at 37 ^° C. 5% CO ₂ conditions (FIG. 7A). Cells are incubated for 24 hours in 24 well tissue culture plates (2 × 10 ⁵ cells). Thereafter, the wound is slowly cut across the center of the well using a lnil pipette tip without changing medium. At this time, use the tip upright with the bottom of the well, cut the wound in one direction, and march twice with medium to remove the fallen cells, and then supplement the new medium. In addition, 25 μM of compounds inhibiting the interaction of the plate group IkBa with AURKC were treated and incubated for 48 hours. This is followed by two marches with IX PBS followed by fixation with 3.7% paraformaldehyde for 30 minutes. The fixed cells were stained for 30 minutes by dissolving 1% crystal vial in 2% ethane and observed using a microscope.

As a result, as shown in FIG. 7, the control group (ΟμΜ) without treatment of the compound that inhibits the interaction of IkBa and AURKC in the breast cancer cell line was narrower than that of the treatment with the compound (25μΜ). Confirmed. In the case of the control group, the cells grew on the wounded portion with the tip to fill the space, but when the compound was treated, the cells did not grow on the wounded portion and the space remained. That is, it was confirmed that the compound which inhibits the interaction of IkBa and AURKC inhibits the proliferation of cells.

 <161>

 <162> <7-2> Transwell Transfer Measurement

The transwell migration assay used a breast cancer cell line MDA-MB-231 incubated with 1 FBS of DMEM (FIG. 7B). To prepare the transwell, 0.5% FBS and 2.6η 1 DMEM were placed in the lower part of the 24 well, and 8 well sized transwell was added. Then, cells (1 × 10 ⁵ cells) were added to the prepared transwells. Then, ΙΟμΜ and 25μΜ were added to the compounds that inhibit the interaction between IkBa and AURKC, and the cells were incubated for 2 hours and 30 minutes at 37 ^° C, 5% C0 ₂ , to move down the filter fitted with the cells. It was. After 2 hours and 30 minutes, remove the filter and the remaining cells on the filter The debris was removed using a cotton swab. The cells under the filter were fixed with 5% glutaraldehyde for 10 minutes and stained with 2% ethanol dissolved in 1% crystal violet for 20 minutes. The crystal violet was removed, observed under a microscope and the cell number in the lower part of the filter was measured. At this time, the other parts of the filter were selected and observed and averaged.

 The results are shown through a graph as shown in FIG. 7. IkBa and Breast Cancer Cell Lines

 When treated with a compound that inhibits the interaction of AURKC it was confirmed that the number of moving cells compared to the control. In addition, when 25 μM of compound was added, the number of cells to be moved decreased more than that of ΙΟ μΜ.

 Thus, it was confirmed that cancer cell proliferation was inhibited by a compound that inhibits the interaction between IkB a and AURKC as in <7-1>.

 <166>

 <167> <7 ~ 3> Annual Agar Analysis

The colony morphology was analyzed by culturing the breast cancer cell line MDA-MB—231 in agar medium prepared using agarose by a Sot agar assay (FIG. 7). First, 4% agar was dissolved and kept warm in a 56 ^° C constant temperature bath, and 10% FBS DMEM was kept at 37 ^° C. In order to make a lower layer of agar medium to culture the cells, DMEM 5 [ii l containing 0.75% agar and 10% FBS was placed in a 60 mm culture dish and waited until it solidified. 10% including 0.36% agar to make the next upstairs

Cells were placed in 3 ml of FBS DMEM by 3 X Ιθ 'ce lls, and 25 μM of compounds inhibiting the interaction between IkB a and AURKC were placed on the solid agar medium and allowed to solidify. Mark on the culture plate and incubate for 3 weeks in a 37 ^° C incubator. Then we observed the number and shape of the colonies.

 As a result, it was confirmed that the shape of colonies in the medium containing the compound that inhibits the interaction between IkB a and AURKC was rounder and smaller than the colonies of the control group not treated with the compound. In addition, it was confirmed that the number of colonies when the compound was treated than the control group was not treated. It was confirmed that cancer cell inhibition was inhibited by a compound that inhibited the interaction between IkB a and AURKC.

 <170>

 Industrial Applicability

<i 7i> The present invention relates to a method for screening a cancer therapeutic agent through the interaction of IkBa and AURKC. More specifically, IkBa and AURKC binding inhibitor compound and the active ingredient It relates to a composition for preventing and treating cancer. The present invention is effective in that cancer therapeutic agents can be screened at the molecular level and the action of the therapeutic agents can be known through intermolecular interactions.

Claims

【Scope of Claim】

【Claim 1】

(a) (i) mobile hairs, a first labeling material, a fusion protein to which IkBa is sequentially bound, a second labeling substance, and AURKC are sequentially bound to express a fusion protein or (Π) mobile hairs, a first labeling material, Preparing cells expressing a fusion protein to which IkBa is sequentially bound, mobile hairs, a second marker, and AURKC are sequentially bound to each other;

(b) adding a cancer treatment candidate;

(c) allowing interaction between the cancer treatment candidate and the fusion protein of step (a);

(d) processing the signal material; and

(e) Contains the step of confirming the interaction between the cancer treatment candidate and the fusion protein within the cell;

A cancer treatment screening method characterized in that a cancer treatment candidate is identified as a cancer treatment when it inhibits the binding of the IkBa protein and the AURKC protein.

【Claim 2】

According to claim 1, the mobile hairs include protein kinase C (PKC), cPKC (classical PKC; PKC— alpha, PKC-beta, PKC-ga画 a), nPKC (novel PKC; PKC-delta, A method characterized in that it is selected from the group consisting of P C-epsilon, P C-eta, PKC-theta).

【Claim 3】

According to item 1, the crab 1 labeling substances are GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFPCRed Fluorescent Protein), mRFPCMonomer ic Red Fluorescent Protein), DsRed (Discosoma sp. red fluorescent protein), and CFP. (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent Protein), YFP (Yel low Fluorescent Protein), AzG (Azami Green), HcR (HcRed, Heteract is crispa red fluorescent protein), and BFP (Blue Fluorescent Protein). A method characterized as selected.

[Claim 4]

The method of claim 1, wherein the second labeling material is different from the first labeling material and is GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein), mRFP (Monomer ic Red Fluorescent Protein), DsRed (Discosoma sp. red fluorescent protein), CFP (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent) Protein), YFP (Yel low Fluorescent Protein), AzG (Azami Green), HcR (HcRed, Heteract ispa red fluorescent protein) and

A method characterized in that it is selected from the group consisting of BFP (Blue Fluorescent Protein).

【Claim 5】

The method of claim 1, wherein the signaling substance is PMMPhorbol 12-myri state 13-acetate, Phorbol ester), TPA (12-otetradecanoylphorbol-13-acetate), PDBu (phorbol 12, 13-dibutyrate), ATP (Adenosine triphosphate), A method characterized in that it is selected from the group consisting of tr idecanoic acid, arachidonic acid, linoleic acid, DiC8, and 130C937.

【Claim 6】

The method according to item 1, wherein the treatment of the signal material is treated with PMMPhorbol 12-myri state 13-acetate, Phorbol ester) at a concentration of 50nM to 5uM.

【Claim 7】

A compound screened through the cancer treatment drug screening method of claim 1.

[Claim 8]

The compound according to claim 7, wherein the compound is represented by the following formula (1).

[Formula 1]

【Claim 9】 The compound according to claim 7, wherein the compound inhibits the binding of IkBa protein and AURKC protein.

【Claim 10】

The compound according to item 7, wherein the compound inhibits the binding of IkBa protein and AURKC protein, thereby preventing cancer and showing effects.

【Claim 11】

A composition for preventing and treating cancer comprising an inhibitor of the interaction between IKBa and AURKC as an active ingredient.

【Claim 12】

The composition for preventing and treating cancer according to claim 11, wherein the inhibitor is a compound represented by the following formula (1).

[Chemical formula

[Claim 13]

The method of claim 11, wherein the cancer is stomach cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, xenograft, breast cancer, sclerosing gonadosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, A composition selected from the group consisting of kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer, lymphoma, psoriasis, or fibroadenoma.

【Claim 14】

Use of inhibitors of the interaction between IKBa and AURKC for the manufacture of cancer prevention and treatment.

【Claim 15】

The method of claim 14, wherein the inhibitor is a compound represented by the following formula (1): Use for manufacturing characterized cancer prevention and treatment.

[Formula 1]

[Claim 16]

The method of claim 15, wherein the cancers include stomach cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, leukoma, breast cancer, sclerosing gonadosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, and kidney cancer. Use for the manufacture of cancer prevention and treatment, characterized in that it is selected from the group consisting of sarcoma, prostate cancer, urethral cancer, bladder cancer, hematological cancer, lymphoma, psoriasis or fibroadenoma.

【Claim 17】

A cancer prevention and treatment method comprising administering an effective amount of an inhibitor of the interaction between IKBa and AURKC to an individual in need thereof.

【Claim 18】

The method of claim 17, wherein the inhibitor is a compound represented by the following formula (1).

[Formula 1]

【Claim 19】

The method of claim 18, wherein the cancer is stomach cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing gonadosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, and parathyroid cancer. ， Kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer, lymphoma, A method for preventing and treating cancer, characterized in that the cancer is selected from the group consisting of psoriasis or fibroadenoma.