KR101738680B1 - IkB AURKC Method for screening anti-cancer agents comprising detecting interactions of IkB and AURKC - Google Patents

Abstract

The present invention relates to a method for screening cancer therapies through interaction of IkB? And AURKC. More particularly, the present invention relates to a binding inhibitor compound of IkBα and AURKC, and a composition for preventing and treating cancer comprising the compound as an effective ingredient. The present invention is effective in that it can screen cancer therapeutic agents at the molecular level and can understand the action of therapeutic agents through intermolecular interactions.

Description

[0001] The present invention relates to a method for screening cancer therapies through the interaction of IkB [alpha] and AURKC,

The present invention relates to a method for screening cancer therapies through interaction of IkB? And AURKC. More particularly, the present invention relates to a binding inhibitor compound of IkBα and AURKC, and a composition for preventing and treating cancer comprising the compound as an effective ingredient.

IkBα (nuclear factor of kappa light polypeptide gene enhancer, B-cell inhibitor, alpha) protein is a class of control subunits that inhibit the activity of NF-κB. IkBα binds to the nuclear locus signal (NLS) site of NF-kB and blocks the nuclear locus signal so that NF-kB remains inactive in the cytoplasm. IkBα also prevents the translational factor of NF-kB from binding to the DNA required for NF-kB to function. The IkBα protein was found to have an IkBα protein mutation in some Hawkins lymphoma cells. This mutation inactivates IkBα to allow NF-kB to be chronically activated. (19) reported that lymphoma tumors are directly related to the development of malignant tumors (Cabannes E et al, Oncogene 18 (20): 306370).

The AURKC (Serine / threonine-protein kinase 13) protein is an enzyme encoded by the AUPKC gene (Bernard M et al., Genomics 53 (3): 4069). This gene encodes the aurora subfamily of serine and threonine protein kinases. The encoded protein is a chromosomal transport protein that can complex with Aurora B and internal centrosomal proteins and play a role in tissue microtubules in association with central and spindle functions during mitosis. This gene is overexpressed in some cancer cell lines, indicating that it is involved in tumor signaling.

On the other hand, the growth, differentiation, migration, and death of cells are made by the interaction between polymer substances such as protein-protein, protein-nucleic acid, and the like. Signals outside the cell pass through the receptor of the cell membrane and are transferred into the nucleus of the cell through various biochemical reactions in the cytoplasm, where they express specific genes. The intracellular delivery of these external signals is mediated by the interactions of proteins at various stages. For example, growth factors and cytokines bind to the corresponding receptors on the cell surface and this binding leads to clustering of the receptors. The ligand-mediated aggregation of these receptors induces the intracellular domains of the receptor to aggregate together, thus inducing interactions with various proteins involved in intracellular signaling processes. This signal transduction system produces an intermediate protein that can transfer signals at various stages through protein phosphorylation by protein kinase, dephosphorylation by protein phosphatase, and ultimately transmits its signal to the transcriptional activator (Helden, CH, Cell 80, 213-223 (1995)). Activated transcription-promoting proteins interact with basal transcriptional machinery, such as RNA polymerase, which binds to DNA and synthesizes mRNA to activate specific genes. Thus, these interactions are regulated to specifically induce transcription in response to specific tissues, embryological processes, and external stimuli. At this time, it is considered that the cause of the disorder is the abnormality such as alteration, inhibition, promotion, or the like of interactions between specific proteins due to intrusion of foreign substances or genetic variation of internal active proteins. Therefore, materials that can regulate these interactions can provide a method of treatment for the diseases caused thereby, and research has been conducted thereon.

Conventional in vitro methods such as crosslinking, affinity chromatography, immunoprecipitation (IP) and the like have been used as methods for analyzing the interactions between biopolymers, However, these methods require the production and separation / purification processes of proteins, and there are problems such as the conditions of the buffer solution in the test tube and the secondary deformation of the extracted protein, which can provide information different from the actual interaction occurring in the living body It has disadvantages.

In order to overcome the disadvantages of these in vitro methods, yeast two hybrids (Y2H), fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (Bi- FC) methods have been developed and used.

The present inventors developed a protein kinase C transfer module, a first construct comprising a first labeling substance and a target substance, a second construct containing a second labeling substance and a labeling substance, (Registration number: 10-0948767) has developed a system that can confirm whether the target substance and the target substance interact with each other. This system provides a useful experimental method in that it can identify interactions between proteins. However, no previous studies have been conducted on screening candidates for therapeutic agents through this system.

Thus, the present inventors have completed a screening method for cancer treatment using IkBα and AURKC based on the system for confirming the interaction between the protein and the protein.

Accordingly, it is an object of the present invention to provide a method for screening cancer therapies through IkB? And AURKC.

It is still another object of the present invention to provide a compound screened by the cancer therapeutic agent screening method.

It is still another object of the present invention to provide a composition for preventing and treating cancer comprising the screened compound as an active ingredient.

In order to achieve the above object, the present invention provides a method for screening cancer therapeutic agents through IkB? And AURKC.

In order to accomplish still another object of the present invention, the present invention provides a compound screened by the screening method for cancer therapeutic agent.

In order to achieve still 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.

Hereinafter, the present invention will be described in detail.

The present invention

(a) expressing a fusion protein in which (i) the transfer module, the first labeling substance, the fusion protein in which IkBα is sequentially bound and the second labeling substance, AURKC are sequentially bound, or (ii) Preparing a cell expressing a fusion protein in which IkB? Is sequentially bound and a fusion protein in which a transfer module, a second labeling substance, and AURKC are sequentially bound;

(b) adding a candidate cancer therapeutic agent;

(c) causing interaction between the candidate cancer therapeutic agent and the fusion protein of step (a);

(d) treating the signal material; And

(e) confirming the interaction through distribution of cancer therapeutic agent candidate and intracellular fusion protein

Wherein the cancer therapeutic agent candidate is identified as a cancer therapeutic agent when the candidate substance inhibits the binding of the IkBα protein to the AURKC protein.

The present invention utilizes intracellular localization of proteins generated by external stimuli or intrinsic signal transduction mechanisms to enable direct and real-time analysis of the interaction between IkBα and AURKC. That is, it is designed to design a moving module in which the position is moved by an external stimulus or an intrinsic signal transmission mechanism, a first construct in which IkBα or AURKC is sequentially combined with a marking substance capable of tracking the movement module, and AURKC or IkBα ≪ / RTI > was designed. The first labeling substance and the second labeling substance should contain one each different from IkBα and AURKC. For example, if the first construct is a transport module, the first label is a IkBα, the second construct is a second label, AURKC, and the first construct is a transport module, the first label, the second construct if AURKC The second labeling substance, IkBa.

The present invention relates to a combination of a first construct and a second construct and distinguishes whether or not it is a cancer treatment agent depending on whether it interferes with the binding of the first construct and the second construct of the cancer treatment agent candidate substance. If the combination of the first construct and the second construct is inhibited, it is determined to be a cancer treatment agent.

In the present invention, the transfer module is a region that functions to transfer the first construct to a specific region in the cell. The movement from the above to a specific region in the cell can be carried out by an external signal or intrinsically, and a specific region within the cell is separated, discreet, identifiable ) Element. The intracellular specific region may preferably be a membrane structure such as a cell membrane, a nuclear membrane, or an intracellular organelle such as an endoplasmic reticulum, Golgi complex, mitochondria, lysosome, and other specific intracellular regions.

The " transfer module " of the present invention comprises a protein kinase C (PKC), a classical PKC (PKC-alpha), a PKC-beta and a PKC- epsilon, PKC-eta, PKC-theta).

All of these have a common C1 domain, and binding to the C1 domain with DAG (diacyl glycerol) or TPA (phorbol ester, PMA) induces localization to the cell membrane. The mutant of PKC may be preferably used as the transfer module of the present invention. It is more preferable that the mutant is a mutant that has eliminated the intrinsic phosphorylation activity of PKC in order to minimize the disturbance due to an inherent signal transduction mechanism.

The " first labeling substance " of the present invention may be any one of GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein), mRFP (Monomeric Red Fluorescent Protein), DsRed (Discosoma sp. , CFP (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), AzG (Azami Green), HcR (HcRed, Heteractis crispa red fluorescent protein) and BFP (Blue Fluorescent Protein) Is selected.

The " second labeling substance " of the present invention is different from the first labeling substance and is different from GFP (Green Fluorescent Protein), Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), Monomeric Red Fluorescent Protein (mRFP) (Cyan Fluorescent Protein), YFP (Yellow Fluorescent Protein), AzG (Maximum Green), HcR (HcRed, Heteractis crispa red fluorescent protein) and BFP (Blue Fluorescent Protein) Fluorescent Protein).

The term "fusion protein" of the present invention refers to a protein or polypeptide having an amino acid sequence derived from two or more proteins. A fusion protein may comprise a region of amino acid connectivity between amino acid portions derived from distinct proteins.

The cell may be an animal, a plant, a yeast, and a cell of a bacterium. Preferably, the cell is capable of accepting a first component and a second component that are introduced from outside in the case of bacteria, and the boundary between intracellular organelles such as cytoplasm and nucleus Lt; / RTI > cells are preferred. More preferably CHO-k1 (ATCC CCL-61, Cricetulus griseus, hamster, Chinese), HEK293 (ATCC CRL-1573, Homo sapiens, human), HeLa (ATCC CCL-2, Homo sapiens, human) ATCC CRL-2266, Homo sapiens, human), Swiss 3T3 (ATCC CCL-92, Mus musculus, mouse), 3T3-L1 (ATCC CL-173, Mus musculus, mouse), NIH / 3T3 ATCC CRL-2256, Rattus norvegicus, rat), MDCK (ATCC CCRL-1, Mus musculus, mouse), Rat2 CCL-34, Canis familiaris). In addition, it may be a variety of stem cells, cells extracted from various tissues, and artificial cell membrane structures.

The cells comprising the first construct and the second construct in the present invention can be produced through molecular biological methods known in the art. After transforming the cells with expression vectors capable of expressing both the first construct and the second construct, or an expression vector capable of expressing both the first construct and the second construct, which are capable of expressing the first construct and the second construct, 1 construct and the second construct are preferably expressed.

Transformation of each expression vector into a cell can be carried out by a transformation method known in the art, for example, a calcium phosphate method, a calcium chloride method, a rubidium chloride method, a microprojectile bombardment method, an electroporation method, Particle gun bombardment, silicon carbide whiskers, sonication, PEG-mediated fusion, microinjection, liposome-mediated method, ), A magnetic nanoparticle-mediated method, or the like.

Meanwhile, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, NY (1984), 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, ; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

As used herein, the term "interaction" refers to the proximity of a particular ligand or compound, or a portion or fragment thereof, and a portion of a second molecule of interest. The interaction may be non-covalent, for example, as a result of hydrogen-bonding, van der Waals interactions, electrostatic or hydrophobic interactions, or may be covalent.

The " signal material " of the present invention may be selected from the group consisting of Phorbol 12-myristate 13-acetate, Phorbol ester, 12-otetradecanoylphorbol-13-acetate, PDBu (phorbol 12,13-dibutyrate), ATP (Adenosine triphosphate) tridecanoic acid, arachidonic acid, linoleic acid, DiC8, and 130C937.

The "treatment of the signal substance" of the present invention is characterized by treating PMA (Phorbol 12-myristate 13-acetate, Phorbol ester) at a concentration of 50 nM to 5 uM. More preferably 1 uM. When the treatment concentration of the PMA is less than 50 nM, the movement of the migration module using PKC is not sufficient, and when the concentration exceeds 5 uM, abnormal phenomena (cell death, signal disturbance, etc.) .

The " step of confirming the interaction " is to detect the distribution of the first labeling substance and the second labeling substance. For example, a coverlip including the cells to be identified is fixed to a perfusion chamber (Carl Zeiss LSM710), and acquiring an image of the constituent vectors before and after the external stimulus.

The present invention provides a compound screened by the cancer therapeutic agent screening method.

The compound of the present invention can be represented by the following formula (1).

[Chemical Formula 1]

The compound of the present invention is characterized by inhibiting the binding of IkB [alpha] protein and AURKC protein.

The above-described fry product of the present invention is characterized by inhibiting the binding of the IkBα protein and the AURKC protein to prevent and effect cancer.

The term " cancer " of the present invention is intended to encompass all types of cancer, including gastric cancer, lung cancer, liver cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, Cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer, lymphoma, psoriasis or fibrosarcoma.

The present invention provides a composition for preventing and treating cancer comprising an inhibitor of interaction between IkB? And AURKC as an active ingredient.

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

[Chemical Formula 1]

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The " treatment " of the present invention refers to ameliorating the symptoms of a particular disease or disorder, which may include curing such disorder, substantially preventing the onset of disorder, or ameliorating the condition of the subject. The term "treatment ", as used herein, refers to the treatment of a full spectrum of a given disorder that afflicts the patient, including alleviating one or more symptoms resulting from the disorder, Healing, or preventing the onset of the disorder.

Accordingly, the present invention provides a method for screening cancer therapies through interaction of IkB? And AURKC. More particularly, the present invention provides a composition for inhibiting and treating cancer comprising an inhibitor compound of IkBα and AURKC and an effective ingredient thereof. The screening method of the present invention can be advantageously used in that cancer treatment can be easily screened depending on whether or not IkB? And AURKC are inhibited from binding.

Figure 1 shows the overexpression of TMD-mRFP-IkBa (the first construct) and EGFP-AURKC (the second construct) in HEK293T cell line and analysis using cell imaging (registration number 10-0948767) The middle panel, the second component, the right panel, the first component and the second component).
FIG. 2 shows the result of overexpression of pTMD-mRFP-C3 plasmid (first construct) and EGFP-AURKC (second construct) in HEK293T cell line followed by treatment with 1 μM PMA (external stimulus) for 3 minutes A first component, a middle panel, a second component, a right panel, a first component and a second component).
3-A) The overexpression of TMD-mRFP-AURKC (first construct) and EGFP-IkBα (second construct) in HEK293T cells were treated with 1 μM PMA (external stimulus) for 3 minutes, (Left panel; first component, middle panel; second component, right panel; first component + second component). Figure 3 shows the binding of two proteins to each other by overexpression of pTMD-mRFP-C3 plasmid (the first construct) and EGFP-IkBα (the second construct) in HEK293T cells for 3 minutes with 1 μM of PMA (Left panel; first component, middle panel; second component, right panel; first component + second component).
FIG. 4 shows the results of overexpression of TMD-mRFP-IkBα (the first construct) and EGFP-AURKC (the second construct) in the CHO-K1 cell line followed by treatment with 1 μM PMA (external stimulus) (Left panel: first constituent, middle panel: second constituent, right panel: first constituent + second constituent).
FIG. 5 shows the results of immunohistochemical staining of the gastric cancer cell line AGS (a), SNU638 (b) and the colon cancer cell line HCT116 (c) and SW620 (d), the cervical cancer cell Hela (e), the breast cancer cell line MDA- 4) and hepatocellular carcinoma cell line HepG2 (a).
Figure 6 shows that overexpression of TMD-mRFP-IkBα (first construct) and EGFP-AURKC (second construct) in HEK293T cells (panel A) and CHO-K1 cell line (panel A) Respectively. (Left panel: first constituent, middle panel: second constituent, right panel: first constituent + prod.), Followed by 3 min of 1 μM PMA (external stimulus) 2 construct).
FIG. 7 shows the results of a wound healing assay (a), a Transwell migration assay (b) and a soft agar assay (c) in a breast cancer cell line MDA-MB- As a result, we confirmed the effect of compounds inhibiting the interaction between IkBα and AURKC on cancer cell proliferation.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Animal cell lines and transformation

≪ 1-1 > Animal Cell Lines and Culture thereof

(ATCC HTB-26, Homo sapiens, ATCC CRL-1573, Homo sapiens, human), CHO-K1 (ATCC CCL-61, Cricetulus griseus, hamster, (ATCC CCL-185 Homo sapiens, human), HepG2 (ATCC HB-8065, HepG2, Homo sapiens, human) The culture conditions of the animal cell lines used in the present invention were determined by the cell culture method of ATCC (American Type Culture Collection), which is a pre-sales institution of each cell line. For the HEK293 and MDA-MB-231 cell lines, DMEM culture medium was used. For the A549 and HepG2 cell lines, RPMI 1640 culture medium was used. Other culture conditions were the same as those used for CHO-K1. On the other hand, a common culture method for each cell is as follows (detailed culturing conditions may vary depending on the purpose of the person skilled in the art). (F-12 and DMEM) containing 25 mM HEPES, 10% FBS (fetal bovine serum, v / v), 100 units / ml penicillin, 100 ug / ml streptomycin And cultured in an incubator maintained at 37 ° C and 5% CO ₂ partial pressure.

<1-2> Transformation of cell line

In the intracellular gene introduction method used in the present invention, Fugene HD (Promega), which is one of commonly used liposome-based methods, was used. All conditions for gene introduction such as gene concentration were followed according to the manufacturer's instructions Respectively. More specifically, the cover slips were placed on a 6-plate, the cells were cultured for one day, and then exchanged with 2 ml of fresh culture medium without penicillin and streptomycin or FBS. Approximately 1 μg of the transformed samples were added to the culture medium without 0.1 ml of penicillin and streptomycin and FBS, mixed thoroughly and added with 3 μl of Fugene HD reagent. The solution was allowed to stand at room temperature for 15 minutes, and then added to each well of a 6-plate containing a coverslip with cells growing therein. After culturing for 4 hours, 200 μl of FBS was added and transformed for 18 hours.

&Lt; Example 2 >

Design and manufacture of first and second components

<2-1> Design and manufacture of the first component

In the present invention, the first construct includes a module (mobile module) capable of moving to a cell membrane when proteins unevenly expressed in the cytoplasm are treated, and a red fluorescent protein capable of being analyzed using these microscopes is labeled And finally a fusion sequence in which the target substance can be bound. The first construct was constructed by using IkBα and AURKC in the TMD-mRFP-empty vector using restriction enzymes EcoRI / KpnI (IkBα) and EcoRI / XmaI (AURKC). The TMD-mRFP-empty vector is a vector comprising Protein Kinase C mutant (TMD) represented by SEQ ID NO: 5 and mRFP represented by SEQ ID NO: 7.

IkBα was prepared by performing PCR using the pcDNA3.1-IkBα vector (NCBI Reference Sequence: NM_020529.2; purchased from Mediscov Inc) as a template and the primers of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, AURKC used a pPTB7-AURKC vector (GenBank: BC002363) as a template and PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4. PCR amplification was performed using a primer and the resultant product was inserted into EcoRI / KpnI (IkB?) And EcoRI / XmaI (AURKC) sites of pTMD-mRFP-C3 vector.

<2-2> Design and manufacture of the second component

The second construct includes a label for analyzing the movement of the target material having the intrinsic property of binding to the target substance bound to the first construct. The second construct uses a green fluorescent protein (EGFP) that is different from the label contained in the first construct. The second construct was constructed using restriction enzymes EcoRI / KpnI (IkBα) and EcoRI / XmaI (AURKC) for IkBα and AURKC in the EFRP-C3 vector (clontech) of a previously registered patent (Registration No. 10-1217718) .

IkBα was prepared by performing PCR using the pcDNA3.1-IkBα vector (NCBI Reference Sequence: NM_020529.2; purchased from Mediscov Inc) as a template and the primers of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, AURKC used a pPTB7-AURKC vector (GenBank: BC002363) as a template and PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4. PCR amplification was performed using a primer and the resultant product was inserted into EcoRI / KpnI (IkB?) And EcoRI / XmaI (AURKC) sites of pTMD-mRFP-C3 vector.

&Lt; Example 3 >

Confirmation of intracellular movement of the first construct and the second construct

A coverslip from which the cells of the first construct and the second construct vector were grown was fixed in a perfusion chamber and mounted on a sacrificial bed of a confocal laser fluorescence microscope (Carl Zeiss LSM710) Images for construct vectors after stimulation (1 [mu] M PMA (Phorbol ester) treatment) were obtained.

The laser of the confocal laser microscope was excited by using a 488 nm Argon laser (EGFP) and a 543 nm HeNe laser (mRFP), and the fluorescence signal generated from each fluorescent label was amplified by a band path filter BP505-530 EGFP), long path filter LP560 or BP560-630 (mRFP) was used, and images were obtained after completely eliminating interference between each fluorescence.

As a result, as shown in FIG. 1, FIG. 3, and FIG. 4, red fluorescent light by the first construct vector including the external stimulus post-migration module (TMD) is moved to the cell membrane using a confocal laser fluorescence microscope, Green fluorescence by the second constituent vector for the target substance is uniformly distributed in the cytoplasm as before the stimulation. Therefore, the second construct vector does not respond to external stimuli, and the movement of the second construct to the cell membrane necessarily assumes the binding of the target substance and the target substance.

<Example 4>

Intracellular binding analysis of IkBα and AURKC using the first construct and the second construct

It is known that the interaction of IkBα protein regulated by TNF-alpha is involved in various cell signaling mechanisms in the cell. Of particular interest among the various mechanisms is the close association with inflammation induction in response to various risk signals generated in vivo. The binding of AURKC interacting with IkBα was examined in real time using living cells.

The first construct and the second construct prepared above were transformed into HEK293T and CHO-K1 cell lines, and fluorescence was confirmed at 0 min and 3 min.

As a result, as shown in Fig. 1, the first construct (TMD-mRFP-IkBα) was distributed evenly in the cells at 0 minutes and then moved to the cell membrane 3 minutes later (left panel in FIG. 1) ) Was also distributed uniformly in the cells at 0 minutes and then moved to the cell membrane with the first construct 3 minutes later (FIG. 1, center panel). This means that the first component and the second component are combined.

On the contrary, the first construct was transformed into TMD-mFRP-AURKC and the second construct was transformed into EGRP-IkBα, and HEK2953T and CHO-K1 cell lines were transformed, and fluorescence was confirmed at 0 min and 3 min.

As a result, the first construct (TMD-mRFP-AURKC) was uniformly distributed in the cells at 0 minutes and then moved to the cell membrane in 3 minutes (Fig. 3-left panel) EGFP-IkBα) was also distributed uniformly in the cells at 0 min, and migrated to the cell membrane with the first construct 3 minutes later (FIG. This means that the first component and the second component are combined.

&Lt; Example 5 >

Screening compounds that inhibit the binding of IkBα and AURKC

Using the above screening method, the first construct and the second construct prepared above were transformed into 6-plate into a coverslip in which HEK293T and CHO-K1 cells grew. The IkBα protein and the AURKC protein binding inhibitor candidate were then treated for 3 hours. The coverslip was fixed in a perfusion chamber and mounted on a sacrifice plate of confocal laser fluorescence microscope (Carl Zeiss LSM710), and for constituent vectors before external stimulation and after external stimulation (1 μM Phorbol ester) Images were acquired.

As a result, a compound represented by the following formula (1) inhibiting the interaction between the IkBα protein and the AURKC protein was screened.

[Chemical Formula 1]

FIG. 6 shows the results of analysis of inhibitory effect of IkBα protein and AURKC protein on the binding of TMD-mRFP-IkBα (first construct) and EGFP-AURKC (second construct) to HEK293T cells and CHO-K1 cell line And 50 [mu] M of the compound was treated for 3 hours. Then, 1 μM of PMA (external stimulus) was treated for 3 minutes to analyze the binding inhibition of the two proteins in real time. The IkBα protein and AURKC protein were not bound by the above compounds. The red fluorescence reflecting the migration module and IkBα migrated to the cell membrane, but the green fluorescence reflecting the AURKC protein was located in the cytoplasm. Therefore, it is shown that the compound represented by the above formula (1) can be used as an inhibitor inhibiting the binding of IkB? With AURKC.

&Lt; Example 6 >

Cytotoxicity measurement of screened compounds

The cytotoxicity of the compounds used in the examples of the invention was determined using a commonly used CCK8 kit (Dojindo Molecular Technologies) and all conditions were followed according to the manufacturer's instructions. More specifically, the cells were cultured on a 96-well plate for one day, treated with 10, 50, and 100 μM of the compound, incubated for 24 hours and 48 hours, and 10 μl of CCK8 solution was added thereto.

As a result, the gastric cancer cell line AGS and SNU638 (Fig. 5A) and the colon cancer cell line HCT116 and SW620 (d), the cervical cancer cell Hela (e), the breast cancer cell line MDA-MB- The cytotoxicity of IKBα and AURKC in cell line A549 and hepG2 cell line HepG2 (a) was increased by the compound which inhibits the interaction of IkBα with AURKC.

&Lt; Example 7 >

Inhibition of cancer cell proliferation of screened compounds

<7-1> Analysis method of cancer cell migration

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 and 5% CO ₂ (FIG. 7A). The cells are cultured in 24-well tissue culture plates (2 x 10 ⁵ cells) for 24 hours. Then, without changing the medium, slowly cut through the center of the well using a 1 ml pipette tip. At this time, the tip is used standing upright from the bottom of the well, and the wound is wound in one direction. Then rinse twice with medium to remove dead cells and replenish fresh medium. Then, 25 μM of a compound that inhibits the interaction between IkBα and AURKC is treated on a test plate, followed by culturing for 48 hours. Then rinse twice with 1X PBS and fix with 3.7% paraformaldehyde for 30 minutes. Fixed cells were stained with 2% ethanol in 1% crystal violet for 30 minutes and observed with a microscope.

As a result, as shown in Fig. 7, it was confirmed that the width of the wound was narrower in the control group (0 μM) not treated with the compounds inhibiting the interaction of IkBα and AURKC in the breast cancer cell line than in the case of treating the compound (25 μM). In the case of the control group, the cells were grown in the tip-wounded area to fill the space, but when the compound was treated, the cells did not grow on the injured area and the space remained intact. In other words, it was confirmed that the compound inhibiting the interaction of IkBα and AURKC inhibited cell proliferation.

<7-2> Transwell movement measurement

Transwell migration assay (MDA-MB-231), a breast cancer cell line cultured in DMEM with 10% FBS, was used (Fig. 7B). To prepare the transwells, 0.5% FBS and 2.6 ml DMEM were added at the bottom of 24 wells, and transwells with an 8 mu m pore size were placed. Cells (1 X 10 ⁵ cells) were then added to the prepared transwells. Then, 10 μM and 25 μM of a compound inhibiting the interaction between IkBα and AURKC were added, and then the cells were cultured at 37 ° C. and 5% CO ₂ for 2 hours and 30 minutes to allow the cells to move toward the lower side of the inserted filter. After 2 hours and 30 minutes, the filter was removed and the remaining cell debris on the filter was removed using a cotton swab. Cells at the bottom of the filter were fixed with 5% glutaraldehyde for 10 minutes and stained with 2% ethanol with 1% crystal violet for 20 minutes. The crystal violet was removed, observed under a microscope and the number of cells in the lower part of the filter was measured. At this time, different parts of the filter were selected and observed and averaged.

The results are shown in the graph as shown in FIG. It was confirmed that the number of cells migrating to the breast cancer cell line decreased when the compounds inhibiting the interaction of IkBα and AURKC were treated. It was confirmed that the number of migrating cells was further reduced when 25 μM of the compound was added to 10 μM.

Therefore, it was confirmed that cancer cell proliferation was inhibited by a compound inhibiting the interaction of IkB? With AURKC as in <7-1> above.

<7-3> Analysis of agar agar

MDA-MB-231, a breast cancer cell line, was cultured on an agar medium prepared by agarose treatment using a soft agar assay (FIG. 7). First, 4% agar was dissolved and kept warm in a constant temperature water bath at 56 ° C, and the temperature was maintained at 37 ° C in 10% FBS DMEM. To make the lower layer of the agar medium to which the cells were to be cultured, 5 ml of DMEM containing 0.75% agar and 10% FBS was placed in a 60 mm culture dish and waited until it hardened. Next, to make the upper layer, 3 x 10 ⁴ cells were added to 3 ml of 10% FBS DMEM containing 0.36% agar, and 25 μM of a compound inhibiting the interaction of IkBα and AURKC was added thereto, and the mixture was placed on a hard agar medium and waited until it hardened . After incubating 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 the colonies on the medium containing the compound inhibiting the interaction of IkBα and AURKC was smaller and smaller than the colony of the control untreated control. It was also found that the number of colonies was lower when the compounds were treated than the control untreated control. It was confirmed that the cell inhibition was inhibited by a compound which inhibits the interaction of IkB? With AURKC.

The present invention relates to a method for screening cancer therapies through interaction of IkB? And AURKC. More particularly, the present invention relates to a binding inhibitor compound of IkBα and AURKC, and a composition for preventing and treating cancer comprising the compound as an effective ingredient. The present invention is effective in that it can screen cancer therapeutic agents at the molecular level and can understand the action of therapeutic agents through intermolecular interactions.

<110> KOREA BASIC SCIENCE INSTITUTE <120> Method for screening anti-          interactions of IkBa and AURKC <130> NP15-0073 <150> KR 10-2014-0094260 <151> 2014-07-24 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> IkBa-Forward primer <400> 1 tggaattcat gttccaggcg gccgagcgcc cc 32 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> IkBa-Reverse primer <400> 2 atggtacctc ataacgtcag acgctggcct cc 32 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AURKC-Forward primer <400> 3 gctgaattct gatgcggcgc ctcacagtcg 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AURKC-Reverse primer <400> 4 gctggatcct aggaagccat ctgagcacag 30 <210> 5 <211> 2028 <212> DNA <213> Artificial Sequence <220> <223> Protein kinase C mutant, TMD <400> 5 atggcgccgt tcctgcgcat cgccttcaac tcctatgagc tgggctccct gcaggccgag 60 gaggaggcga accagccctt ctgtgccgtg aagatgaagg aggcgctcag cacagagcgt 120 gggaaaacac tggtgcagaa gaagccgacc atgtatcctg agtggaagtc gacgttcgac 180 gcccacatct atgaggggcg cgtcatccag attgtgctaa tgcgggcagc agaggagcca 240 gtgtctgagg tgaccgtggg tgtgtcggtg ctggccgagc gctgcaagaa gaacaatggc 300 aaggctgagt tctggctgga cctgcagcct caggccaagg tgttgatgtc tgttcagtat 360 ttcctggagg acgtggattg caaacagtct atgcgcagtg aggacgaggc caagttccca 420 acgatgaacc gccgcggagc catcaaacag gccaaaatcc actacatcaa gaaccatgag 480 tttatcgcca ccttctttgg gcaacccacc ttctgttctg tgtgcaaaga ctttgtctgg 540 ggcctcaaca agcaaggcta caaatgcagg caatgtaacg ctgccatcca caagaaatgc 600 atcgacaaga tcatcggcag atgcactggc accgcggcca acagccggga cactatattc 660 cagaaagaac gcttcaacat cgacatgccg caccgcttca aggttcacaa ctacatgagc 720 cccaccttct gtgaccactg cggcagcctg ctctggggac tggtgaagca gggattaaag 780 tgtgaagact gcggcatgaa tgtgcaccat aaatgccggg agaaggtggc caacctctgc 840 ggcatcaacc agaagctttt ggctgaggcc ttgaaccaag tcacccagag agcctcccgg 900 agatcagact cagcctcctc agagcctgtt gggatatttc agggtttcga gaagaagacc 960 ggagttgctg gggaggacat gcaagacaac agtgggacct acggcaagat ctgggagggc 1020 agcagcaagt gcaacatcaa caacttcatc ttccacaagg tcctgggcaa aggcagcttc 1080 gggaaggtgc tgcttggaga gctgaagggc agaggagagt actttgccat cagggccctc 1140 aagaaggatg tggtcctgat cgacgacgac gtggagtgca ccatggttga gaagcgggtg 1200 ctgacacttg ccgcagagaa tccctttctc acccacctca tctgcacctt ccagaccaag 1260 gaccacctgt tctttgtgat ggagttcctc aacggggggg acctgatgta ccacatccag 1320 gacaaaggcc gctttgaact ctaccgtgcc acgttttatg ccgctgagat aatgtgtgga 1380 ctgcagtttc tacacagcaa gggcatcatt tacagggacc tcaaactgga caatgtgctg 1440 ttggaccggg atggccacat caagattgcc gactttggga tgtgcaaaga gaacatattc 1500 ggggagagcc gggccagcac cttctgcggc acccctgact atatcgcccc tgagatccta 1560 cagggcctga agtacacatt ctctgtggac tggtggtctt tcggggtcct tctgtacgag 1620 atgctcattg gccagtcccc cttccatggt gatgatgagg atgaactctt cgagtccatc 1680 cgtgtggaca cgccacatta tccccgctgg atcaccaagg agtccaagga catcctggag 1740 aagctctttg aaagggaacc aaccaagagg ctgggagtga cgggaaacat caaaatccac 1800 cccttcttca agaccataaa ctggactctg ctggaaaagc ggaggttgga gccacccttc 1860 aggcccaaag tgaagtcacc cagagactac agtaactttg accaggagtt cctgaacgag 1920 aaggcgcgcc tctcctacag cgacaagaac ctcatcgact ccatggacca gtctgcattc 1980 gctggcttct cctttgtgaa ccccaaattc gagcacctcc tggaagat 2028 <210> 6 <211> 676 <212> PRT <213> Artificial Sequence <220> <223> Protein kinase C mutant, TMD <400> 6 Met Ala Pro Phe Leu Arg Ile Ala Phe Asn Ser Tyr Glu Leu Gly Ser   1 5 10 15 Leu Gln Ala Glu Asp Glu Ala Asn Gln Pro Phe Cys Ala Val Lys Met              20 25 30 Lys Glu Ala Leu Ser Thr Glu Arg Gly Lys Thr Leu Val Gln Lys Lys          35 40 45 Pro Thr Met Tyr Pro Glu Trp Lys Ser Thr Phe Asp Ala His Ile Tyr      50 55 60 Glu Gly Arg Val Ile Gln Ile Val Leu Met Arg Ala Ala Glu Glu Pro  65 70 75 80 Val Ser Glu Val Thr Val Gly Val Ser Val Leu Ala Glu Arg Cys Lys                  85 90 95 Lys Asn Asn Gly Lys Ala Glu Phe Trp Leu Asp Leu Gln Pro Gln Ala             100 105 110 Lys Val Leu Met Ser Val Gln Tyr Phe Leu Glu Asp Val Asp Cys Lys         115 120 125 Gln Ser Met Arg Ser Glu Asp Glu Ala Lys Phe Pro Thr Met Asn Arg     130 135 140 Arg Gly Ala Ile Lys Gln Ala Lys Ile His Tyr Ile Lys Asn His Glu 145 150 155 160 Phe Ile Ala Thr Phe Phe Gly Gln Pro Thr Phe Cys Ser Val Cys Lys                 165 170 175 Asp Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Lys Cys Arg Gln Cys             180 185 190 Asn Ala Ala Ile His Lys Lys Cys Ile Asp Lys Ile Ile Gly Arg Cys         195 200 205 Thr Gly Thr Ala Asp Ser Arg Asp Thr Ile Phe Gln Lys Glu Arg     210 215 220 Phe Asn Ile Asp Met Pro His Arg Phe Lys Val His Asn Tyr Met Ser 225 230 235 240 Pro Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Leu Val Lys                 245 250 255 Gln Gly Leu Lys Cys Glu Asp Cys Gly Met Asn Val His His Lys Cys             260 265 270 Arg Glu Lys Val Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu Leu Ala         275 280 285 Glu Ala Leu Asn Gln Val Thr Gln Arg Ala Ser Arg Arg Ser Serp Ser     290 295 300 Ala Ser Ser Glu Pro Val Gly Ile Phe Gln Gly Phe Glu Lys Lys Thr 305 310 315 320 Gly Val Ala Gly Glu Asp Met Gln Asp Asn Ser Gly Thr Tyr Gly Lys                 325 330 335 Ile Trp Glu Gly Ser Ser Lys Cys Asn Ile Asn Asn Phe Ile Phe His             340 345 350 Lys Val Leu Gly Lys Gly Ser Phe Gly Lys Val Leu Leu Gly Glu Leu         355 360 365 Lys Gly Arg Gly Glu Tyr Phe Ala Ile Arg Ala Leu Lys Lys Asp Val     370 375 380 Val Leu Ile Asp Asp Asp Val Glu Cys Thr Met Val Glu Lys Arg Val 385 390 395 400 Leu Thr Leu Ala Ala Glu Asn Pro Phe Leu Thr His Leu Ile Cys Thr                 405 410 415 Phe Gln Thr Lys Asp His Leu Phe Phe Val Met Glu Phe Leu Asn Gly             420 425 430 Gly Asp Leu Met Tyr His Ile Gln Asp Lys Gly Arg Phe Glu Leu Tyr         435 440 445 Arg Ala Thr Phe Tyr Ala Glu Ile Met Cys Gly Leu Gln Phe Leu     450 455 460 His Ser Lys Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Leu 465 470 475 480 Leu Asp Arg Asp Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys Lys                 485 490 495 Glu Asn Ile Phe Gly Glu Ser Arg Ala Ser Thr Phe Cys Gly Thr Pro             500 505 510 Asp Tyr Ile Ala Pro Glu Ile Leu Gln Gly Leu Lys Tyr Thr Phe Ser         515 520 525 Val Asp Trp Trp Ser Phe Gly Val Leu Leu Tyr Glu Met Leu Ile Gly     530 535 540 Gln Ser Pro Phe His Gly Asp Asp Glu Asp Glu Leu Phe Glu Ser Ile 545 550 555 560 Arg Val Asp Thr Pro His Tyr Pro Arg Trp Ile Thr Lys Glu Ser Lys                 565 570 575 Asp Ile Leu Glu Lys Leu Phe Glu Arg Glu Pro Thr Lys Arg Leu Gly             580 585 590 Val Thr Gly Asn Ile Lys Ile His Pro Phe Phe Lys Thr Ile Asn Trp         595 600 605 Thr Leu Leu Glu Lys Arg Arg Leu Glu Pro Pro Phe Arg Pro Lys Val     610 615 620 Lys Ser Pro Arg Asp Tyr Ser Asn Phe Asp Gln Glu Phe Leu Asn Glu 625 630 635 640 Lys Ala Arg Leu Ser Tyr Ser Asp Lys Asn Leu Ile Asp Ser Met Asp                 645 650 655 Gln Ser Ala Phe Ala Gly Phe Ser Phe Val Asn Pro Lys Phe Glu His             660 665 670 Leu Leu Glu Asp         675 <210> 7 <211> 674 <212> DNA <213> Artificial Sequence <220> <223> mRFP <400> 7 tggcctcctc cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc atggagggct 60 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca 120 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc 180 tgtcccctca gttccagtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg 240 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg 300 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca 360 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca 420 tgggctggga ggcctccacc gagcggatgt accccgagga cggcgccctg aagggcgaga 480 tcaagatgag gctgaagctg aaggacggcg gccactacga cgccgaggtc aagaccacct 540 acatggccaa gaagcccgtg cagctgcccg gcgcctacaa gaccgacatc aagctggaca 600 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgcgcc gagggccgcc 660 actccaccgg cgcc 674 <210> 8 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> mRFP <400> 8 Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val   1 5 10 15 Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu              20 25 30 Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val          35 40 45 Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln      50 55 60 Phe Gln Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro  65 70 75 80 Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val                  85 90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser             100 105 110 Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn         115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu     130 135 140 Ala Ser Thr Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu 145 150 155 160 Ile Lys Met Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu                 165 170 175 Val Lys Thr Thr Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Ala             180 185 190 Tyr Lys Thr Asp Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr         195 200 205 Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly     210 215 220 Ala 225

Claims (13)

(a) expressing a fusion protein in which (i) the transfer module, the first labeling substance, the fusion protein in which IkBα is sequentially bound and the second labeling substance, AURKC are sequentially bound, or (ii) Preparing a cell expressing a fusion protein in which IkB? Is sequentially bound and a fusion protein in which a transfer module, a second labeling substance, and AURKC are sequentially bound;
(b) adding a candidate cancer therapeutic agent;
(c) causing interaction between the candidate cancer therapeutic agent and the fusion protein of step (a);
(d) treating the signal material; And
(e) confirming the interaction through distribution of cancer therapeutic agent candidate and intracellular fusion protein
Wherein the cancer therapeutic agent candidate is identified as a cancer therapeutic agent when the candidate substance inhibits the binding of the IkBα protein to the AURKC protein.
2. The method according to claim 1, wherein the transfer module is selected from the group consisting of protein kinase C (PKC), cPKC (classical PKC), PKC-beta, PKC-gamma, nPKC -epsilon, PKC-eta, PKC-theta).
The method of claim 1, wherein the first labeling substance is selected from the group consisting of GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein), mRFP (Monomeric Red Fluorescent Protein), DsRed ), Cyan Fluorescent Protein (CFP), Cyan Green Fluorescent Protein (CGFP), Yellow Fluorescent Protein (YFP), AzG (Azami Green), HcR (HcRed, Heteractis crispa red fluorescent protein) and BFP (Blue Fluorescent Protein) &Lt; / RTI &gt;
The method of claim 1, wherein the second labeling material is different from the first labeling material and is selected from the group consisting of GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein), mRFP (Monomeric Red Fluorescent Protein) (Cyan Fluorescent Protein), CFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), AzG (Maximum Green), HcR (HcRed, Heteractis crispa red fluorescent protein) and BFP Blue Fluorescent Protein). &Lt; / RTI &gt;
2. The method of claim 1 wherein the signal material is selected from the group consisting of Phorbol 12-myristate 13-acetate,
Phorbol ester), TPA (12-otetradecanoylphorbol-13-acetate), PDBu (phorbol 12,13-dibutyrate), ATP (Adenosine triphosphate), tridecanoic acid, arachidonic acid, linoleic acid, DiC8, 130C937 Lt; / RTI &gt;
2. The method of claim 1, wherein the treatment of the signal material is a treatment of PMA (Phorbol 12-myristate 13-acetate, Phorbol ester) at a concentration of 50 nM to 5 uM.
delete delete delete delete A composition for preventing and treating cancer, comprising an inhibitor of interaction between IkBα and AURKC represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

delete 12. The method of claim 11, wherein the cancer is selected from the group consisting of gastric cancer, lung cancer, liver cancer, colon cancer, small bowel cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, scleroderma, uterine cancer, cervical cancer, head and neck cancer, Kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer, lymphoma, psoriasis or fibroin adenoma.

Images