KR20070114660A - Plk1-based protein chip, method for high throughput screening of anticancer agents using the protein chip and anticancer agents preparing thereby - Google Patents

Abstract

A Plk1-based protein chip is provided to screen anticancer agents capable of inhibiting binding of carcinogen to its substrate protein rapidly and conveniently by using SPRI(surface plasmon resonance imaging) bioassay technique, so that it is useful for developing a new anticancer agent capable of inducing apoptosis of cancer cells. A Plk1-based protein chip contains a substrate protein of Plk1(polo-like kinase 1) on a substrate, wherein the substrate protein of Plk1 is selected from stathmin, katanin p80, TCTP(translationally controlled tumor protein), Cdc25C(cell division cycle 25C), NudC, Myt1, katanin p60, APC(anaphase promoting complex), Myt1-2, grasp65, cohesin, Mklp1, Mklp2, Asp, N1p, Weel, Pin1, BRCA2(breast cancer type 2 susceptibility protein) and p53; and PBD(polo-box domain) of Plk1 is immobilized on the substrate. A method for high throughput screening of anticancer agents comprises the steps of: (a) reacting a candidate material inhibiting binding of Plk1 substrate protein with Plk1 PBD on the Plk1-based protein chip; and (b) analyzing and selecting the candidate material as an anticancer agent by using SPRI(surface plasmon resonance imaging) system.

Description

PLK1-Based Protein Chip, Method for High Throughput Screening of Anticancer Agents Using the Protein Chip and Anticancer Agents Preparing Thereby}

FIG. 1 is a schematic diagram illustrating an ultrafast screening method using a protein chip and an unlabeled SPRI bioassay for discovering a candidate for inhibiting binding between a Plk1 protein and a matrix protein.

2A is agarose gel photographs of genes encoding substrate proteins of Plk1 (Polo-like kinase 1) produced by PCR from cDNA libraries of animal cells.

Figure 2b shows the expression and thrombin in bacterial cells of the PBD (polo-box domain), which is the site of binding to the substrate protein in glutathione-S transferase (GST) fused form Plk1. SDS-PAGE gel photograph showing establishment of conditions for removing GST-tag from PBD protein.

FIG. 2C is a SDS-PAGE gel photograph showing the expression of various tag-protein fused matrix proteins in bacterial cells. FIG.

3 is a photograph showing a binding image of Plk1 and a substrate protein using SPRI.

Figure 4a is a photograph showing the discovery results of the binding inhibitor candidate in the primary screening by the ultra-fast screening method using a protein chip and the unlabeled SPRI.

FIG. 4B shows the results of SPRI screening and numerical analysis of the results according to the concentrations of the primary inhibitors of binding screening screened in FIG. 4A.

Figure 4c shows the results of the third screening to confirm whether the binding inhibitory candidates selected in Figure 4b specific for the inhibition of binding of Plk1 protein and substrate protein.

FIG. 5 confirms and analyzes the binding inhibitory effect between Plk1 substrate protein NudC and Cdc25c, which were used for screening PBD of Plk1 by GST pull-down method of the binding inhibitory candidates selected in FIG. 4C. (A) shows PBD and NudC. , (B) shows the results of experiments using PBD and Cdc25C protein.

The present invention relates to a Plk1-based protein chip, a high throughput screening method of an anticancer substance using the same, and an anticancer substance obtained by the method. More specifically, Plk1 (polo-like kinase) is a carcinogenic protein on a substrate. Protein chip for detecting interaction between Plk1 substrate protein and PBD, wherein the substrate protein of 1) or PBD (polo-box domain), which is a substrate protein binding site of Plk1, is immobilized, between the PBD of Plk1 and the substrate protein on the protein chip The present invention relates to a method for screening an anticancer substance and an anticancer substance screened by the method, wherein the candidate substance for inhibiting binding is selected as an anticancer substance.

In the present invention, the protein Plk1 (Polo-like kinase 1), which is a target material of the protein chip, is a Ser / Thr kinase protein and plays a key role in regulating cell division in the cell cycle of eukaryotic organisms. Plk1 is activated by phosphorylating several kinds of proteins. The activated Plk1 substrate proteins play an essential role in cell division. Plk1 is composed of N-terminal kinase domain and C-terminal polo-box domain (PBD). The kinase domain is activated by phosphorylating target protein, and polo-box domain (PBD) is a substrate protein. And the intracellular location of Plk1 (Cell 2003, 115, 3-4). On the other hand, excessive expression of Plk1 in mice results in cancer, and it is also found that Plk1 is excessively expressed in various cancer cells such as colorectal cancer and prostate cancer (FASEB J. 2004, 18, 5-7, Cancer sci. 2003, 94, 148-152), suggesting that Plk1 is associated with the development of cancer. Specifically, it was found that depletion of Plk1 by RNAi in cancer cells induces apoptosis of cancer cells (PNAS, 2003, 100, 5789-5794). The above results demonstrate that screening for substances that inhibit the activity of Plk1 protein may be a strategy of anticancer drug development.

In addition to protein-protein binding, protein chip technology provides a measurement technique that can analyze the binding of nucleic acids, sugars, and lipids to proteins. This includes not only biological research related to proteins such as intracellular signal transduction and proteomics, but also various screening such as environmental monitoring, screening for harmful environmental hazards, and medical diagnosis of disease and detection of new drug candidates. Has a width. In addition, it provides a technique for measuring a large amount of libraries in a short time, and a technique for reducing the amount of sample that has always been a problem when screening because only a small amount of sample is used.

For conventional drug development, screening methods required protein-protein interactions to label fluorescent, radioactive isotopes and the like. However, there are problems such as difficulty in labeling proteins specifically and uniformly, expensive labeling materials, and risk of radiation exposure when using radioisotopes.

In addition, U.S. Patent Application Publication No. 2005-85626 discloses binding pockets of the polo domain, and describes that a high-efficiency large-capacity search can be performed using the same. However, this problem still has not been solved.

Accordingly, the present inventors have made diligent efforts to develop a method for screening new drug candidates in a large amount quickly and conveniently in developing an anticancer drug. As a result, an unlabeled protein interaction detection method, "SPR image protein chip system," We have developed a high throughput screening method for anticancer substances.

After all, the main object of the present invention, the Plk1 substrate protein, characterized in that the substrate protein of Plk1 (polo-like kinase 1) or PBD (polo-box domain), which is a substrate protein binding site of Plk1, is immobilized on the substrate. To provide a protein chip for detecting the interaction of the PBD (polo-box domain) of Plk1.

Another object of the present invention to provide a method for screening an anticancer substance using the protein chip.

Still another object of the present invention is to provide an anticancer substance screened by the above method.

In order to achieve the above object, the present invention is a protein chip and PBD (polo-like kinase 1) of Plk (polo-like kinase 1) on the substrate, characterized in that the substrate protein is fixed on the substrate Plk1 (polo-like kinase 1) It provides a protein chip characterized in that the -box domain) is fixed.

In the present invention, the substrate protein of Plk1 is stasmin, katan p80 (Katanin p80), TCTP (Translationally Controlled Tumor Protein), Cdc25C (Cell division cycle 25C), NudC, Myt1, catanine p60 (Katanin p60), APC8 (Anaphase Promoting Complex8), Myt1-2, Grasp65, Cohesin, Mklp1, Mklp2, Asp, Nlp, Wee1, Pin1, BRCA2, p53 may be selected from the group consisting of, but not limited to, Plk1 Any substrate protein can be used.

In the present invention, the protein chip comprises (a) coating a substrate with a binder, and then depositing a metal thin film to form and activate a self-assembled monomolecular film; And (b) immobilizing the substrate protein of Plk1 on the activated substrate, wherein the self-assembled monomolecular film comprises a Piranha solution; 70% H ₂ SO ₂ , 30% H ₂ O ₂ ), and then immersed in a 10mM MUA (11-mercapto-1-undercanoic acid) solution can be characterized in that it is produced.

In the present invention, the step (b) may be characterized in that to fix the protein binding to the affinity material tag on the substrate, and then to fix the substrate protein of Plk1 tagged with an affinity material, the substrate It may be characterized in that it is selected from the group consisting of glass, silicon, silicon oxide (silicon oxide), silicon nitride (silicon nitride), polycarbonate, polystyrene and polypropylene.

In the present invention, the metal is gold (Au), molybdenum (Mo), silver (Ag), aluminum (Al), calcium (Ca), cadmium (Cd), iron (Fe), nickel (Ni), platinum ( Pt), zinc (Zn) and copper (Cu), characterized in that the binder is selected from the group consisting of chromium (Cr), titanium (Ti), titanium tungsten (TiW) and nickel chromium (NiCr) It may be characterized in that it is selected from the group consisting of.

In the present invention, the affinity tag is glutathione-S transferase (GST), 6xHis (hexa histidine), maltose binding protein (MBP), thioredoxin (thioredoxine), di Dihydrofolate reductase (DHFR), cellulose binding domain (CBD), galactose-binding protein, calmodulin binding protein (CBP), staphylo Cocal protein A, staphylococcal protein G, stacylococcal protein G, polycysteine, polyphenylalanine, poly-argin, flag-FLAG, streptoavidin; STA), Biotin, c-myc, s-peptide and chitin-binding domain, characterized in that it is selected from the group consisting of The protein binding to the group affinity tag may be characterized in that it is selected from the group consisting of glutathione (GSH), nickel-nitrilo-triacetic acid (Ni-NTA) and amyloase (amyloase).

The present invention comprises the steps of (a) reacting a substrate substance of Plk1 (polo-like kinase 1) on the protein chip and a candidate substance that inhibits the binding of PBD (polo-box domain) of Plk1; And (b) selecting a candidate substance that inhibits the binding of the substrate protein of Plk1 and PBD of Plk1 as an anticancer substance.

In the present invention, the candidate substance that inhibits the binding between the PBD of Plk1 and the substrate protein may be characterized in that it is analyzed by Surface Plasmon Resonance Imaging (SPRI) system, and the fluorescent substance or radioisotope is analyzed. It may be characterized by using the analysis, but is not limited thereto.

The method for screening an anticancer substance according to the present invention further comprises the steps of (c) analyzing the animal cancer cell selected by step (b) with a Tetrazolium-based colorimetric (MTT) assay in animal cells. It may be characterized by including as.

The present invention also relates to extracts of Typahe pollen, Amico Cardamomi Fructus extract, Dioscoreae Rhizoma extract, Torricellia angulata var.intermedia extract, Copaiba, Copaifera It provides an anticancer composition containing any one or more extracts selected from the group consisting of Lansdorifii ) extract and Buchinha ( Luffa operculata ) extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention spots a carcinogen and a candidate for inhibiting binding between the carcinogen and the matrix protein on a protein chip prepared by immobilizing a matrix protein of the carcinogen, and then, a non-labeled agent for detecting the binding inhibitor between these proteins. Ultrafast screening using SPRI, a detection method, and anticancer candidates obtained from more than 10,000 material libraries using the same (FIG. 1). FIG. 1 is a schematic diagram showing the present invention, and shows an ultrafast screening method using a protein chip and an unlabeled SPRI bioassay for discovering candidates for inhibition of binding between a Plk1 protein and a substrate protein.

First, in order to secure genes encoding carcinogens and matrix proteins of the carcinogens, and to manufacture protein chips in which the matrix proteins are immobilized, the genes are redesigned to have appropriate tags, and they are expressed in bacteria to be expressed at the N-terminal or A protein in the form of a tag fused to the C-terminus is used. In one embodiment of the present invention, by using a protein fused with glutathione-S transferase (GST), the protein is selectively bound to glutathione (GSH) treated on a chip. Was fixed on the chip.

On the other hand, the affinity tag for fixing the protein on the chip (tag) is not limited to GST can be used in various ways. The types include glutathione-S transferase (GST), 6xHis (hexahistidine), maltose binding protein (MBP), thioredoxine, and dihydrofolate reductase ( dihydrofolate reductase (DHFR), cellulose binding domain (CBD), galactose-binding protein, calmodulin binding protein (CBP), staphylococcal protein A (staphylococcal protein) A), staphylococcal protein G, polycysteine, polyphenylalanine, polyarginine (Poly-Arg), flag (FLAG), streptoavidin (STA), biotin ), C-myc, s-peptide and chitin-binding domain.

In addition, since the protein is immobilized on the chip by selective binding, the protein chip of the present invention can be used as it is a bacterial extract containing a target protein having an appropriate tag. Therefore, when the protein is fixed on the substrate, there is an advantage that the protein purification step can be omitted.

In the present invention, mainly used proteins that are easily expressed in bacteria. Among the carcinogens, the PBD (polo-box kinase 1) of Plk1 (polo-like kinase 1), which plays a key role in regulating cell division in the eukaryotic cell cycle, is used as Ser / Thr kinase protein. For the production of protein chips, if Plk1 and substrate proteins are not expressed in bacteria, all methods of expressing them in insect cells, yeast or animal cells using a baculovirus vector are possible. Do. In addition, bacteria are not limited to types of expression cells, expression conditions and methods, such as a method of improving expression conditions or refolding insoluble proteins.

Substrate proteins of Plk1, which are carcinogens, include stasmin, katanine p80 (Katanin p80), TCTP (Translationally Controlled Tumor Protein), Cdc25C (Cell division cycle 25C), NudC, Myt1, and catanine p60 (Katanin p60). , APC8 (Anaphase Promoting Complex8), Myt1-2, Grasp65, Cohesin, Mklp1, Mklp2, Asp, Nlp, Wee1, Pin1, BRCA2, may be selected from the group consisting of, but not limited to, substrate of Plk1 Any protein can be used.

On the other hand, there are some substrate proteins that do not bind to Plk1 in vitro or do not have strong binding in vitro, some of which may require structural modifications such as phosphorylation. The problem may be solved by using proteins expressed in cells.

The protein chip used in one embodiment of the present invention may be a glass substrate coated with a gold thin film as a gold chip. The substrate serves as a support for the chip, and in addition to glass, silicon, silicon oxide, silicon nitride, polycarbonate, polystyrene, polypropylene, and the like may be used. . In general, since the bonding strength between gold and the substrate is weak, in order to improve the bonding between them, a metal is first coated on the substrate with a binder such as chromium, titanium, titanium tungsten, or nickel chromium, and then gold is attached to one side of the substrate to form a thin film layer. It is desirable to have a.

Surface treatment of the gold chip can be accomplished through the formation of self assembled monolayers (SAMs), which firstly convert the substrate into a Piranha solution (70% H ₂ SO ₂ , 30% H ₂ O ₂ ). Treated, and immersed in a 10 mM MUA (11-mercapto-1-undercanoic acid) solution to form a single granulated monomolecular film and prepared by treating reduced L-glutathione (GSH).

In the next step, the GST-fusion substrate protein is selectively bound to the surface of the gold thin film of the chip formed by glutathionylation by dropping a bacterial extract overexpressing the GST-tag fused substrate protein onto the surface-treated chip, A mixture of PBD and binding inhibitory candidate was again dropped onto it, then washed off to remove unbound material and analyzed using SPRI. SPRI is capable of identifying existing protein separations such as SDS-PAGE and Western blot for protein analysis, as well as protein-protein or protein-nucleic acid binding assays. have.

Plk1 (polo-like kinase 1), a cancer-related cell cycle control protein obtained in the present invention, and a candidate for inhibiting binding of substrate proteins binding thereto were obtained from plant extracts, and among them, those obtained from herbal extracts used as herbal medicines, This, by the present invention reveals the mechanism of inhibiting the binding of the intracellular Plk1 and substrate protein gives an important fact that can add a new anti-cancer efficacy in addition to the description of the known efficacy.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

The high throughput screening method of the anticancer substance of the present invention may be used for screening an anticancer substance that inhibits the binding of a carcinogen and a substrate protein that binds the carcinogen, but in the following examples, a cell-related cell cycle control protein Only a method of screening for the inhibition of binding of phosphorus Plk1 (polo-like kinase 1) and substrate proteins that bind to it will be described.

In addition, in the following examples, only a method for analyzing a candidate substance that inhibits the binding between the PBD of Plk1 and the substrate protein of the present invention by a Surface Plasmon Resonance Imaging (SPRI) system is described. It will be apparent that analysis using elements is also easy for those skilled in the art.

Example 1: Securing Plk1 (polo-like kinase 1) and Substrate Proteins

1-1: Recombinant Cloning and Protein Expression of Plk1 and Substrate Proteins

A cDNA library prepared using reverse transcriptase from total RNA extracted from human cell lines was used as a template, and Plk1 (the entire Plk1 and the PBD moiety) and various substrate proteins (stasamine, catanine p80, TCTP, Cdc25C, NudC, Myt1, and ka) 5'-end and 3'-end of each primer specific for the nucleotide sequence of each gene of tannin p60, APC8, Myt1-2, Grasp65) and designed to contain restriction enzymes suitable for cloning (Table 1). After the PCR product (Fig. 2a) obtained using the respective restriction enzymes, a recombinant clone was prepared by reacting the expression vector cut by the same restriction enzyme with T4 DNA ligase for 5 hours at 16 ° C. It was. At this time, three types of expression vectors were used depending on the type of each tag protein. These include a pET21a vector (Novagen, USA) and glutathione-S transferase (GST) protein with 6xHis-tag. PGEX4T-1 vector (Amersham Bioscience, USA), and the maltose binding protein (Maltose binding protein (MBP) is a vector that is designed to fuse (New England Biolabs, USA).

Nucleotide sequence of the primer used for PCR and restriction enzyme contained in the primer primer Primer sequencing Restriction enzyme PBD 5'-Terminal 5'-cggatccgaattctttcgattgctcccagc EcoR I PBD 3'-end 5'-aataaaaagcttttaggaggccttgag Hind III Stathmin 5'-end 5'-cttggatccaccatggcttcttct BamH I Stathmin 3'-end 5'-tctcaagcttgtcagcttcagtct Hind III Katanin p80 5'-end 5'-agcggatccatggccacccct BamH I Katanin p80 (kataninp80) 3'-end 5'-ctgctcgaggtccagactggc Xho I TCTP 5'-end 5'-gtcggatccatgattatctacc BamH I TCTP 3'-end 5'-gccaagcttacatttttccattt Hind III Cdc25C 5'-Terminal 5'-tcgggatccatgtctacgga BamHI Cdc25C 3'-end 5'-gctaagctttgggctcatgtcc Hind III NudC 5'-end 5'-aatggatccatgggcggagag BamHI NudC 3'-end 5'-gcgctcgaggttgaatttagcct Xho I Myt1-1 5'-end 5'-aggggatccatgctagaacgg BamHI Myt1-1 3'-end 5'-gtctaagcttggttgggtcta Hind III Grasp65 5'-end 5'-gcgaattcatgggcctgggcgtca EcoR I Grasp65 5'-end 5'-gcaagcttttctgtggtagagatc Hind III Katanin p60 5'-end 5'-gcggatccatgagtcttcttatga BamH I Katanin p60 (kataninp60) 3'-end 5'-gcctcgaggcatgatccaaactca Xho I APC8 5'-end 5'-gcggatccatggtcccggtggctg BamH I APC8 3'-end 5'-gcgtcgactggcgtgacagaagac Sal I Myt1-2 5'-end 5'-gcggatccatgctagaacggcctc BamH I Myt1-2 3'-end 5'-gcaagcttggtccagggcatccct Hind III

 2A is agarose gel photographs of genes encoding substrate proteins of Plk1 (Polo-like kinase 1) produced by PCR from cDNA libraries of animal cells.

For protein expression, E. coli BL21 / DE3 (Novagen, USA) transformed by the above-recombinant clones was shaken at 37 ° C. until the OD (Optical density, A600 nm) became 0.6, followed by IPTG (Isoprophyl -beta-D-thiogalactopyranosid) was added to a final concentration of 1 mM, followed by incubation at 25 ° C. for 16 hours to induce protein expression. Next, the cultured cells were centrifuged to suspend the precipitated cells in PBS buffer solution (pH7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ ), and then ultrasonically (Branson, Sonifier 450, 3 kPa, 3 W, 5 min) and crushed and centrifuged to obtain a recombinant protein solution dissolved in a soluble fraction. The expression and amount of protein were confirmed by SDS-PAGE for both supernatant (Soluble fraction) and precipitate (Insoluble fraction) obtained by centrifugation of the crushed bacterial solution.

2B and 2C show the expression patterns of the bacterial clones BL21 / DE3 (Novagen, USA) of the recombinant clones. That is, FIG. 2B shows the results of establishing conditions for removing GST-tag from the PBD (polo-box domain) protein using SDS-PAGE results (A) and thrombin after expressing the Plk1 protein fused with the GST protein (B). ) Is a picture showing.

The present inventors divided the whole gene, kinase domain and polo-box domain (PBD) of Plk1, cloned each to have 6xHis-tag and GST-tag, and induced protein expression in the bacteria, but only GST-PBD was in a water-soluble state. (soluble form) (Fig. 2b, A). In addition, thrombin was treated to cut the thrombin cleavage site present between PBD and GST in the clone to remove GST-tag protein from the PBD protein for screening (FIG. 2B, B). As a result, it was found that GST-tag was efficiently removed when 0.06 unit of thrombin was treated for 16 hours.

Figure 2c is a picture showing the expression of the substrate proteins, 6xHis-tag substrate protein with the stamina, TCTP, Cdc25C, APC8 is expressed in a soluble form (A), glutathione-s transferase (glutathione) -S transferase; GST) fused protein was expressed stasmin, TCTP, Cdc25C, NudC, Myt1 and APC8 (B), maltose binding protein (MBP) fused protein is stasmin and It was found that NudC was expressed (C).

1-2: Purification of Plk1 and Substrate Proteins

The protein obtained in 1-1 above was used as the cell extract or purified according to each purpose. Purification methods were performed differently for each tag protein. Specifically, Ni-NTA (nickel-nitrilo-triacetic acid) resin (resin) is included when 6xHis-tag is included, and glutathione agarose bead (GSH agarose bead) is included when GST-tag is included. If column, MBP-tag is included, purification was performed using amyloase resin.

Specifically, i) When His-tag is included, the solution containing the expressed protein is mixed with activated Ni-NTA (nickel-nitrilo-triacetic acid) resin after washing with distilled water and PBS buffer solution. The reaction was allowed to slowly react at 4 ° C. for 1 hour to allow the protein to bind to the resin. After 1 hour, the resin was washed with PBS buffer and 10 mM imidazole buffer, and finally, the target protein was recovered from the resin with 100 mM imidazole buffer. The recovered protein was placed in a Dialysis bag and dialyzed with Tris buffer for one day.

ii) When GST-tag is included, the column containing glutathione agarose bead is washed with PBS buffer and activated, and then the protein is expressed by passing the solution containing the expressed protein. It was allowed to bind to the beads. Next, the target protein was collected by eluting from the beads with a buffer solution containing glutathione (pH 7.5, 20 mM GSH, 50 mM Tris, 200 mM NaCl).

iii) When MBP-tag is included, the solution containing the expressed protein is washed with distilled water and PBS buffer and slowly mixed with the activated amylose resin, and then reacted at 4 ° C. for 5 hours to react the protein. It was bound to. After 5 hours, the supernatant was removed and washed with PBS buffer containing 10 mM maltose for 10 minutes, followed by elution buffer (PBS buffer containing 500 mM maltose; elution buffer). 2 hours, the target protein was recovered from the resin.

Example 2: Manufacture of Protein Chip

2-1: Fabrication of gold chip coated with gold thin film

To fabricate gold chips coated with gold thin films, thin (22 mm x 22 mm x) using commercially available electron-beam evaporators (Dada, Korea) 0.3 t) On the glass plate, the binder chromium was coated with 2 nm, and then a 47 nm gold thin film was attached to prepare a gold chip. The gold chip coated with the thin film of the glass substrate was treated with a Piranha solution (Piranha solution; 70% H ₂ SO ₂ , 30% H ₂ O ₂ ) at 65 ° C. for 30 minutes, and then 10 mM dissolved in ethanol. It was immersed in the solution of 11-mercapto-1-undecanoic acid (MUA) for 16 hours to form self assembled monolayers (SAMs).

In addition, in order to activate the gold chip formed with the self-assembled monolayer, 0.4 M sodium hydroxide solution and 2-methoxy-ethyl-ether solution were mixed at a volume ratio of 1: 1, and epichlorohydrin was 0.6 M in the mixed solution. After making it to react with the chip at room temperature for 4 hours. The activated chip surface was coated with dextran by reacting with dextran solution dissolved in 0.3 mg / ml in 0.1 M sodium hydroxide solution at room temperature for 20 hours. The surface of dextran was activated using ephichlorohydrin and immersed in 100 mM phosphate buffer (pH 7.0) of reduced L-glutathione (GSH) dissolved at 44 mM concentration, as in the above method. After reacting for 20 hours at, the reaction solution was discarded and the chips were washed with distilled water. In addition, in order to block the unreacted activator on the chip surface, 1 M ethanol amine solution was treated on the chip surface and reacted at 37 ° C. for 4 hours.

2-2: Immobilization of GST-tag Protein on Gold Chip Surface

An experiment was performed to confirm whether the obtained proteins bind to the gold chip on the glutathione surface. Bacterial cell extracts containing 20% glycerol overexpressed with each of the substrate proteins (NudC, Cdc25c, Stathmin, TCTP) fused with GST-tag obtained in Example 1 were dispensed into 384 microwell plates Using an automated robot arrayer (Proteogen, CM-1000), spotting was performed on a gold chip surface-treated with glutathione with a pin of 335 μm in diameter under conditions of 75% internal humidity. The control group of the experiment used BSA (1 mg / ml). By specific reaction of glutathione on the chip surface and GST protein, only the substrate protein having GST-tag was selectively arrayed on the chip. After reacting for about 30 minutes at room temperature, the unreacted extract was washed three times with PBST buffer (10 mM phosphate buffer, 150 mM NaCl, 0.05% Tween20, pH7.4), and then twice with distilled water. In addition, blocking with GST protein, and then immersed in a solution containing 1 mg / ㎖ purified PBD, and reacted for about 1 hour at room temperature so that the binding between the PBD and the substrate protein. After 1 hour of reaction, the reaction product was washed with PBS buffer to remove unreacted product, dried over nitrogen, and observed by SPRI (FIG. 3). 3 shows whether the substrate proteins bind to Plk1. As shown in FIG. 3, the control BSA did not bind to the chip surface at all, and NudC except for stasmin, whose binding was not known, In case of Cdc25c and TCTP, signal intensity was increased. Therefore, it was confirmed that the screening conditions by the detection of the PBD of Plk1, the protein chip of SPM and the SPRI were established.

Example 3: Screening Candidates for Inhibiting Binding Between Plk1 and Substrate Proteins

NudC and Cdc25c, which showed the best imaging results on SPRI among the substrate proteins bound to PBD of Plk1 in Example 2, were mainly used for screening. As a library of candidate candidates for binding inhibition, more than 10,000 chemical extracts from 6,300 chemical libraries distributed by the Korea Research Institute of Chemical Technology and 4,200 plant extracts from the Korea Plant Institute of Biotechnology were used. The screening process was carried out in the same manner as in Example 2 except for mixing the binding inhibitor candidate with the PBD of plk1, and then spotting on the protein chip on which the substrate protein was loaded.

That is, a sample was prepared by mixing 1 mg / ml of purified PBD protein and 50 μM chemicals (chemical library) in a 1: 1 (v / v) ratio on a protein chip to which substrate proteins were immobilized. Samples were prepared by mixing a plant extract (plant extract library) at a concentration of 1: 1 (v / v), and then mixing each sample well in a 384 microwell plate and reacting at room temperature for 1 hour, Spotting. As a negative control, BSA was used as a positive control, and a PBD protein that did not react with chemicals or plant extracts was used. After reacting for about 30 minutes at room temperature, the unreacted materials were washed three times with distilled water, dried using nitrogen gas, and analyzed in an SPRI system to screen candidate binding inhibitors (FIG. 4A). Figure 4a is an excerpt of the primary screening results showing the discovery results of the binding inhibitor candidate by the ultra-fast screening method using a protein chip and unlabeled SPRI. All screening groups were duplicated for screening and PBD protein was not reacted with chemicals or plant extracts as a positive control. By comparing the signal intensity with the positive control, we chose the one with no or weak intensity. The lower signal intensity indicates that the PBD is unable to bind to the substrate protein by binding to a PBD or a substrate inhibitor. In FIG. 4A, the y-axis represents the number of rows, and the number indicated on the x-axis represents the number of spots. In addition, the bottom two spots are the control and the squares above indicate that they were selected at screening compared to the control. That is, in FIG. 4A, it can be seen that one sample is selected.

In addition, Figure 4b is the SPRI image of the water extract of the SPRI secondary screening results of the binding inhibition candidates selected in the primary screening of Figure 4A again according to the concentration of the inhibitor using the software installed in the program of SPRI The signal intensity is quantified. In the photograph, 15 selected binding inhibitory substances selected in the primary screening were shown at 10x (1 mg / ml), 100x (0.1 mg / ml) and 1000x (0.01 mg / ml) according to the concentration, as indicated on the y-axis. Diluted and compared the inhibitory effect of the binding of the substrate protein and PBD according to the concentration of the inhibitor. As the dilution ratio of the inhibitor increases, the signal intensity increases. Six water extracts from 1, 6, 7, 8, 9 and 11, with no or low signal intensity due to the large inhibitory effect at 10x dilution, were selected for the second screening (1: sulphur, 6: baekgu (sec) ), 7: Mui, 8: Mountain medicine, 9: Tangerine core, 11: 3).

Secondary screening was performed using SPRI in the same manner again according to the concentration of the binding inhibitory candidate detected in the first screening. Finally, different types of proteins (IgG-proteinG, or RB-E7) were used to analyze whether the binding inhibition candidates detected in the secondary screening were specific for inhibition of binding between PBD and PBD of Plk1. Third screening was performed (FIG. 4C). The image of FIG. 4C shows the results of confirming whether or not the 6 water extracts selected as binding inhibitor candidates of 4b and 4 methanol extracts selected in the primary screening are specific for inhibiting binding of PBD protein and substrate protein. . In the case of methanol extract, only 4 extracts were selected in the primary screening. Therefore, the results of the experiment conducted simultaneously with the secondary screening which is confirmed according to the concentration and the tertiary screening which analyzes whether the binding inhibition is specific between target proteins are shown. 4c is shown together. The left side of FIG. 4C is an image showing the inhibitory effect of binding of the PBD to the substrate protein NudC according to the concentration of the inhibitor (0.1, 0.05, 0.02, 0.01 mg / ml) as in the second screening as a control, and the right side is the selected binding. SPRI was used to determine whether the inhibitory effect between these proteins was demonstrated using immunoglobulin G (IgG) and Protein G, which is known as a protein that binds to it, to see if the inhibitory substance binds to all proteins. Inspected using. As a result, the tangerine core and the three-chile in the six water extracts (1: turmeric, 2: baekdu (cho), 3: radish, 4: powder (formula), 5: tangerine nucleus, 6: samchil) signal the binding of IgG and Protein G. By showing an image of decreasing intensity, it was determined that these two substances had a nonspecific inhibitory effect, and these two candidates were excluded from binding inhibitor selection. Also, among the four methanol extracts (1: splendid epiphysis, 2: copaiba, 3: Bukinha, 4: white locust), the fourth locust tea was found to have a weak binding inhibitory effect between PBD and the substrate protein NudC. Binding inhibitors were excluded from selection.

In experiments with PBDs and substrate proteins, NudC and Cdc25c, the final screening by SPRI revealed that, over 6,300 chemicals were not found to bind, and 4,200 plant extracts were 6 types (water Substances showing inhibition of binding of extract 1: sulphate, 2: baekdu (cho), 4: acid (formula); methanol extract 1: spliced phalange, 2: copiva, 3: under Bukin) were detected (FIG. 4C).

Example 4: Apoptosis Analysis in Animal Cells of Screened Binding Inhibitors

Through the procedure of Examples 1 to 3, it was examined by MTT analysis in animal cells whether the six binding inhibitory substances detected in the plant extracts show the ability of cancer apoptosis.

MTT screening method was carried out by dehydrogenase reaction, and the yellow water-soluble matrix MTT tetrazolium was converted into non-aqueous MTT formazan (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide). It is a test that takes advantage of the ability of mitochondria in reducing cells. The absorbance of MTT formazan peaks at a wavelength of 540 nm, and the absorbance measured at this wavelength reflects the concentration of living, metabolized cells. Cancer cells cultured after the anticancer agent or the candidate substance is administered to the cell culture medium have a smaller proportion of surviving cells, depending on the concentration of the treated substance, compared to the cancer cells of the untreated control group. The rate of reduction depends on the type and concentration of the anticancer agent or candidate and on the sensitivity of the cancer cell to the substance. The susceptibility to anticancer agents or candidates is compared as a percentage of the absorbance of the untreated material in the well treated with the material. Cells that are not killed and have metabolic activity can be seen indirectly by measuring absorbance. The concentration of a substance that can reduce the concentration of live cancer cells to 50% compared to the untreated group (control) after treatment is called IC _50.In actual experiments, the average value of three or more experiment results is calculated to reduce the error. .

The present inventors put HeLa, HEK-293, SKBR-3, MCF-7, BT-474 and MDA-MB-231 cells as animal cells in a 96 well plate so that the cell number is 500-1000 / well, and then 37 ° C. Incubated at 5% CO ₂ for one day. The plant extract screened in Example 3 was treated to cells cultured at concentrations of 0, 0.02, 0.2, 1.0 mg / ml. The treated cells were incubated for 2 to 3 days at 37 ° C. and 5% CO ₂ , and then MTT compounds were added to each well to a concentration of 1 mg / ml. The cells to which the MTT compound was added were reacted for 4 hours at 37 ° C. and 5% CO ₂ , and then the cell medium was removed, and MTT formazan was dissolved in DMSO and absorbance was measured at 540 nm.

Table 2 shows the results of MTT analysis of plant extracts in animal cells with IC ₅₀ values. In the MTT search, three kinds of water extracts and three kinds of methanol extracts showed a slight IC ₅₀ value per cell and cell death, but cell death according to each extract showed similar tendency. The number of pulmonary extracts from water extracts showed IC ₅₀ values at concentrations of 0.1-0.5 mg / ml in most cells. However, water extracts No. 2 and No. 4 extracts were more apoptotic than other extracts, and most of the cells were unable to obtain IC ₅₀ values. Meanwhile, in the case of methanol extract, cell death was performed at a much lower concentration than water extract. Extracts 1, 3, and 4 were able to obtain IC ₅₀ values below the concentration of 0.1 mg / ml. However, the second extract showed slightly different results for each cell. Cell death was performed at a higher concentration than the other extracts, and some cells could not obtain IC ₅₀ values at the tested concentration. On the other hand, IC ₅₀ values of water extracts were obtained at concentrations of 10 to 100 times higher than methanol extracts. This is due to differences in the properties of extracts dissolved in different solvents such as hydrophilicity and charge. It is anticipated that this may have affected the migration of binding inhibitory substances into cells. Thus, for water extracts, more effective IC ₅₀ values are expected to be obtained by applying the methods used for drug delivery into cells.

Example  5: Screened  Binding inhibitory substances GST pull - down By way Plk1 of PBD And temperament Protein liver  Binding inhibitory effect analysis

GST pull-down method is one of several approaches that reveals the interrelationship between proteins. The GST pull-down method separates GST-fused proteins using glutathione (GSH) beads having affinity to the GST protein. It is used to find or confirm the binding between proteins.

       In the present invention, using the GST pull-down method, it was once again confirmed whether the extracts obtained by high throughput screening (HTS) inhibit the mutual binding between the PBD domain of Plk1 and the substrate protein. Gd-PBD expressed in Escherichia coli was attached to glutathione (GSH) Bead, and NudC (NudC) fused with Cdc25C (Cdc25C-His) and MBP-tag fused with His-Tag, which was used for screening with respective extracts and substrate proteins. -MBP) protein was reacted with each other. After reaction at 4 ° C. for 2 to 4 hours, the reaction mixture was removed with PBS solution and electrophoresed on 12% SDS-PAGE Gel. After electrophoresis, proteins were transferred from gel to nitrocellulose filter through 'Transfer' process (90 minutes at 200mA), and Western Blotting was performed using Anti-His antibody, Anti-MBP antibody and Anti-GST antibody.

FIG. 5 analyzes the binding inhibitory effect between PBD and substrate protein of Plk1 by the GST pull-down method of the binding inhibitory candidates selected in FIG. 4C. The results were almost identical to those of the screening using SPRI. That is, the extracts 1, 2, and 4 in the water extract results showed the inhibition of the binding between PBD-Cdc25C and PBD-NudC. On the other hand, as shown in Figure 4c extract 3 showed a weaker binding inhibitory effect than the other 1, 2, 4 extract in the screening, even at a concentration of 0.05mg / ㎖ did not show a binding inhibitory effect Is the same result as the GST pull-down result. Since GST pull-down requires a large amount of protein and samples to carry out extracts at various concentrations, it is difficult to experiment. However, the screening using SPRI can be seen as a slight difference in these results because it can be easily confirmed on the same chip with the inhibitory effect of each concentration. In methanol extract, extracts 1, 2 and 3 inhibited the binding between PBD-Cdc25C and PBD-NudC. On the other hand, in case of extract 4, HTS screening (FIG. 5) showed no binding inhibition effect compared to the other extracts 1, 2, 3, GST pull-down also showed the same result. Through the GST pull-down results, we can confirm that HTS screening using the protein chip and SPRI in the present invention is a very effective and accurate screening method.

Table 3 summarizes the results obtained through Examples 3 and 5.

Chemical library Plant extract library Number of libraries used for SPRI analysis 6300 4200 No. of binding inhibition candidates detected by SPRI analysis 0 6 Results of GST Pull-down Analysis 0 6

As shown in Table 3, no substances showing binding inhibition were detected in 6300 chemicals, and six kinds of binding inhibitors were detected in 4200 plant extracts.

Table 4 shows the names of the plant extracts detected, Chinese characters / English names, Latin herbal drugs, extract solvents.

Product Name Kanji / English name Latin Medicine menstruum Huang 蒲黃 Typahe pollen water Baekdugu (sec) 白 豆寇 (炒) Amomi Cardamomi Fructus water Acid medicine (meal) 山藥 Dioscoreae Rhizoma water Bowl 大 接骨 丹 Torricellia angulata var. intermedia Methanol Copaiba Copaiba Copaifera Iansdorfii Methanol Bukinha Buchinha Luffa operculata Methanol

As described in detail above, according to the present invention, a protein chip having a substrate protein of Plk1, which is a carcinogenic protein or a substrate protein binding site of Plk1, is immobilized, a method for screening an anticancer substance using the protein chip, and the screening method. It has the effect of providing an anticancer substance screened by.

The ultra-fast screening method of anti-cancer material according to the present invention is a high-throughput screening (HTS) in a large amount of fast and convenient screening inhibitors between carcinogens and their substrate proteins using protein chips and SPRI bioassay technology. It can be useful for discovering new anticancer drugs that can induce apoptosis of cancer cells.

<110> Korea research institute of bioscience and biotechnology <120> Plk1-Based Protein Chip, Method for High Throughput Screening of          Anticancer Agents Using the Protein Chip and Anticancer Agents          Preparing Thereby <130> P07-B088 <150> KR10-2006-0048372 <151> 2006-05-29 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cggatccgaa ttctttcgat tgctcccagc 30 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aataaaaagc ttttaggagg ccttgag 27 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cttggatcca ccatggcttc ttct 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tctcaagctt gtcagcttca gtct 24 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agcggatcca tggccacccc t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctgctcgagg tccagactgg c 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtcggatcca tgattatcta cc 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gccaagctta catttttcca ttt 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcgggatcca tgtctacgga 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctaagcttt gggctcatgt cc 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aatggatcca tgggcggaga g 21 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gcgctcgagg ttgaatttag cct 23 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 aggggatcca tgctagaacg g 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gtctaagctt ggttgggtct a 21 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcgaattcat gggcctgggc gtca 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcaagctttt ctgtggtaga gatc 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gcggatccat gagtcttctt atga 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gcctcgaggc atgatccaaa ctca 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcggatccat ggtcccggtg gctg 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gcgtcgactg gcgtgacaga agac 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gcggatccat gctagaacgg cctc 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gcaagcttgg tccagggcat ccct 24

Claims (9)

A protein chip characterized in that a substrate protein of Plk1 (polo-like kinase 1) is immobilized on a substrate. The method of claim 1, wherein the substrate protein of Plk1 is stasmin, katanin p80 (Katanin p80), TCTP (translationally controlled tumor protein), Cdc25C (cell division cycle 25C), NudC, Myt1, catanine p60 ( Katanin p60), APC (Anaphase promoting complex), Myt1-2, Grasp65, Cohesin, Mklp1, Mklp2, Asp, Nlp, Wee1, Pin1, BRCA2, p53. A protein chip characterized in that a PBD (polo-box domain) of Plk1 (polo-like kinase 1) is immobilized on a substrate. Screening method of anticancer substance comprising the following steps: (a) reacting a substrate protein of Plk1 (polo-like kinase 1) and a candidate substance that inhibits binding of Plk (polo-box domain) of Plk1 on the protein chip of claim 1; And (b) selecting a candidate substance that inhibits the binding of the substrate protein of Plk1 and PBD of Plk1 as an anticancer substance. Screening method of anticancer substance comprising the following steps: (a) reacting a substrate protein of Plk1 (polo-like kinase 1) and a candidate substance that inhibits binding of Plk (polo-box domain) of Plk1 on the protein chip of claim 3; And (b) selecting a candidate substance that inhibits the binding of the substrate protein of Plk1 and PBD of Plk1 as an anticancer substance. The method of claim 4 or 5, wherein the candidate that inhibits the binding between the PBD of Plk1 and the substrate protein is characterized in that it is analyzed by Surface Plasmon Resonance Imaging (SPRI) system. The method according to claim 4 or 5, wherein the candidate which inhibits the binding between the PBD of Plk1 and the substrate protein is analyzed using a fluorescent substance or a radioisotope. The method according to claim 4 or 5, further comprising: (c) analyzing an animal cell with a Tetrazolium-based colorimetric (MTT) assay to determine whether the anticancer substance selected in step (b) shows cancer cell death ability. Method comprising the as. (Typahe pollen) extract, baekdu (cho) ( Amomi Cardamomi Fructus ) Extract, Herbal Medicine ( Dioscoreae ) Rhizoma ) Extract, Periphyllium ( Torricellia) angulata var . intermedia ) extract, Copaiba, Copaifera Lansdorifii ) Extract and Buchinha, Luffa operculata ) anticancer composition containing any one or more extracts selected from the group consisting of the active ingredient.

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