JP2008096397A - Method for screening anti-carcinogenic substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening an anti-carcinogenic substance easily and quickly. <P>SOLUTION: The method for screening anti-carcinogenic substance candidates contacts Cdt1 protein with Geminin protein and measures binding of the Cdt1 protein and the Geminin protein, and then selects a material to be tested that reduces the binding as an anticarcinogenic substance, in the presence of a material to be tested. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a simple and rapid screening method for anticancer substances.

  Cdt1 is an essential protein for replication of chromosomal DNA and plays a role in binding the MCM complex, which is a replication helicase, to the chromosome in cooperation with ORC and CDC6. This MCM is essential for the DNA replication reaction, and loading of the MCM is performed in the G1 phase preceding the DNA synthesis phase (S phase). In order to double the chromosomal DNA exactly once (ie, to suppress reloading and re-replication of MCM after S phase), the activity of Cdt1 must be sufficiently suppressed after S phase. Initially, the suppression was thought to be due to the binding of a protein called Geminin to Cdt1 (Non-patent Documents 1 and 2). Therefore, Geminin was initially thought to be suppressive to cell proliferation and possibly a tumor suppressor gene. Actually, when Geminin is excessively expressed, cell proliferation is suppressed (Non-patent Document 3).

  On the other hand, the present inventor has considered that Cdk plays a central role in re-replication suppression from previous studies. That is, it has been known that Cdk phosphorylates ORC and CDC6 to suppress the function at the same time as turning on replication and suppressing re-replication. Therefore, Cdt1 was also studied by considering that it was controlled by Cdk independently of Geminin coupling. And it was clarified that Cdt1 is also phosphorylated by Cdk, and as a result, the DNA-binding ability is lowered, and it is also led to proteolysis via ubiquitination by SCF-Skp2 (Non-patent Document 4). Furthermore, it has also been clarified that it is proteolytically mediated through ubiquitination by Cul4-DDB1-PCNA in a form coupled with replication (Non-patent Document 5).

  In other words, Cdt1 is strictly suppressed and controlled by a plurality of mechanisms after the S phase, which seems to indicate that Cdt1 control is central in cell cycle control of replication. Then, in order to investigate the effect of Cdt1 deregulation, when Cdt1 was overexpressed in normal human cells, it was found that mild but significant chromosomal damage was caused and chromosomal instability was induced (Non-patent Document 6). . No such phenomenon is observed in the overexpression of ORC1 and CDC6, indicating the importance of Cdt1. And when considered together with a report that Cdt1 can promote canceration of cells (Non-patent Document 7), it can be said that deregulation of Cdt1 is a new molecular mechanism from chromosomal instability to canceration. Let's go.

  On the other hand, cancer is one of the three leading causes of death, and many therapeutic agents and treatment methods have been studied. However, current therapeutics or treatment methods may be effective for certain patients but not for some patients, and some may have fatal side effects. For example, the mainstream of therapeutic agents up to now uses substances that show nonspecific toxicity to DNA and cells as anticancer agents, and have large side effects. In recent years, a number of so-called molecular targeting agents have been developed, which clarify the difference between cancer cells and normal cells at the molecular level and develop anticancer agents targeting them, and have become effective anticancer agents. Thus, it is required to search for various types of therapeutic agents and treatment methods and develop effective means for as many patients as possible.

Wohlschlegel, J. A. et al. Science290, 2309-2312, 2000 Tada, S. et al. Nat.Cell Biol. 3, 107-113, 2001 Shreeram, S. et al. Oncogene 21, 6624-6632, 2002 Sugimoto, N. et al. J. Biol. Chem. 279, 19691-19697, 2004 Nishitani, H. et al. EMBO J. 25, 1126-1136, 2006 Tatsumi, Y. et al. J. Cell Sci., 119, 3128-3140, 2006 Arentson, E. et al. Oncogene 21, 1150-1158, 2002

  An object of the present invention is to provide a simple and rapid screening method for anticancer agents.

  As a result of intensive studies to solve the above problems, the present inventor has paid attention to the DNA replication mechanism, and binds Cdt1 protein necessary for DNA replication to Geminin protein, which is one of the proteins responsible for the function regulation. Based on the idea that inhibiting cancer can damage cancer cells, the inventors have obtained the knowledge that a substance that inhibits the binding between Cdt1 protein and Geminin protein can be screened as an anticancer drug candidate, and the present invention It came to be completed.

  That is, the present invention is characterized in that Cdt1 protein and Geminin protein are brought into contact with each other in the presence of a test substance, the binding between Cdt1 protein and Geminin protein is measured, and the test substance that reduces the binding is selected as an anticancer drug candidate. This is a screening method for anticancer drug candidates.

  In the screening method described above, for example, either Cdt1 protein or Geminin protein is preferably immobilized.

  The screening method may further include administering the selected anticancer drug candidate to a cancer cell and observing a change in proliferation of the cancer cell.

  The present invention also provides a screening kit for candidate anticancer agents, comprising Cdt1 protein and Geminin protein.

  The present invention provides a screening method for anticancer agents. Such a screening method is useful as a primary screening for screening a large number of test substances at high throughput because it can easily and rapidly screen for anticancer drug candidates.

The present invention will be described in detail below.
The present invention relates to a screening method comprising measuring the binding between Cdt1 protein and Geminin protein in the presence of a test substance, and selecting a test substance that inhibits the binding as an anticancer drug candidate. The present invention focuses on the difference in the mechanism of chromosomal DNA replication control between cancer cells and normal cells, and aims to develop a new molecular target anticancer agent.

  As described above, Cdt1 hyperfunction is an adverse event for normal cells, but it did not cause rapid cell damage. However, at the same time, it was reported that reducing the protein content of Geminin with siRNA caused significant chromosomal damage / re-replication to cell death (Melixetian, M. et al. J. Cell Biol. 165, 473-482, 2004; Zhu, W. et al. Mol. Cell Biol. 24, 7140-7150, 2004). This seemed strange at first glance considering that Cdt1 is functionally suppressed by multiple mechanisms and that Cdk also has multiple suppression pathways. In fact, as described above, even when Cdt1 is overexpressed in normal cells, no cell damage up to that point is observed. Thus, almost all of these siRNA experiments have been performed using cancer cell lines, and it has been found that ORC1, CDC6, Cdt1, and MCM proteins are overexpressed in many cancer cell lines. (Tatsumi, Y. et al. J. Cell Sci., 119, 3128-3140, 2006), we thought that the influence of Geminin reduction would be extremely strong in cancer cell lines. From such a background, the present inventor considered that a compound that inhibits the binding between Cdt1 and geminin may be an anticancer agent that selectively damages cancer cells. For example, as described above, when the expression of Geminin protein in cancer cells is reduced using siRNA, chromosomal damage / re-replication is caused and the cells die (Melixetian, M. et al., Supra, Zhu , W. et al., Supra), which is thought to be due to the excessive function of Cdt1 due to the decrease in Geminin. Therefore, cancer cells can be expected to die if the binding between Geminin and Cdt1 is inhibited so that the function of Cdt1 becomes excessive. On the other hand, since other control mechanisms function normally in normal cells, even if the binding between Geminin and Cdt1 is similarly inhibited and the function of Cdt1 becomes excessive, the effect is expected to be small. In fact, when Cdt1 is overexpressed in normal cells, only relatively mild damage occurs, whereas when it is overexpressed in cancer cell lines, more remarkable chromosomal damage and cytotoxicity are observed. (Unpublished data and Vaziri, C. et al. Mol. Cell 11, 997-1008, 2003).

  Therefore, in the present invention, the binding between Cdt1 protein and Geminin protein in the presence of a test substance is measured, and a test substance that inhibits the binding is selected as a candidate for an anticancer agent. Specifically, the screening method of the present invention comprises a step of contacting Cdt1 protein and Geminin protein in the presence of a test substance, a step of measuring the binding between Cdt1 protein and Geminin protein, and the absence of the test substance. And selecting a test substance that reduces the binding between Cdt1 protein and Geminin protein as a candidate for an anticancer agent as compared with the case where it is measured in the presence of other substances.

  Cdt1 protein can be obtained by any method known in the art. For example, based on the nucleotide sequences registered in public databases (GenBank accession numbers NM_030928 (cDNA) and NP_112190.1 (protein); SEQ ID NOs: 1 and 2), methods known in the art (for example, gene sets) It can be manufactured using a replacement method. In addition, the Cdt1 protein may be a protein fragment as long as it maintains the ability to bind to the Geminin protein. Such a protein fragment can be easily prepared by preparing an arbitrary peptide based on the above base sequence and confirming the binding ability of the peptide to Geminin protein.

  Similarly, Geminin protein can also be obtained by any method known in the art. For example, base sequences registered in public databases (GenBank accession numbers NM_015895 (cDNA) and NP_0567979.1 (proteins) ); Can be prepared based on SEQ ID NOs: 3 and 4). Similarly, the Geminin protein may be a protein fragment as long as it has the ability to bind to the Cdt1 protein.

  The Cdt1 protein and / or Geminin protein may be prepared as a fusion protein as long as these proteins bind to each other for the sake of ease of purification or for the convenience of the screening test described later. For example, a fusion protein with a known affinity tag, specifically glutathione-S-transferase (GST), His tag, FLAG tag, etc. can be prepared.

  Further, the Cdt1 protein and / or Geminin protein may be in an isolated form or in a form such as in a cell extract.

  The kind of the test substance used in the present invention is not particularly limited. For example, the test substance can be any material factor, specifically a naturally occurring molecule such as an amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (such as sugar), steroid, glycopeptide Synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (aptamers, antisense nucleic acids, double-stranded RNA (RNAi), etc.), etc .; non-naturally occurring molecules, such as And low molecular organic compounds (inorganic and organic compound libraries, combinatorial libraries, etc.) prepared using combinatorial chemistry techniques, and the like, and mixtures thereof. The test substance may be a single substance, a complex composed of a plurality of substances, a transcription factor, or the like. When considering use as an anticancer agent, it is preferably a small compound that is easily taken up by cells, for example, an inorganic compound or an organic compound.

  In addition, as a test substance, a single test substance may be tested independently, or a mixture of several candidate test substances (including a library) may be tested. Examples of the library containing a plurality of test substances include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library).

  When the Cdt1 protein and Geminin protein are contacted in the presence of the test substance, the contact conditions are not particularly limited, and either a liquid phase system or a solid phase system can be used. A person skilled in the art can appropriately determine the contact conditions according to the type of test substance and the form of Cdt1 protein or Geminin protein (isolated protein, fusion protein, immobilized protein, protein-expressing cell, etc.). it can. For example, the Cdt1 protein and the Geminin protein may be added to a solution containing the test substance and brought into contact with each other, or the test substance and the Geminin protein (or the immobilized Cdt1 protein (or Geminin protein) may be contacted. Cdt1 protein) may be contacted at the same time.

  In addition, the effect of the test substance can be examined under several conditions. Such conditions include the time, amount, number of times, etc. for which the test substance is present. For example, a plurality of doses can be set by preparing a dilution series of the test substance. The existence time of the test substance can also be set as appropriate. For example, it can exist over a period of several minutes to several hours.

  Furthermore, when examining the additive action and synergistic action of a plurality of substances, test substances may be used in combination.

  The binding between the Cdt1 protein and the Geminin protein after contact can be measured according to any method known in the art. For example, immunological techniques (methods such as immunoprecipitation, Western blot analysis, and sandwich method) can be used.

  When performing immunoprecipitation, Cdt1 protein and Geminin protein are contacted in the presence of a test substance in the liquid phase, and then these binding complexes are immunoprecipitated using anti-Cdt1 antibody or anti-Geminin antibody. . Next, Western blot analysis is performed on the precipitated complex using an antibody against the other protein, and the binding between the Cdt1 protein and the Geminin protein is evaluated.

  When performing the sandwich method, either Cdt1 protein or Geminin protein is immobilized on a solid support (for example, a membrane, a filter, a microtiter plate, beads, etc.), and the test is performed with Geminin protein or Cdt1 protein. After adding and reacting the substance, unbound protein / test substance is washed / removed, followed by reacting with anti-Cdt1 antibody or anti-Geminin antibody, and binding of unbound or unbound anti-Cdt1 antibody or anti-Geminin antibody. By measuring the amount, the binding between the Cdt1 protein and the Geminin protein can be evaluated. In this case, the amount of binding can be measured, for example, by directly labeling the anti-Cdt1 antibody or anti-Geminin antibody and detecting the label, or reacting the labeled secondary antibody against the anti-Cdt1 antibody or anti-Geminin antibody. It is also possible to measure by detecting the label. Such labels include, but are not limited to, radioactive labels, enzyme labels, fluorescent labels, biotin-avidin systems, and the like. A person skilled in the art can select and use an appropriate label depending on the reaction conditions. Detection of the labeled signal can also be performed according to methods known in the art. For example, in the case of using an enzyme label, a substrate that develops color by degradation by enzymatic action is added, and the enzyme activity is obtained by optically measuring the amount of degradation of the substrate. The amount of antibody is calculated from the comparison. The fluorescent label can be detected and quantified using, for example, a fluorescence microscope or a plate reader. When a radioactive label is used, the radiation dose emitted by the radioactive label is measured with a scintillation counter or the like.

  As the substances and reagents used in the above-described measurement methods, commercially available products may be used, or those skilled in the art can appropriately prepare them. For example, anti-Geminin antibody is available from Santa Cruz Biotechnology Inc.

  Alternatively, the binding between Cdt1 protein and Geminin protein can be measured using another method known in the art, such as two-hybrid method, detection of protein binding using surface plasmon resonance (BIACORE, etc.), etc. .

  When the binding between the Cdt1 protein and the Geminin protein in the presence of the test substance is reduced as compared with the case where it is measured in the absence of the test substance or in the presence of another substance, The test substance is selected as an anticancer drug candidate. Here, the other substance includes a negative control.

  Further, in order to further evaluate the activity of the anticancer drug candidate selected as described above as an anticancer drug, the anticancer drug candidate may be administered to a cancer cell, and a change in the proliferation of the cancer cell may be observed. Furthermore, the effect of anticancer drug candidates on normal cells may be examined.

  Furthermore, the screening method can be more easily performed using a kit. The screening kit of the present invention comprises the above-mentioned Cdt1 protein and Geminin protein. The form of Cdt1 protein and / or Geminin protein is not particularly limited as long as they bind and the binding can be evaluated.

  The form of the screening kit is not particularly limited, but a container containing Cdt1 protein and / or Geminin protein, a solid phase on which Cdt1 protein and / or Geminin protein are immobilized (membrane, filter, microtiter plate, beads) Etc.), and can take the form of a container containing freeze-dried Cdt1 protein and / or Geminin.

  Moreover, the other component for measuring the coupling | bonding of Cdt1 protein and Geminin protein may be included, For example, an anti- Cdt1 antibody, an anti- Geminin antibody, a labeled secondary antibody etc. are mentioned.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Reference Example 1]
(1) Preparation and Purification of GST (Glutathione S-Transferase) -Cdt1 Fusion Protein Human Cdt1 cDNA (SEQ ID NO: 1) is based on reported sequence information (cDNA: NM — 0300928, protein: NP — 112190.1). Cloned from human cell mRNA. This cDNA was inserted into pGEX-6P-1 (Amersham), which is a commercially available GST fusion protein expression vector. After introducing pGEX-6P-1-Cdt1 into E. coli BL21 and culturing at 37 ° C. in 1 L culture solution until OD ₆₀₀ is about 0.7 to 0.8, IPTG (isopropyl-β-D-galactopyra Noside) 0.5 mM was added, and expression induction was performed at 25 ° C. for 2 to 3 hours.

  E. coli was collected by centrifugation and suspended in 40 ml of lysis buffer (20 mM Tris.HCl, pH 7.4, 200 mM NaCl buffer). Phenylmethylsulfonyl fluoride (PMSF, final concentration 1 mM), dithiothreitol (DTT, final concentration 1 mM) and protease inhibitor mix (Nacalai) were added, followed by lysis by sonication (BRANSON SONIFIER) while cooling. Triton X-100 was added to a final concentration of 0.1% and left on ice for 15 minutes. Thereafter, centrifugation was performed at 10,000 rpm and 4 ° C. for 10 minutes, and the supernatant was passed through a 0.45 μm filter (Whatman) to prepare a cell extract.

  Glutathione-Sepharose 4B (Amersham) 1.5 ml was equilibrated with 5 ml of A buffer (20 mM Tris.HCl, pH 7.4, 200 mM NaCl, 1 mM DTT, 0.1% Triton X-100) at 4 ° C. for 30 minutes. Made. The equilibrated glutathione-sepharose 4B and the cell extract were mixed and stirred at 4 ° C. for 2 hours for binding. Thereafter, the beads were washed three times with 10 ml of A buffer.

For elution, 1 ml of elution buffer (20 mM reduced glutathione, 20 mM Tris · HCl, pH 7.4, 200 mM NaCl, 1 mM DTT, 0.05% TX-100) was added to the beads, stirred at room temperature for 10 minutes, 2000 rpm, The supernatant was collected by centrifugation at 3 ° C. for 3 minutes. This was repeated 5 times. Dialysis was performed using B buffer (20 mM Tris.HCl, pH 8.0, 200 mM NaCl, 1 mM DTT, 0.05% Triton X-100, 5% glycerol) to obtain a final sample.
(2) Preparation and purification of His-geminin fusion protein The cDNA of human geminin (SEQ ID NO: 3) was cloned from human cell mRNA based on the reported sequence information (cDNA: NM — 015895, protein: NP — 0567979.1). This cDNA was inserted into pIVEX2.4b Nde (Roche), which is a commercially available 6 × histidine fusion protein expression vector. This vector was mixed with RTS500, an in vitro protein synthesis system from Roche, to express His-geminin protein.

  Take 0.5 ml of HIS-Select HC Nickel Affinity Gel (Sigma) on the column, equilibrate with 4 ml of C buffer (50 mM sodium phosphate, pH 8.0, 250 mM NaCl, 10 mM imidazole), add the above reaction solution, and then 1 hour Untied and bound through the column. Thereafter, washing was performed with 10 ml of C buffer.

  Elution was performed with 3 ml of C buffer containing 250 mM imidazole. Dialysis was performed using D buffer (10 mM Tris.HCl, pH 7.5, 100 mM NaCl, 25% glycerol) to obtain a final sample.

Cdt1-Geminin Binding Inhibitor Screening Method In this example, about 50 types of inhibitor candidate substances were used to screen for drugs that inhibit Cdt1-Geminin binding.

First, GST-Cdt1 diluted with 50 mM NaHCO ₃ —Na ₂ CO ₃ (pH 9.3) buffer or control GST (20 μg / ml) was added to a commercially available 96-well plastic plate (Becton Dickinson, Falcon 35-3912). 50 μl / well in concentration). Then, GST-Cdt1 was fixed to the plate by leaving still at room temperature for 2 hours. Then, the plastic plate was turned upside down to discard the liquid and wiped with a paper towel.

  1% skim milk was placed in a plastic plate (100 μ / 1 hole) and allowed to stand at room temperature for 1 hour (or overnight at 4 ° C.) to block the surface of the plate. The plastic plate was turned upside down, the liquid was discarded, washed once with PBS, and then wiped with a paper towel.

  After mixing His-geminin diluted with PBS (at a concentration of 0.125 μg / ml, 45 μl / well) and an inhibitor candidate substance dissolved in DMSO or DMSO alone (5 μl / well) as a control, It was allowed to stand at room temperature for 5 minutes (preincubation). In addition, the inhibitor candidate substance prepared the dilution series of 15 micromol, 150 micromol, and 1500 micromol, and was made to react, respectively.

  The solution after the pre-incubation was put into the plastic plate prepared as described above (50 μl / 1 hole) and allowed to stand at room temperature for 1 hour to form a complex of Cdt1 and Geminin. Thereafter, the plastic plate was turned over to discard the liquid, washed twice with PBS, and then wiped with a paper towel.

  Subsequently, a commercially available anti-Geminin antibody (Santa Cruz Biotechnology Inc., FL-209: SC-13015) diluted 2000 times with 1% skim milk is placed in a plastic plate (50 μl / well) and allowed to stand at room temperature for 1 hour. Thus, Geminin and anti-Geminin antibody were bound. Thereafter, the plastic plate was turned over to discard the liquid, washed once with PBS containing 1% Tween-20, washed twice with PBS, and then wiped with a paper towel.

  A commercially available peroxidase (HRP) -labeled anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology Inc., SC-2370) diluted 1000 times with 1% skim milk was placed in a plastic plate (50 μl / well) and allowed to stand at room temperature for 1 hour. By placing, the anti-Geminin antibody and the secondary antibody were bound. Next, the plastic plate was turned over, the liquid was discarded, washed twice with PBS containing 1% Tween-20, washed three times with PBS, and then wiped with a paper towel.

  Subsequently, color was developed using an ELISA kit (Nippon EnviroChemicals, 304-13161), and colorimetric determination was performed at 515 nm.

The results are shown in FIG. In FIG. 1, AJ are the following controls or inhibitor candidates:
A: negative control B: cis-oleic acid C: ursolic acid D: 12-OH-cis-oleic acid E: nervonic acid F: positive control G: trans-oleic acid H: MGDG (monogalactosyl diacylglycerol)
I: DGDG (digalactosyl diacylglycerol)
J: SQDG (sulfoquinovosyl diacylglycerol)

Geminin was not added to the negative control of A. In the positive control for F, no inhibitor candidate substance was added, and only Geminin was used. It has been confirmed that GST does not react with His-Geminin. The structure of this inhibitor candidate substance is shown in FIG. In FIG. 2, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently a fatty acid having 16 to 18 carbon atoms and containing 0 to 3 double bonds.

  From these results, it was found that SQDG, which is one of cis-oleic acid, ursolic acid and glycolipid, inhibits Cdt1-Geminin binding.

  In this example, SQDG that was confirmed to inhibit Cdt1-Geminin binding in Example 1 was used to examine its human cell proliferation inhibitory activity.

The cells used in this experiment are human uterine cancer-derived HeLa-S3 cells (purchased from Human Science Promotion Foundation (Chuo-ku, Tokyo)). As the medium, Eagle's minimum essential medium (MEM) (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with fetal bovine serum 10% (v / v), 250 μg / ml fungizone, 300 μg / ml L-glutamic acid was used. . The culture was performed at 37 ° C. in a 5% CO ₂ incubator.

  SQDG was dissolved in the medium shown above. However, since SQDG is hardly soluble in water, it was once dissolved in DMSO (dimethyl sulfoxide) and dissolved in the above medium. In addition, the final concentration of DMSO in the culture medium is 1% or less in all the test groups, and the possibility that DMSO is involved in suppressing the growth of the HeLa-S3 cells used in this measurement example is in a state that can be denied. .

The culture for this test was performed in a 96-well microplate. Each well was inoculated with 2.0 × 10 ³ cells, giving 5 wells for each test concentration. As a positive control, a medium containing 1% DMSO was used.

After adding the compound, the cells were cultured for 24 hours at 37 ° C. in a 5% CO ₂ incubator, and the cell viability of each test group was determined. The determination of survival rate is described in the literature ["Rapid Colorimetric Assay for Cellular Growth and Surviva1: Application to Proliferation and Cytotoxicity Assays", Tim Mosmann, J. Immunol. Methods, 65, 55 (1983)]. The assay method was used. That is, after 24 hours, the tetrazolium salt MTT was added and further cultured for 4 hours. The amount of formazan produced through reduction by viable cells was considered to be proportional to viable cells and was quantified with an optical density (OD) of 570 nm.

Cell viability was calculated by the following formula:
Cell viability (%) = O. D. [570 nm] / O. D. [570 nm]
The obtained results are shown in Table 1. The data shown in Table 1 is an average value of 5 wells.

〔ＳＱＤＧが癌細胞増殖を阻害するという表１の実験結果は、Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K. Action of a new mammalian DNA polymerase inhibitor, sulfoquinovosyldiacylglycerol. Biol Pharm Bull. 1999, 22 (2) 111-116.において発表済である〕

[The experimental results in Table 1 that SQDG inhibits cancer cell growth are the results of Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K. Action of a new mammalian DNA polymerase inhibitor, (Presented in sulfoquinovosyldiacylglycerol. Biol Pharm Bull. 1999, 22 (2) 111-116.)

  From these results, it was shown that SQDG that inhibits the binding between Cdt1 and Geminin actually exerts a cell growth inhibitory effect on human cancer cell lines and can be used as an anticancer agent.

In this example, the binding inhibitory effect between Cdt1 and Geminin was tested using coenzyme Q group, and the cancer cell proliferation inhibitory activity was tested.
(1) Binding inhibition test between Cdt1 and Geminin In the same manner as in Example 1, an experiment was performed on the coenzyme Q group. The structure of the coenzyme Q group used is as follows.

〔式中、
コエンザイムＱ_１において、ｎ＝１であり、
コエンザイムＱ_２において、ｎ＝２であり、
コエンザイムＱ_４において、ｎ＝４であり、
コエンザイムＱ_６において、ｎ＝６であり、
コエンザイムＱ_１０において、ｎ＝１０である〕。

[Where,
In coenzyme Q ₁ , n = 1,
In coenzyme _{Q 2,} it is n = 2,
In coenzyme _{Q 4,} a n = 4,
In coenzyme _{Q 6,} a n = 6,
In coenzyme _{Q 10,} which is n = 10].

These five coenzyme Qs were used at concentrations of 1000 μM, 100 μM, 10 μM and 1 μM. In the positive control, no inhibitor candidate substance was added, and only Geminin was used. As negative controls, those using only GST (negative control 1) and those not containing GST-Cdt1 (negative control 2) were used.
The experimental results are shown in FIG.

From this result, it was found that CoQ ₆ and CoQ ₁₀ inhibit the binding between Cdt1 and Geminin.

(2) Confirmation of human cancer cell growth inhibitory activity CoQ ₁₀ that showed suppression of Cdt1-Geminin binding in (1) above was evaluated using the method described in Example 2 (MTT assay). did.

  The obtained results are shown in Table 3. The data shown in Table 3 is an average value of 5 wells.

From this result, it was shown that CoQ ₁₀ that inhibits the binding between Cdt1 and Geminin actually exerts a cell storage inhibitory effect on human cancer cell lines and can be used as an anticancer agent.

(3) Effect on human normal cells The effect of CoQ ₁₀ on human normal cells was evaluated using the following method.
The cells used in this experiment are human umbilical vein endothelial cell-derived HUVEC cells (purchased from Kurabo Corp. (Osaka)). As the medium, HuMedia EG2 (Kurabo Co., Ltd.) to which fetal bovine serum 2% (v / v) was added was used. The culture was performed at 37 ° C. in a 5% CO ₂ incubator.

CoQ ₁₀ was dissolved in the medium shown above. However, since CoQ ₁₀ is hardly soluble in water, it was once dissolved in DMSO (dimethyl sulfoxide) and dissolved in the above medium. In addition, the final concentration of DMSO present in the medium in the medium is 1% or less in all the test sections, and the possibility that DMSO is involved in the suppression of the growth of the HUVEC cells used in this measurement example can be denied. State.

The culture for this test was performed in a 96-well microplate. Each well was inoculated with 2.0 × 10 ³ cells, giving 5 wells for each test concentration. As a positive control, a medium containing 1% DMSO was used.

After the addition of the compound, the cells were cultured for 7 days at 37 ° C. in a 5% CO ₂ incubator, and the cell viability of each test group was determined. The determination of survival rate is described in the literature ["Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations", Altman SA, Randers L, Rao G., Biotechnology Progress, Vol. 9, No. 6, pp. 671-674 (1993 The trypan blue staining method described in)] was used. That is, after 7 days, 0.3% trypan blue solution was added, and the cell suspension was appropriately diluted and immediately put into the calculation panel. Cells stained blue are dead cells, and live cells and dead cells were counted, respectively, and the total number of cells and the ratio of live cells were determined.
Table 4 shows the obtained results. The data shown in Table 4 is an average value of 5 wells.

From this result, it was found that CoQ ₁₀ that inhibits the binding between Cdt1 and Geminin does not show cell growth suppression against human normal cell lines.

  The present invention provides a screening method for anticancer agents. Such a screening method is useful as a primary screening for screening a large number of test substances at high throughput because it can easily and rapidly screen for anticancer drug candidates.

The result of having combined Cdt1 protein and Geminin protein in presence of an inhibitor candidate substance is shown. The structure of the inhibitor candidate substance used in the Example is shown. The result of having combined Cdt1 protein and Geminin protein in presence of coenzyme Q group is shown.

Claims (4)

  Cdt1 protein and Geminin protein are contacted in the presence of a test substance, the binding between Cdt1 protein and Geminin protein is measured, and the test substance that reduces the binding is selected as an anticancer drug candidate screening Method.   The method according to claim 1, wherein either one of Cdt1 protein or Geminin protein is immobilized.   The method according to claim 1 or 2, further comprising administering the selected anticancer drug candidate to a cancer cell and observing a change in proliferation of the cancer cell.   A kit for screening an anticancer drug candidate, comprising a Cdt1 protein and a Geminin protein.

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