KR101771070B1

KR101771070B1 - Methods for screening anti-cancer agents inhibiting interactions between MRS and CDK4

Info

Publication number: KR101771070B1
Application number: KR1020150159368A
Authority: KR
Inventors: 권남훈; 이진영; 김성훈
Original assignee: 재단법인 의약바이오컨버젼스연구단
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2017-08-24
Also published as: WO2017082494A1; KR20170056146A

Abstract

The present invention relates to an anticancer agent screening method for inhibiting the binding of MRS and CDK4, and more particularly, to a method for screening an anticancer agent screening method which inhibits the binding of MRS (methionyl-tRNA synthetase) or its fragment and CDK4 (cyclin-dependent kinase 4) Contacting the fragment; Measuring the binding of MRS to CDK4; Determining a change in the binding level of MRS and CDK4 by the test substance; Selecting a test substance that reduces the binding level of MRS and CDK4; And identifying an anticancer activity of the selected test substance in a cell or an animal, and an anticancer composition comprising the anticancer agent selected by the method as an active ingredient.
The present invention can be used to develop an anticancer agent of completely new mechanism based on the discovery that MRS is a novel regulator of CDK4 that stabilizes and binds directly to CDK4 which is important for cancer development and progression. The anticancer composition comprising the selected anticancer agent according to the present invention as an active ingredient is effective for inhibiting the expression of MRS or inhibiting the binding of MRS and CDK4 to lower the level of CDK4 protein and effectively inhibiting cancer progression can be useful for developing cancer therapeutic agents that do not express p16 ^INK4a .

Description

Methods for screening anti-cancer agents inhibiting interactions between MRS and CDK4 < RTI ID = 0.0 >

The present invention relates to an anticancer agent screening method for inhibiting the binding of MRS to CDK4. More particularly, the present invention relates to a method for screening anticancer agents that inhibit the binding of MRS to CDK4, ) Or a fragment thereof; (b) measuring the binding of MRS and CDK4 in the presence or absence of the test substance; (c) comparing the binding of MRS and CDK4 in the presence of the test substance and the binding of MRS and CDK4 under the absence of the test substance to determine the change of the binding level of MRS and CDK4 by the test substance; (d) selecting a test substance that reduces the binding level of MRS and CDK4; And (e) identifying an anticancer activity of the selected test substance in a cell or an animal, and an anticancer composition comprising the anticancer agent selected by the method as an active ingredient.

Methionyl-tRNA synthetase (MRS) is an essential enzyme that binds methionine to the initiator and elongator tRNA ^Met for translation. Since Met-tRNA ^Met is essential for initiation of protein polymerization and extension of the polypeptide, it is in a crucial position to regulate translation as well as potentially other biological processes. For example, MRS modulates the specificity for tRNA in response to oxidative stress and allows more methionine to be inserted into the polypeptide chain being synthesized, allowing increased methionine to remove reactive oxygen species (Lee et al., 2014 ; Wiltrout et al., 2012). Upon UV irradiation, the enzymatic activity of MRS is inhibited by the GCN2-dependent phosphorylation of the S662 site and the initiation of translation is inhibited (Kang et al., 2012; Kwon et al., 2011). When MRS is phosphorylated, it is separated from AIMP3, a tumor suppressor that binds to MRS. AIMP3, which has been isolated, moves into the nucleus to recover DNA. In this context, MRS plays a role as a linker to regulate DNA damage response and translational inhibition by UV irradiation (Kwon et al., 2011). These results suggest that MRS is capable of modulating the biological processes of cytoplasm and nucleus in response to various cell stimuli.

In regulating the cell cycle, it is very important that the cyclin and cyclin dependent kinases (CDKs) form a timely complex, activate and deactivate. CDK4 and CDK6, which form a complex with D-type cyclin, particularly Cyclin D1, are responsible for metastasis of the G1 / S cell cycle and are important for the determination of growth and resting (Musgrove et al., 2011). Two CDK inhibitors play an important role in regulating cell cycle arrest. These two types are INK4 type is a ^{(p16 INK4a, p15 INK4b, p18} INK4c, p19 INK4d) and KIP / CIP type ^{(p21 CIP1 / WAF1, p27 KIP1} , p57 KIP2) (Sherr and Roberts, 1995; Witkiewicz et al,. 2011; Zou et al., 2002). While KIP / CIP inhibitors form broad CDKs and cyclin and ternary complexes, INK4 inhibitors specifically bind to CDK4 or CDK6 and inhibit CDK4 / 6 and Cyclin D complex formation (Franklin et al., 1998).

CDK4 has been reported to be very important in Ras-activated mutants of mouse embryonic fibroblasts to cancer cells, tumoral tumors induced by Erbb2 or Hras, carcinogens and Myc-induced skin cancer (Berthet and Kaldis, 2007; Malumbres and Barbacid, 2009; Reddy et al., 2005; Yu et al., 2006; Zou et al., 2002). In addition, the CDK4-Cyclin D1 complex is sufficient to cause and spread breast cancer (Yu et al., 2006). These findings strongly demonstrate the importance of CDK4 in cancer, but it is not fully understood as to how the level of CDK4 remains high in cancer through any mechanism.

Therefore, it is necessary to identify the molecular mechanism that can regulate the activity of CDK4, which plays an important role in the development and progression of cancer by abnormal regulation of the cell cycle, and to develop a new action mechanism anticancer drug that inhibits CDK4 .

Therefore, the present inventors have found that MRS binds directly to CDK4 to stabilize the CDK4 protein and allow the cell cycle to proceed smoothly. When the binding of MRS and CDK4 is inhibited, the cell cycle does not progress, resulting in the division and proliferation of cancer cells And thus the present invention has been completed.

It is therefore an object of the present invention

(a) contacting a fragment of MRS (methionyl-tRNA synthetase) or a fragment thereof with CDK4 (cyclin-dependent kinase 4) or a fragment thereof in the presence or absence of the test substance;

(b) measuring the binding of MRS and CDK4 in the presence or absence of the test substance;

(c) comparing the binding of MRS and CDK4 in the presence of the test substance and the binding of MRS and CDK4 under the absence of the test substance to determine the change of the binding level of MRS and CDK4 by the test substance;

(d) selecting a test substance that reduces the binding level of MRS and CDK4; And

(e) identifying an anticancer activity of the selected test substance in a cell or an animal.

Another object of the present invention is

And to provide an anticancer composition comprising the anticancer agent selected by the method of screening the anticancer agent as an active ingredient.

To achieve these and other advantages and in accordance with the purpose of the present invention,

In order to achieve another object of the present invention,

There is provided an anticancer composition comprising an anticancer agent selected by the method for screening an anticancer agent as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

(a) in the presence or absence of the test substance MRS ( 메 - tRNA synthetase ) Or fragments thereof and CDK4 ( cyclin - dependent kinase 4) or a fragment thereof;

(b) the presence or absence of the test substance MRS Wow CDK4 Measuring a binding of the antibody;

(c) in the presence of the test substance MRS Wow CDK4 And the presence of the test substance MRS And the binding of CDK4 was compared MRS Wow CDK4 Change in the level of Judge A step;

(d) MRS Wow CDK4 Selecting a test substance that reduces the binding level of the test substance; And

(e) identifying an anticancer activity of the selected test substance in a cell or an animal.

In addition to the original function that MRS performs in protein translation, the present inventors have found that there is a new function of binding to CDK4 to stabilize the CDK4 protein so as not to be degraded, and to enable smooth progression of cell cycle progression due to CDK4. Factors capable of inhibiting the expression of MRS and reducing the level of MRS that can react with CDK4 or directly inhibiting the response of MRS and CDK4 are those that decrease CDK4 protein levels and result in cell division and G0 / G1 Let it be stopped in the cell cycle. Inhibition of MRS and CDK4 binding inhibits cancer cell proliferation and tumor growth, especially in cancer patients, indicating a strong correlation between MRS and CDK4 expression levels and that MRS levels are also implicated after cancer progression . Based on the discovery of the functional relationship between MRS and CDK4, the present inventors first disclose an anticancer drug screening method capable of screening a drug capable of inhibiting the binding of MRS and CDK4 and selecting them as anticancer drugs.

The step (a) is a step of contacting MRS (methionyl-tRNA synthetase) or a fragment thereof with CDK4 (cyclin-dependent kinase 4) or a fragment thereof in the presence or absence of the test substance.

In the present invention, 'protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to a polymer of amino acid residues as commonly found in natural state proteins. The 'fragment' means a portion of the protein. In the present invention, "polynucleotide" or "nucleic acid" refers to deoxyribonucleotide (DNA) or ribonucleotide (RNA) in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' is the RNA that transfers the genetic information of the base sequence of a specific gene to the ribosome that forms the polypeptide during protein synthesis.

In the present invention, 'MRS (methionyl-tRNA synthetase)' refers to a methionyl-thione synthetase which functions to link a methionine amino acid to a tRNA. In humans, MARS And the sequence information of MRS is known as a genbank accession number such as NM_004990 (mRNA), NP_004981.2, P56192.2 (protein). The human MRS protein consists of 900 amino acids and contains a GST-like domain (1 to 266th amino acid), a catalytic domain (267 to 598th amino acid), a tRNA binding domain (599th to 900th amino acid ).

The MRS protein for practicing the method of the present invention may preferably be derived from a mammal, including a human, and most preferably comprises the amino acid sequence of a human MRS protein as shown in SEQ ID NO: 1.

Also, the fragment of MRS means a fragment containing a portion necessary for binding with CDK4 in MRS. The present inventors confirmed that the catalytic domain and the tRNA binding domain of MRS can bind CDK4 and that the GST-like domain inhibits binding by domain mapping. Furthermore, in the catalytic domain of MRS, the portion of the methionine activation reaction (ATP-binding to methionine and pyrophosphate release) occurs. Specifically, the 596th lysine (K596) of human MRS protein amino acid sequence is essential for the binding of MRS and CDK4 . Therefore, the fragment of MRS for carrying out the present invention preferably contains the 596th lysine (K596) in the human MRS amino acid sequence represented by SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid sequence of 1 to 266 (SEQ ID NO: 3) which does not contain the GST-like domain.

CDK4 (cyclin-dependent kinase 4) is a cyclin-dependent kinase, also known as cell division protein kinase 4, belonging to the Ser / Thr protein kinase family and important for the progression of G1 / S in the cell cycle. CDK4 is regulated so that its activity is exerted only by the G1 / S cycle by D type cyclin and its activity is inhibited by p16 ^INK4a . The protein that CDK4 phosphorylates is retinoblastoma protein (Rb). Human CDK4 is encoded by the CDK4 gene, and the sequence information is known as a genbank accession number such as NM_000075.3 (mRNA) and NP_000066.1 (protein).

The CDK4 protein for carrying out the method of the present invention may preferably be derived from a mammal including a human, and most preferably the amino acid sequence or sequence of the human wild type (WT) CDK4 protein of SEQ ID NO: The amino acid sequence of human CDK4 R24C mutant protein represented by SEQ ID NO: 15. The CDK4 R24C mutation is a mutation that does not bind p16 ^INK4a , an endogenous inhibitor of CDK4 in which the 24th arginine of the CDK4 amino acid sequence is replaced by cysteine. According to the present invention, MRS directly binds not only wild type CDK4 but also CDK4 R24C mutation.

Also, the fragment of CDK4 means a fragment containing a necessary part for binding with MRS. The present inventors confirmed that MRS binds to the N-terminal region of CDK4. Specifically, it was revealed that a fragment containing the 1st to 102nd amino acid (SEQ ID NO: 17) in the amino acid sequence of human CDK4 (SEQ ID NO: 13) can bind to MRS. Thus, the fragment of CDK4 for carrying out the present invention may be a fragment comprising the amino acid sequence of SEQ ID NO: 13 (SEQ ID NO: 17).

The MRS or fragments thereof according to the invention and CDK4 or fragments thereof also include functional equivalents thereof. Refers to at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i. E. Identity) to the amino acid sequence of MRS and its fragments and CDK4 or fragments thereof. Refers to a polypeptide having substantially the same physiological activity as the MRS represented by SEQ ID NO: 1 and the polypeptide represented by SEQ ID NO: 13 by CDK4. Here, "substantially homogenous physiological activity" means that a direct and specific binding between the wild-type MRS and wild-type CDK4 can be reproduced. That is, the functional equivalent of a fragment of MRS has an activity capable of binding to full-length CDK4 or a fragment thereof, particularly a K596 of human MRS, or a lysine amino acid at a position corresponding thereto, An equivalent means to have an activity capable of binding to a site that binds to CDK4 in the full-length MRS or MRS. The functional equivalents may result from the addition, substitution or deletion of a portion of the amino acid sequence. The substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Also included in the functional equivalents are variants in which some of the amino acids are deleted in the amino acid sequence. The deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. Also, deletion of the amino acid is preferably located at a site that is not directly involved in the physiological activity of the MRS or CDK4 polypeptide. Also included are variants in which some amino acids are added at both ends or sequences of the amino acid sequence. Also included within the scope of the functional equivalents of the present invention are polypeptide derivatives in which some of the chemical structures of the polypeptides are modified while maintaining the basic skeleton of the polypeptide according to the present invention and its physiological activity. This includes, for example, structural modifications to alter the stability, shelf stability, volatility, or solubility of the polypeptides of the present invention.

"Contact" in the methods of the present invention is a general term and refers to binding two or more agents (eg, two polypeptides) or binding a agent to a cell (eg, a protein and a cell) It says. Contacting is in vitro (in in vitro . For example, two or more agents may be combined in a test tube or other container, or the test agent may be combined with the cell or cell lysate and the test agent. In addition, or in-situ contacting the cells (in situ . For example, two polypeptides are contacted in a cell or cell lysate by co-expression in a cell of a recombinant polynucleotide encoding two polypeptides. The protein to be tested may be a protein chip or protein array arranged on the surface of the immobilized phase.

When the method of the present invention is carried out in a cell, MRS and CDK4 may be expressed intracellularly, or overexpression may be carried out by introducing a nucleic acid encoding MRS or its fragment, CDK4 or a fragment thereof into a cell, have. Also in such a method of the invention and within the array or protein in vitro For situ , MRS or fragments thereof and CDK4 or fragments thereof can be prepared by extraction from natural sources or by genetic engineering methods. For example, a recombinant expression vector can be prepared by a conventional method and a nucleic acid encoding the polypeptide or a functional equivalent thereof, and the expression can be obtained by expression in a suitable host cell. The polypeptide necessary for carrying out the method of the present invention can also be produced by a chemical synthesis method known in the art.

Also, in the method of the present invention, the 'test substance' is a substance which can be used interchangeably with a test agent or agent and includes any substance, molecule, element, compound compound, entity, or a combination thereof. For example, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or chemical compound, or a combination of two or more substances.

Specifically, the anticancer agent selected through the method of the present invention is not limited to the properties of the substance as long as it can inhibit the protein binding of MRS and CDK4 or lower the expression level of MRS protein capable of binding to CDK4, for example, a methionine amino acid An antibody, an aptamer, a peptide, a natural extract, a chemical substance, and the like. Preferably a shRNA or siRNA capable of lowering the expression of MRS, and a methionine analogue that inhibits the CDK4 binding activity of MRS. The term "mimetics or analog" refers to a substance that is structurally similar to a reference molecule but whose target or control method has been modified by substitution of a specific substituent of the reference material. Analogs have the same, similar, or improved utility as would be expected by those skilled in the art when compared to standard materials.

The step (b) is a step of measuring the binding of MRS and CDK4 that are contacted in the presence or absence of the test substance in step (a).

The step of measuring the binding of MRS and CDK4 may be selected without limitation as long as it is commonly used in the art in order to measure the degree of binding of the two proteins. For example, two hybrid methods, co-immunoprecipitation assay (co-IP), tissue immuno staining and co-localization assay, scintillation proximity assay (SPA) ), UV or chemical crosslinking methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), fluorescence polarization assays (FPA) Full-down essay ( in The binding between MRS and CDK4 can be measured by an appropriate selection from an in vitro pull-down assay, an enzyme linked immunosorbent assay (ELISA), a protein chip or array, Venus BiFC (biomolecular fluorescence complementation) have.

In a specific example of the present invention, co-IP experiments are performed by overexpressing MRS and CDK4 appropriately labeled with HA, Strep, radioactive isotope or the like, or CDK4 binding to MRS using GST pull- The level of protein was measured or, conversely, the level of MRS protein bound to CDK4 was measured. In addition, the pattern of binding of MRS and CDK4 in cells was observed using the BiFC method, Venus fluorescence complementation method by binding of two proteins.

(C) comparing the binding of MRS and CDK4 in the absence of the test substance to the binding of MRS and CDK4 in the presence of the test substance measured in (b), and determining the change of the binding level of MRS and CDK4 by the test substance to be.

That is, the step of determining the influence of the test substance on the binding of MRS and CDK4 by determining the difference in the binding level of the protein of MRS and CDK4 that are contacted in the presence or absence of the test substance derived in step (b).

The step (d) is a step of selecting a test substance that reduces the binding level of MRS and CDK4.

The inventors of the present invention found that MRS and CDK4 are specifically bound directly to each other in a cell, that CDK4 is stabilized not to be degraded through binding with MRS, and consequently, cell cycle progression by CDK4 is smoothly performed. Respectively. Stabilization of the CDK4 protein is achieved through direct binding with the MRS protein. When the CDK4 protein is dissociated from the MRS protein, the CDK4 protein becomes unstable and degraded to decrease its level. As a result, Cells do not divide and become stuck in the G0 / G1 cycle. These effects could be confirmed using siRNA or shRNA, which inhibits the expression of MRS, and FSMO, an analogue of methionine that inhibits the binding of MRS and CDK4 proteins, and an inhibitor of activity. It is possible to inhibit cell division by inhibiting the binding between MRS and CDK4, thereby inhibiting the development and proliferation of abnormally cell-dividing cancer cells.

In another embodiment, inhibition of the expression level of MRS through tumor-associated cell experiments and xenograft animal studies leads to a decrease in CDK4 protein level and inhibition of cancer cell proliferation, tumor formation and growth , And the protein expression analysis of tumor tissues in breast cancer patients showed that MRS is closely related to cancer development or cancer prognosis through interaction with CDK4 protein.

Through the discovery by the inventors of the present invention, those skilled in the art can understand that a substance that decreases the binding level of MRS and CDK4 can inhibit cell cycle progression by making CDK4 unstable and have anticancer activity. The substance that decreases the binding level of MRS and CDK4 may be a substance that typically inhibits the binding between MRS and CDK4 itself or a substance that decreases the amount of MRS protein capable of binding CDK4. A specific example of the anticancer agent selected according to the anticancer agent screening method of the present invention is described in the description of the anticancer composition according to the present invention.

The step (e) is a step of confirming the anticancer activity of the substance selected in step (d) in a cell or an animal.

(e) is a step of confirming whether the test substance selected by decreasing the binding level of MRS and CDK4 in step (d) specifically has anticancer activity predicted by destabilization of CDK4 protein in a cell or animal of a cancer or tumor model to be. The anticancer activity means that the abnormal cell division is increased, the cell is transformed into a cancer cell, the cell division and proliferation of the cancer cell, and the development and growth of the tumor are inhibited.

Cells or animals of the cancer or tumor model may be appropriately selected if they are commonly used in the art, and the anticancer activity of the selected material in step (d) can be confirmed. In a specific example of the present invention, the transformability of cells was measured using anchorage independent soft agar assay, and xenografts injecting human cancer cells into mice were used And the formation and formation process of the tumor was observed.

The method for screening an anticancer agent according to the present invention may further comprise steps (d) and (e)

(1) contacting the test substance with cells expressing CDK4;

(2) measuring the CDK4 protein level in the control cells not in contact with the test substance; And

(3) selecting a test substance that reduces CDK4 protein levels compared to control cells.

According to the present invention, since it is necessary to directly bind MRS to stabilize CDK4 not to be degraded, it is understood that when the binding level of MRS and CDK4 decreases, the protein level of CDK4 decreases. The above steps (1) to (3) further confirm whether the test substance selected by decreasing the binding level of MRS and CDK4 in step (d) reduces the CDK4 protein level. The additional step is selected as the agent that reduces the binding level of MRS and CDK4 in step (d), but is false-positive, or when the level inhibiting the binding of MRS to CDK4 is insufficient to cause destabilization of CDK4 Can be carried out in order to determine and remove.

In step (1), the test substance selected in step (d) is contacted with cells expressing CDK4.

The cell expressing CDK4 may be a cell that innately expresses CDK4 or may be a cell that is transformed with a recombinant expression vector containing a polynucleotide encoding CDK4 to overexpress CDK4. For example, CDK4 destabilizing effect of the test substance can be verified by appropriately selecting among various cancer cell lines expressing CDK4. The meaning of the 'contact' is as described above.

In step (2), the level of CDK4 protein is measured in cells contacted with the test substance in step (1) and control cells not in contact with the test substance.

To determine the level of the CDK4 protein, a protein detection method commonly used in the art may be selected without limitation, for example, Western blotting, dot blotting, enzyme immunoassay immunoassay, enzyme-linked immunosorbent assay (ELISA), radioactive immunoassay (RIA), radioimmunoprecipitation, Oucheroton immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, Analysis method (FACS) or protein chip method can be used.

The step (3) is a step of selecting a test substance that reduces CDK4 protein level as compared with a control cell.

In the step (3), the CDK4 protein level measured in the control cell not in contact with the cell contacted with the test substance measured in the step (2) previously is compared, and the test substance showing that the CDK4 protein level is actually decreased is further selected do.

It is preferable that the cell for carrying out the method for screening an anticancer agent according to the present invention does not express p16 ^INK4a .

The p16 ^INK4a is a protein that is expressed intrinsically in cells to inhibit CDK4 activity and stabilize CDK4. The present inventors ^confirmed that p16 ^INK4a competitively ^binds to MRS in the examples. p16 ^INK4a and MRS do not bind to the same amino acid position in CDK4, but are thought to bind at similar or close distances on CDK4 to such an extent that they competitively bind. When p16 ^INK4a binds to CDK4, it inhibits the binding to Cyclin D1, which is necessary for CDK4 activity. However, MRS does not inhibit the binding of CDK4 and Cyclin D1, suggesting that the functional nature of the binding is different.

However, since p16 ^{INK4a competes} with CDK4 competitively with MRS, the binding efficiency of MRS and CDK4 is relatively decreased in an environment where p16 ^INK4a is present at a high level, and the effect of reduction of CDK4 protein level due to inhibition of binding of MRS and CDK4 Is difficult to be demonstrated. In the examples of the present invention, it has been observed that the destabilizing effect of CDK4 protein due to inhibition of MRS expression appears more clearly in cells that do not express p16 ^INK4a . Therefore, it is preferable that the cell for carrying out the method of the present invention is a cell that does not express p16 ^INK4a .

Cells that do not express the p16 ^INK4a are cells of the original or cells that do not express p16 ^INK4a, or expressing p16 ^INK4a but genetic mutation or other operations expression reasons may be a cell that is not expressing the p16 ^INK4a, in the p16 ^INK4a in Or a cell transformed with siRNA or shRNA for inhibition. ^Examples of cells that do not express p16 ^INK4a internally and that can reduce the CDK4 protein level by inhibiting the binding of MRS and CDK4 include A549, H460, H1299 (an abnormal lung cancer-derived cell line), MDA-MB -231, MDA-MB-453, BT20, T47D, MCF7 (breast cancer-derived cell lines), HT-29, SW620 and HCT116 (colon cancer derived cell lines).

For the same reason as described above, it is preferable that the method for screening an anticancer agent according to the method of the present invention is for a cancer that does not express p16 ^INK4a that competitively binds CDK4 with MRS.

The cancer that does not express p16 ^INK4a specifically includes breast cancer, lung cancer, colon cancer, anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, angioma, Osteosarcoma, prostate cancer, colon cancer, pancreatic cancer, renal cancer, stomach cancer, skin cancer, basal cell carcinoma, melanoma, squamous cell carcinoma, oral squamous cell carcinoma, large intestine Rectal cancer, glioblastoma, endometrial cancer and malignant brain glioma, and may be preferably breast cancer, lung cancer or colon cancer.

Meanwhile, the present inventors found that MRS directly binds not only to wild-type CDK4 but also to CDK4 R24C mutation which is not ^under the control of p16 ^{INK4a in} the course of identifying that MRS and p16 ^INK4a competitively bind to CDK4. The CDK4 R24C mutation is the result of substitution of the 24th arginine of the CDK4 amino acid sequence with cysteine, which does not bind p16 ^INK4a but has always been phosphorylated and has been observed in human skin cancer tissues. In addition, when the R24C mutation was expressed in the mouse, it was observed to induce various types of cancer such as sarcoma, endocrine system cancer, epithelial cell cancer, lung cancer, liver cancer, and lymphoma for a short period of time (Sotillo et al., 2001).

It can be seen that the activity of CDK4 R24C, which is not regulated by p16 ^INK4a , can be inhibited by using an agent that inhibits the expression of MRS or inhibits the binding of MRS and CDK4 R24C because the MRS binds to CDK4 R24C. That is, a new therapeutic strategy can be attempted using the combination of MRS and CDK4 R24C for the above-mentioned various kinds of cancer including CDK4 mutated R24C skin cancer. Since CDK4 R24C does not bind p16 ^INK4a , Unlike wild-type CDK4, the modulation effect by MRS may not be affected by the expression of p16 ^INK4a .

The present invention also provides an anticancer composition comprising an anticancer agent selected by the method of screening an anticancer agent according to the present invention as an active ingredient.

The anticancer agent selected according to the method of the present invention is not particularly limited to the characteristics of the substance if it reduces the binding level of MRS and CDK4 and can inhibit cell cycle progression by destabilizing the CDK4 protein. A substance that inhibits the binding between two proteins of MRS and CDK4 or a substance that inhibits the expression of MRS and lowers the level of MRS protein capable of binding to CDK4 can be considered.

The anticancer agent selected by the method of the present invention specifically includes a methionine amino acid analogue, an siRNA, a shRNA, a miRNA, a ribozyme, a DNAzyme, a PNA (peptide nucleic acid), an antisense oligonucleotide, an antibody, an aptamer, Lt; / RTI >

The present inventors have found that inhibiting the expression of MRS in a breast cancer cell line or a lung cancer cell line using siRNA (si-MRS) or shRNA (sh-MRS) specific to MRS reduces the level of CDK4 bound to MRS and degrades the CDK4 protein . As a result, it was confirmed that CDK4-induced cell cycle progression was suppressed, resulting in reduction of BrdU insertion rate and arrhythmia of G0 / S period. The effect of such an MRS expression inhibitor was more clearly observed in the absence of p16 ^INK4a .

Furthermore, the MDA-MB-231 breast cancer cells inhibited the expression of MRS, compared with the control breast cancer cells in which the expression of MRS was not inhibited in the adhesion-independent soft cloth test, And the growth rate of the tumor was greatly decreased when injected into the mouse by xenotransplantation. This indicates that agents capable of inhibiting the expression of MRS such as siRNA or shRNA can be effectively used for the prevention and treatment of cancer diseases.

Therefore, the anticancer agent selected by the screening of the anticancer agent according to the present invention may be MRS-specific siRNA or shRNA. The siRNA or shRNA is composed of 10 to 30 nucleotide sequences capable of specifically binding to MRS mRNA and inducing degradation of mRNA, preferably siRNA comprising the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21 Lt; RTI ID = 0.0 > shRNA. &Lt; / RTI >

The present inventors also confirmed that methionine mimetics capable of inhibiting the binding of MRS to CDK4 have an effect of destabilizing CDK4. Among the 13 commercially available methionine analogs, FSMO, which is selected based on the protein translation inhibition effect and the CDK4 protein reduction effect, is an MRS-specific activity inhibitor, and specifically binds ATP to methionine and releases amino acid of MRS releasing PPi (pyrophosphate) Lt; RTI ID = 0.0 > aminoacylation < / RTI > The above aminoacylation reaction is a step of activating methionine before binding to tRNA. It is important for FSMO to inhibit the methionine activation reaction of MRS to inhibit the binding of MRS and CDK4. As the present inventors have demonstrated through the domain mapping of CDK4 and MRS binding and the MRS amino acid substitution mutation experiment that 596th lysine (K596), which is important for the methionine activation reaction, is essential for the binding of CDK4 and MRS to be. That is, the substance which is important for the activation of methionine in MRS, in particular, a substance which binds to K596 or a substance which inhibits the methionine activation reaction are highly likely to be selected as the anticancer agent of the present invention.

In a specific embodiment of the present invention, the FSMO directly inhibits the binding of MRS and CDK4 to various in- cells such as pull-down, co-IP, In vitro or in cell experiments, the cells treated with FSMO showed a marked decrease in the BrdU incorporation rate, similar to that of cells inhibiting MRS expression, and that cell division did not occur smoothly due to cell cycle arrest.

Therefore, the anticancer agent selected by the screening of the anticancer agent according to the present invention may be a methionine analogue capable of binding to MRS and inhibiting binding with CDK4, and specifically, Boc-S-trityl-L-homocysteine, Fmoc-DL-selenomethionine, Fmoc -Methyl-DL-methionine, and preferably Fmoc-Sec (Mob) -OH (FSMO).

The anticancer composition according to the present invention may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intracerebral, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration And may preferably be intravenous administration.

In addition, the composition for preventing or treating cancer according to the present invention may be formulated variously according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " refers to a nontoxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like . Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

When the anticancer composition of the present invention is administered parenterally, the composition of the present invention may be formulated together with a suitable parenteral carrier according to methods known in the art in the form of injection, transdermal drug delivery, and nasal inhalation . In the case of the injections, they must be sterilized and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferred examples of suitable carriers include Hank's solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or isotonic solutions such as sterilized water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride.

Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. By " transdermal administration " as used herein, it is meant that the composition of the present invention is locally administered to the skin, whereby an effective amount of the active ingredient contained in the composition is delivered into the skin. For example, the composition of the present invention may be prepared in a spiral form and administered by pricking the skin lightly with a 30 gauge thin needles or by directly applying it to the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, which is a commonly known formulary in pharmaceutical chemistry.

In the case of an inhalation dosage form, the compositions according to the present invention can be prepared from a pressurized pack or sprayer using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in aerosol spray form. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators may be formulated to contain the compound and a powder mixture of a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The anticancer composition according to the present invention may also contain one or more buffers (e.g., saline or PBS), a carbohydrate (e.g., glucose, mannose, sucrose or dextran), an antioxidant, a bacteriostatic agent, (For example, EDTA or glutathione), an adjuvant (e.g., aluminum hydroxide), a suspending agent, a thickening agent, and / or a preservative.

The anti-cancer composition of the present invention may also be variously formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The composition of the present invention may also be administered in combination with a known compound having an effect of preventing or treating cancer.

Therefore, the present invention provides a method of reducing the binding level of MRS and CDK4 by using MRS or a fragment thereof and CDK4 or a fragment thereof, based on the finding that MRS directly binds to and stabilizes CDK4, And an anticancer agent selected by the above method as an active ingredient. The siRNA, shRNA, and the like, which inhibit the expression of MRS according to the present invention, and the methionine analogues that inhibit the binding of MRS and CDK4, specifically lower the level of CDK4 protein in cancers that do not express p16 ^INK4a , Is excellent.

FIG. 1 shows experimental results for inhibiting the expression of MRS or confirming cell cycle progression under the condition of deficiency of methionine.
Figure 1A shows the effect of BrdU incorporation (left panel) of MDA-MB-231 cells stably expressing shRNA (sh-MRS) or control shRNA (sh-Cont) on MRS and cells using PI staining and flow cytometry The results of the periodic analysis are shown (Proportion of cells (%), right panel). Data were expressed as means ± standard error (mean ± SEM). Figure 1B shows the BrdU incorporation rate and cell cycle analysis results of H460 cells cultured for 9 hours in a methionine-deficient (-Met) condition. Data were expressed as mean ± standard deviation (mean ± SD). In Figs. 1A and 1B, ** indicates p < 0.05, *** indicates p < 0.001. Figure 1C shows the results of measuring the insertion rate of [ ³⁵ S] Met labeled methionine in MDA-MB-231 cells or H460 cells expressing shRNA.
FIG. 2 shows a protein array experiment and an immunoblot experiment to find a cell cycle-related protein affected by MRS.
Figure 2A shows changes in Cyclin D1, p21, CDK4, CDK3 and c-ABL protein levels in MDA-MB-231 cells stably expressing shRNA and in Figure 2B in H460 cells in a methionine-deficient condition. Figure 2C shows immunoblot results using MDA-MB-231 cells (left panel) and H460 cells (right panel) and cell lysates of each control group, which were deficient in methionine for 9 hours. FIG. 2D shows the results of co-immunoprecipitation (co-IP) experiments using H460 cells co-expressing HAK-labeled CDK4 or CDK3 with streptavidin-labeled MRS. WCL means whole cell lysate.
Figure 3 shows the results of a Venus BiFC experiment to observe the binding of intracellular MRS and CDK4. Blue fluorescence (DAPI) stained with blue fluorescence, VN or VC (green fluorescence) conjugated with VN or VC, and blue fluorescence (Alexa fluor 647) with HA labeled MRS, red fluorescence (Alexa fluor 594) with Flag labeled CDK4 or WRS And a Venus fluorescent protein formed by binding of the protein.
Fig. 4 shows binding mapping experiment results for confirming structural features of MRS and CDK4 binding.
4A shows co-IP experiment results for confirming the binding of MRS and CDK4 in MDA-MB-231 cells deficient in methionine for 6 hours. 4B is a schematic diagram showing fragments of the MRS and CDK4 proteins. MRS is divided into the GST-like domain (1-266 amino acids (aa)), the catalytic domain (267-597 aa) and the tRNA binding domain (598-900 aa) Five different fragments, MRS and F1 (1-266 aa), F2 (267-597 aa), F3 (598-900 aa), F4 (1-597 aa) and F5 (267-900 aa) . Two types of fragments, CDK4 and N (1-102 aa) and C (103-303 aa), of full length (Full, 1-303 aa) were used in the experiment, while CDK4 was divided into N-terminus and C- . Figure 4C is an autoradiography showing the results of GST pull-down experiments using radioactively labeled fragments of CDK4 and GST-MRS. Figure 4D is an autoradiography showing the results of GST pull-down experiments using radioactively labeled MRS fragments and GST-CDK4. Figure 4E shows co-IP experimental results using H460 cell lysates co-overexpressed with Flag labeled CDK4 and Strep labeled MRS (WT, wild type; HA, H560A mutation; KQ, K596Q mutation). FIG. 4F shows immunoblotting results of a lysate of H460 cells treated with cyclohexamide (CHX, 120 μg / ml) for 0, 4, and 8 hours to confirm the stability of the CDK4 protein.
Figure 5 shows the results of screening experiments for screening for methionine analogues (Met analogs).
5A is in using, firefly luciferase to determine the effect of 13 different analog of methionine (1mM) on the protein translation vitro translation experiment results. Data were expressed as mean ± standard deviation (mean ± SD). Figure 5B shows the results of immunoblot experiments confirming the effect of each methionine analog on CDK4-related protein levels. -Met means a methionine deficiency condition.

FIG. 6 shows experimental results for confirming the effect of the methionine-like FSMO on the activity of MRS.
6A is a graph showing the effect of inhibiting protein translation in FSMO (1 mM) in vitro (Fig. 6A) by adding methionine (M), serine (S), cysteine (C), leucine (L) The results of the translation experiment are shown. 6B is in to determine the effect of FSMO (150μM) on the catalytic activity of the MRS, CRS, SRS and KRS (each cysteinyl-, seryl-, and lysyl-tRNA synthetase) vitro aminoacylation experiments. Data were expressed as means ± standard error (mean ± SEM). FIG. 6C shows the results of ATP-PPi exchange experiment to confirm the effect of FSMO (0, 150, 300 μM) on the methionine activation action of MRS. The abscissa is time (minutes) and the data are expressed as mean ± standard error (mean ± SEM). 6D shows immunoblot results of H460 cells treated with various concentrations of FSMO or borrelidin. 6E are in the FSMO (top) and a borrelidin (lower) showing the effects of protein translation by the concentration vitro translation experiment results.
FIG. 7 shows the results of experiments in which FSMO affects the binding of MRS and CDK4 and cell cycle progression.
7A shows the results of GST pull-down experiments to confirm the effect of FSMO on the binding of MRS and CDK4. FIG. 7B shows co-IP experimental results using H460 cell lysate co-overexpressed with Myc-labeled MRS and HA-labeled CDK4. FSMO was treated at a concentration of 25 or 50 μM for 4 hours. FIG. 7C shows the result of co-IP experiment to confirm the effect of FSMO (50 μM) on the binding of MRS and CDK4 which are expressed intrinsically in H460 cells. 7D shows the Venus BiFC test results for observing the binding of intracellular MRS and CDK4. The blue fluorescence is the DAPI stained nucleus, the green fluorescence is the two proteins conjugated with VN or VC, respectively, while the purple fluorescence (Alexa fluor 647) represents the HA labeled MRS, the red fluorescence (Alexa fluor 594) represents the Flag labeled CDK4 protein, Of Venus fluorescence protein. FSMO was treated at a concentration of 50 μM for 6 h and the methionine deficiency (-Met) lasted for 6 h. And pretreated with MG132 (50 μM) for 2 hours to prevent protein degradation. FIG. 7E shows the BrdU incorporation rate of H460 cells treated with FSMO (50 μM), and FIG. 7F shows the cell cycle distribution trend using PI staining and FACS. ** was p <0.05, *** was p <0.001 and data were expressed as mean ± standard error (mean ± SEM).
8 is a p16 ^INK4a Expression and MRS-mediated regulation of CDK4.
8A is a ^graph showing the ^effect of siRNA (si-MRS) on MRS on the expression level of CDK4 in cells expressing p16 ^INK4a (p16 ^INK4a- ) and p16 ^INK4a (p16 ^INK4a + The experimental results are shown. Fig. 8B shows the immunoblot experiment results confirming the effect of siRNA (si-p16 ^INK4a ) on FSMO and p16 ^INK4a on the protein level of CDK4 in WI-26 cells expressing p16 ^INK4a . Figure 8C is FSMO an impact on the protein levels of CDK4 normal cells (gray, n = 6), cancer cell lines expressing the p16 ^INK4a (blue, n = 4) and cancer cell lines that do not express p16 ^INK4a (green, n = 11). Protein levels of CDK4, CDK2, MRS, and p-eIF2α were determined by immunoblot and quantified using the Image J program, indicating that the higher the color, the higher the expression level.
FIG. 9 shows experimental results for confirming the relationship between p16 ^INK4a and MRS.
Figure 9A shows the results of a GST pull-down experiment in which binding to CDK4 was confirmed by increasing the level of MRS protein (left panel) or increasing the level of p16 ^INK4a (right panel). FIG. 9B shows co-IP experiment results confirming the effect of si-p16 ^INK4a on the binding of Flag-labeled CDK4 overexpressed in WI-26 cells and Strep-labeled MRS. (PDB ID: 1BI7) at the top of FIG. 9C and the co-expression of co-expression of the HA-labeled CDK4 (WT or R24C mutation) and the 293T cell lysate coexpressed with Strep- IP experimental results are shown. The blue arrow indicates the position of R24C. Figure 9D is an autoradiography showing the effect of MRS on the binding of radiolabeled Cyclin D1 to GST-CDK4.
FIG. 10 shows the results of experiments in which cell and animal models confirm the correlation between the level of MRS and cancer.
FIG. 10A is a ^{graph showing the} levels of MRS and CDK4 protein determined by immunoblot analysis in breast cancer cell lines using Image J and showing that p16 ^INK4a And a dark color indicates a high level of expression. Fig. 10B shows the results of the adhesion-independent soft cloth experiment of MDA-MB-231 cells stably expressing shRNA. Figure 10C shows tumorigenic tendency in mice injected with MDA-MB-231 cells stably expressing shRNA. FIG. 10D shows changes in body weight (Body weight (g)) of mice in the xenotransplantation experiment over time. Figure 10E shows the change in tumor size (Tumor size (mm ³ )) over time as measured in mice in xenotransplantation experiments. FIG. 10F shows immunohistochemical staining photographs confirming MRS, CDK4, and Ki-67 protein expression levels in tumor tissues formed by xenotransplantation.
Figure 11 shows the results of an experiment in which the correlation between the level of MRS and the cancer disease was confirmed in cancer patients.
FIG. 11A is a Kaplan-Meier plot showing the non-recurrence-free survival rate (RFS) of breast cancer patients and FIG. 11B is the overall survival rate (OS) of lung cancer patients. Figure 11C shows the criteria for protein expression assay scores using a breast cancer tissue array. MRS, CDK4, cyclin D1, p-Rb and p16 ^INK4a were classified as 0, 1+, 2+ or 3+ depending on the protein staining signal intensity. FIG. 11D shows the correlation between CDK4 and MRS levels in cancer tissues that do not express p16 ^INK4a and FIG. 11E shows cancer tissues that express p16 ^INK4a . Figure 11F is in cancer tissues that do not express p16 ^INK4a, Figure 11G is MRS-level analysis in cancer tissue expressing p16 ^INK4a; represents the difference between the summing point (weighted sum of score) according to the (low, MRSL High, MRSH) . Data were expressed as means ± standard error (mean ± SEM).

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.

<Experimental Method>

Cell culture and transformation

Cell lines of MDA-MB-231, 293, 293T, NIH3T3, WI-26, WI-38, CCD18C0, HeLa, BT20, MCF7, HT29 and T47D used in the present invention were obtained from American Type Culture Collection or Korean Cell Line Bank) and MDA-MB-231 cell line expressing GFP was purchased from Cell Biolabs. Each cell line was cultured in DMEM (Hyclone) medium containing 10% fetal bovine serum (FBS) heat-inactivated with 5% CO ₂ , 50 μg / ml penicillin / streptomycin at 37 ° C. HCC1937, MDA-MB-453, MDA-MB-436, MDA-MB-468, A549, H460, HCT116, H1299, Panc10.05, SW620 cell line was purchased from the Korea Cell Line Bank, 5% CO _2, at 37 ℃ And cultured in RPMI supplemented with 10% FBS and 50 μg / ml penicillin / streptomycin. BT474, HCC70, HCC1569, HCC1954, HCC1419, HS578T, HCC2218 and HCC1395 cells were purchased from American Type Culture Collection or Korean Cell Line Bank and cultured in the same manner. The MCF10A cell line was treated with 5% CO ₂ , 10 μg / ml insulin at 37 ° C., 100 ng / ml cholera toxin, 25 ng / ml epidermal growth factor, 500 ng / ml hydrocortisone, 0.2% v / v amphotericin B, 5% horse serum, and 50 μg / ml penicillin / streptomycin.

Plasmids and siRNAs for gene overexpression were introduced into cells according to the manufacturer's instructions using Fugene HD (Roche) and Lipofectamine 2000 (Invitrogen), respectively. The sequence of the siRNA used to inhibit gene expression is as follows: si-MRS, 5'-CUACCGCUGGUUUAACAUUUCGUUU-3 ', SEQ ID NO: 19; si-p16 ^INK4a , 5'-CGCACCGAAUAGUUACGGU-3 ', SEQ ID NO: 20. The control siRNA (si-Control) was purchased from Invitrogen's stealth RNAi negative control duplex (medium GC).

Stable cell line production

MDA-MB-231 cells expressing GFP were transfected with shRNA (sh-MRS, 5'-GGCGAAACTCTTGGATCTA-3, SEQ ID NO: 21) or control shRNA (sh-Control; SMART choice inducible non-targeting control hEF1a / turboRFP VSC6573 , Thermo Scientific). &Lt; / RTI > Cells transformed with lentivirus were screened for suitable colonies using puromycin (1 μg / ml). Expression of sh-MRS and sh-Control was induced by treatment with doxycycline (1 μg / ml).

Cell cycle analysis using FACS

The cultured cells were obtained by treating trypsin, washed twice with cold PBS, and fixed with 70% ethanol at 4 ° C for 2 hours. Washed twice with cold PBS and treated with 500 μl PI staining solution containing 1 × PBS, 100 μg / ml RNase A, 50 μg / ml PI (propidium iodide, Sigma) for 30 min at 37 ° C in the dark. PI stained cells were analyzed by flow cytometry (BD Biosciences). The percentage of cells in the G0 / G1, S, and G2 / M phases of the cell cycle was analyzed using Cell Quest acquisition software (BD Biosciences).

Cell survival analysis using IncuCyte

Cells were inoculated into 96-well plates with 2000 cells per well. The next day, cells were replaced with DMEM or RPMI containing various concentrations of FSMO and 5% FBS, and cell growth was monitored using the IncuCyte Kinetic Live Cell Imaging System (Essen BioScience). Transfected with a pcDNA3-empty vector comprising pcDNA3-CDK4, pEXPR-IBA105-MRS, pEXPR-IBA105-CDK4, pcDNA3-MRS or pcDNA3-LRS containing a gene to express MDA-MB-231 cells or H460 cells, Cell growth rate was measured.

Firefly Luciferase in vitro translation

In vitro translation (protein translation) analysis was performed using rabbit reticulocyte lysate (Promega). The reaction solution contained 1 template (0.5 mg / ml firefly luciferase DNA), 1 μl compound, 1 μl methionine (1 mM stock), 1 μl distilled water, 6 μl rabbit reticulocyte lysate. The reaction solution was incubated at 30 캜 for 1.5 hours. In order to observe translation recovery by overloading with amino acids, each amino acid (Met, Ser, Cys and Leu, 25 mM stock) was added to the reaction sample and incubated, and then 2 x luciferase substrate (10 μl) was added to the mixture . Each sample was transferred to a 96 well plate and the level of chemiluminescence was measured using an EnVision Multilabel Reader (PerkinElmer). Borrelidin and methionine analogs were purchased from Sigma and Anaspec, respectively.

Cell cycle protein array

Expression of sh-RNA in MDA-MB-231 cells transformed to stably express shRNA (sh-Cont, sh-MRS) was induced by treatment with doxycycline for 4 hours. H460 cells were cultured in Met-free DMEM medium containing 10% dialyzed FBS for 9 hours to induce a methionine deficiency state. Protein lysates were prepared according to the guidelines of the cell cycle protein array kit (Full Moon Biosystems). Briefly, biotin was bound to protein and incubated on array slides with antibody specific for the signaling-related protein. After conjugation to Cy3-streptavidin and drying, fluorescence levels were measured and analyzed at 532 nm using a Genepix 4100A microarray scanner (Molecular devices).

In vitro Aminoacylation ( aminoacylation ) analysis

A549 cells were treated with trypsin, diluted with cold PBS, and then sonicated five times at 5 seconds intervals on ice. Aminoacylation assays were performed at 37 ° C with 30 mM Hepes pH 7.4, 100 mM potassium acetate, 10 mM magnesium acetate, 2 mM ATP, 100 / ml yeast tRNA and 25 μCi of [ ³⁵ S] Met, [ ³⁵ S] Cys , PerkinElmer) or [ ³ H] Ser, [ ³ H] Lys (40 Ci / mmol, PerkinElmer) and 20 protein lysate. The aminoacylation reaction was stopped on a 3MM filter paper soaked with 5% trichloroacetic acid, washed with 5% trichloroacetic acid and dried, and radioactivity was detected using a liquid scintillation counter (PerkinElmer) .

Methionine Insertion rate analysis

MDA-MB-231 and H460 cells were cultured in 12-well plates. Cells were treated with FSMO at concentrations of 0, 25, 50 and 100 μM for 8 h, respectively, and replaced with Met-free media containing [ ³⁵ S] Met. After incubation for 2 hours, the cells were lysed and the level of radioactive protein was measured using a liquid scintillation counter.

BrdU Insertion rate analysis

MDA-MB-231 and H460 cells were inoculated into 96 well plates at a concentration of 4000 cells per well. After cell attachment, the cells were treated with media containing 50 μM FSMO and 2% serum for 8 hours or with Met-free DMEM media containing FBS containing 10% dialysed for 9 hours to induce a methionine deficiency state . BrdU solution (100 μl) contained in each medium was added to each well and cultured for 2 hours. The BrdU-embedded cells were fixed and the antibodies were treated according to the instructions of the cell proliferation assay kit (Cell Signaling). Finally, the plate was washed three times with wash buffer, and then 100 μl of tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 30 seconds. The amount of BrdU introduced into the cells was measured at 450 nm using ELISA (Tecan).

In vitro pull - down assay

The fusion protein linked to GST was expressed in E. coli Rosetta 2 by induction with 1 mM IPTG at 37 < 0 > C for 16 hours. Cells were harvested and sonicated and the cell lysates were reacted with glutathione Sepharose 4B (GE Healthcare) overnight at 4 ° C in lysis buffer (PBS containing 0.5% Triton X-100 with protease inhibitors) . The protein extracts, which were overexpressed in the radioactively labeled protein or 293 cells synthesized using the TNT-coupled translation kit (Promega), were reacted with the GST fusion protein at 4 ° C for 2 hours to overnight. The beads were washed three times with lysis buffer and the eluted proteins were separated by SDS-PAGE and detected by autoradiography or immunoblotting.

Quantitative Real Time PCR (qRT-PCR)

RNA was extracted from the cells using the miRNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Total 2 μg of RNA was reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen) and random hexamer. The PCR cycles for synthesizing cDNA from the extracted RNA are 65 ° C / 5 min, 37 ° C / 2 min, 25 ° C / 10 min, 37 ° C / 50 min, and 70 ° C / 15 min. qRT-PCR was performed using the POWER SYBR GREEN master mix (Applied Biosystems) according to the manufacturer's instructions. The nucleotide sequences of the primers of MRS and CDK4 used in the RT-PCR are shown in SEQ ID NOS: 22 to 25.

BiFC Analysis Immunofluorescence Imaging

MRS was cloned into p-BiFC-VC155 vector containing HA tag, and tryptophanyl-tRNA synthetase (WRS) and CDK4 were cloned into p-BiFC-VN173 vector containing Flag tag . H460 cells were seeded on a cover slip and co-transformed with p-BiFC-VC155-MRS to p-BiFC-VN173-WRS or p-BiFC-VN173-CDK4 and cultured for 24 hours. Cells were treated with MG132 (50 [mu] M) for 2 hours before treatment with FSMO (50 [mu] M) and further incubated for 6 hours in the presence of 2% FBS. To induce a methionine deficiency, the cells were cultured in Met-free DMEM medium containing MG132 (50 [mu] M) and 10% dialyzed FBS for 6 hours. The cultured cells were fixed with pre-chilled 100% methanol for 7 minutes, blocked with 3% CAS at room temperature for 15 minutes, reacted with Flag antibody and HA antibody for 1 hour or overnight, respectively, and Alexa fluor 594 or Alexa and reacted with secondary antibody conjugated with fluor 647 for 1 hour. Nuclei were stained with DAPI solution for 10 min in the absence of light. Fluorescence was observed with a confocal microscope at 60 × magnification.

Complex modelling

CDK4-p16 ^INK4a The schematic model of the complex was generated using PyMOL after manual assembly using Coot (Emsley and Cowtan, 2004) with reference to the known structure of the protein. The known structures of the proteins used in the complex modeling are as follows: CDK4-Cyclin D1 complex, 2W96; ^CDK6 -p16 ^INK4a complex, 1BI7.

Attach Non-dependence Light cloth analysis( anchorage - independent soft agar assay )

One ml of a basic agar mixture containing 0.6% low-melting agar was added to each well of a 12 well plate and allowed to cool for 30 minutes at room temperature. Stably transformed MDA-MB-231 cells were mixed at a concentration of 3 × 10 ³ cells / well in a volume such as DMEM containing 0.6% low-melting agar and 10% serum and added to the well-aged basic agar . To confirm the growth of nonadherent cells, 10% DMEM was added to each well. Cells cultured for 1 month were fixed with 10% methanol / 10% acetic acid for 10 minutes and stained with 0.01% crystal violet for 1 hour.

In vivo Xenotransplantation experiment xenograft )

Animal experiments were conducted in accordance with the guidelines of the University Animal Care and Use Committee at Seoul National University. MDA-MB-231 cells expressing GFP on the flank of 6-week-old female BALB / c Slc-nu / nu SP nude mice and stably transformed with sh-RNA (sh-MRS or sh-Cont) ⁶ cells) were subcutaneously inoculated (5 mice per group). To induce the expression of siRNA, a solution of doxycycline hydrochloride (5% sucrose, 2 mg / ml) was provided. Mice receiving xenotransplantation were observed daily and their body weight and tumor size were measured every 3 days. After 3 weeks, the fluorescence signal emitted from the tumor site formed in the mouse was photographed using Optix MX3 (ART), and the mouse was sacrificed to obtain tumor tissue. Tumor tissues were weighed and immunohistochemically stained.

group Microarray Manufacture of blocks

The tissue blocks fixed with formalin and embedded in paraffin were arrayed using an Accu Max Array tissue array instrument (Petagen). Briefly, representative breast lesions were selected on the H & E slide for analysis by a breast pathologist. Specified regions of each donor tissue block were punched with tissue cylinders of 3 mm in diameter to obtain tissue and transferred to the receive block in a lattice pattern.

Immunohistochemical staining

Immunohistochemistry (IHC) was performed by preparing a tissue slice embedded in paraffin with a thickness of 4 mm and performing standard H & E staining. The sections were removed with paraffin with xylene and rehydrated with a gradient ethanol aqueous solution. A 3% (v / v) hydrogen peroxide solution was treated for 10 minutes to inhibit the intrinsic peroxidase of tissue sections and a 2x solution of epitomic retrieval at 100 ° C for 20 minutes (Leica Biosystems, pH 6.0) was treated before staining. ^Were reacted with primary antibodies against MRS (Neomics), p16 ^INK4a (Santa Cruz), Cyclin D1 (Santa Cruz), CDK4 (Santa Cruz), pRb (Abcam) Biosystems) according to the manufacturer ' s instructions. Images of stained tissue sections were taken using IP Lab software (BD Biosciences Clontech) and Nikon TE2000 inverted microscope. Slides were contrasted with Harris haematoxylin. Normal breast tissue and appropriate control tissue included in tissue sections were used as positive control. H & E stained slides were examined by a breast pathologist. The IHC staining results of IHC staining were analyzed by IHC staining of MRS, p16 ^INK4a , Cyclin D1, CDK4, and pRb in the range of 0 to 100, and the high-power field (HPF ) Were classified as '0', '1+', '2+', or '3+'. High levels of MRS expression were assigned scores of '2+' and '3+', and scores of '1+' to '3+' were assigned to other marker expression at high levels. The interpretation of the IHC results was made in a blinded state where no information was provided about the medical indicators or the experimental results. Criteria for giving a weighted sum of score (WSS) are shown in Table 2.

< Example 1>

Regulation of cell cycle progression and CDK4 protein levels by MRS

<1-1> Effect of MRS inhibition and methionine deficiency on cell cycle progression

The eukaryotic MRS plays a pivotal role in life events such as transcription and rRNA synthesis in the nucleus (Ruggero and Pandolfi, 2003). Since transcription and rRNA synthesis are very important for cell cycle regulation, we examined whether MRS is involved in DNA synthesis and cell cycle regulation.

In order to examine the effect of inhibition of MRS expression on cell cycle progression, MDA-MB-231 cells, a breast cancer cell line, were stably expressed with shRNA (sh-MRS) or control shRNA (sh-Cont) ), And cell cycle analysis using BrdU insertion rate and flow cytometry (FACS) was performed ( FIG. 1A ). The expression of BrdU in the cells suppressed the expression of MRS was significantly lower than that of the control, and the ratio of cells in the GO / G1 phase of the cell cycle was remarkably higher than that of the control, and the ratio of cells in the S phase was remarkably lower Respectively. The same results were also observed in experiments using siRNA against H460 cells and MRS, a lung cancer cell line (data not shown).

In addition to the inhibition of MRS expression, cell cycle analysis was performed in the presence of methionine (Met) deficiency, which affects the activity of MRS ( FIG. 1B ). In the methionine-deficient state (-Met) H460 cells, the BrdU insertion rate was greatly decreased and the percentage of arrested cells in the GO / G1 phase was significantly increased compared with the control (control) . Similar results were observed with MDA-MB-231 cells in the methionine-deficient state (data not shown).

The inhibition of MRS expression and the condition of methionine depletion have in common that the activated MRS induces a depleted state. We have investigated whether protein translation (translation) is nonspecifically inhibited by deficiency of activated MRS which is important for initiation of translation. One measures the methionine insertion rate of inhibiting the MRS expression of cell (sh-MRS) measuring the cell total protein translation reaction results, compared to the control (sh-Cont) protein translation reaction there was no significant difference (Fig. 1C ).

These results indicate that the cell cycle progresses from the GO / G1 phase to the S phase and activated MRS is essential for inducing cell division.

<1-2> Identification of cell cycle proteins regulated by MRS

In order to understand the molecular mechanisms by which MRS regulates cell cycle, we examined the target or lower signaling pathways directly affected by MRS.

Proteins whose cell levels were greatly changed compared to the control group were examined using a protein array related to the cell cycle under conditions of inhibition of MRS expression ( Fig. 2A ) or methionine deficiency ( Fig. 2B ). Of the 60 cell cycle-related proteins analyzed, 18 proteins were unchanged, and 42 proteins were significantly altered by MRS expression inhibition or methionine deficiency, 31 of which inhibited MRS expression The level changes only in one condition of the methionine deficiency. The six types showed the opposite direction in the two conditions, and the other five changed in the same direction in both conditions (data not shown). Of these five proteins, p21 ^WAF1 , CDK3, CDK4, and cyclin D1 decreased protein levels under both MRS inhibition and methionine deficiency. p21 ^WAF1 is a complex regulatory protein involved in not only the cell cycle but also various signal transduction systems, and CDK3 is known to promote the progression from the GO to the G1 phase of the cell cycle. On the other hand, CDK4 and Cyclin D1 jointly regulate the progression from G1 to S, and Cyclin D1 is a protein closely related to CDK4 functionally such as easily degraded by inherent instability in the absence of CDK4.

Among the proteins selected through the protein array, CDK3 and CDK4, which are considered to be more related to the cell cycle regulation by MRS, were expressed in the cells and the interaction with MRS was confirmed. CDK4 significantly decreased protein levels in MDA-MB-231 cells deficient in methionine, but CDK3 did not significantly change in actual cells unlike the protein array results ( Fig. 2C , MDA-MB-231 cells on the left panel, Right panel is H460 cells). In addition, co-immunoprecipitation (co-IP) of CDK3 and CDK4 overexpression in H460 cells resulted in immunoprecipitation of CDK4 with MRS, but CDK3 was found to be almost immune-precipitated with MRS ( Fig. 2D ). Therefore, it was thought that CDK4 actually interacts with MRS in cells.

In order to investigate whether MRS regulates the expression of CDK4 in the transcription stage, expression of MRS was inhibited by siRNA in MDA-MB-231 cells, and transcription level of CDK4 was confirmed by qRT-PCR. As a result, expression of MRS (Si-MRS) showed very similar mRNA expression levels of the control (si-Cont) and CDK4 ( Fig. 2E ). In other words, MRS was not involved in the transcription level of CDK4.

These results suggest that CDK4 is the most important target and effector in regulating cell cycle in MRS. As shown in <Example 1-1>, since the protein level of CDK4 is decreased, while the overall protein translation is not greatly changed under the condition of suppressing the expression or activity of MRS ( FIG. 1C ), MRS is CDK4 protein specific , Respectively.

&Lt; Example 2 >

Direct coupling of MRS and CDK4

<2-1> Measurement of binding level of MRS and CDK4 in cells

In order to understand the mechanism by which MRS regulates CDK4 protein levels, we examined whether MRS and CDK4 bind directly in the cells.

Venus BiFC (Biomolecular fluorescence complementation) was used to observe intracellular binding of MRS and CDK4 (Kwon et al., 2011; Shyu et al., 2008). We constructed a construct in which the C-terminal (VC) of the fluorescent protein Venus was bound to MRS and the N-terminal (VN) of Venus was bound to CDK4 and expressed in H460 cells to bind MRS and CDK4, Green fluorescence was generated ( Fig. 3 ). In the cells co-expressed with MRS and CDK4, strong green fluorescence was observed. However, in the cells co-expressing WRS with MRS instead of CDK4, green fluorescence was not observed at all, confirming that MRS and CDK4 specifically bind to each other in the cells.

<2-2> MRS Wow CDK4 Structural features of coupling

To understand the mechanism of the binding of MRS and CDK4, we observed changes in the binding of MRS and CDK4 in a deficient state of methionine, and produced a fragment of MRS and CDK4, and confirmed the binding, confirming the structural basis of MRS and CDK4 binding saw.

In Example 1, MRS and CDK4 were immunoprecipitated together, and it was confirmed that MRS and CDK4 are highly likely to be directly bound to each other. As a result of repeating the co-IP experiment in the absence of methionine in MDA-MB-231 cells, the amount of MRS bound to CDK4 was greatly reduced, while the total amount of MRS was not changed in the methionine-deficient condition ( FIG. 4A ). This demonstrates the importance of binding to MRS to stabilize the CDK4 protein and maintain its level.

The functional fragments of MRS and CDK4 were constructed and identified for the binding site of MRS and CDK4. MRS is a GST-like domain (1-266 amino acids), a catalytic domain (267-597 amino acids), and a tRNA binding domain (598-900 amino acids) F2, 267-597 amino acids, SEQ ID NO: 5, F3, 598-900 amino acids, SEQ ID NO: 7, F4, 1-597 amino acids, SEQ ID NO: 9, F5, 267- CDK4 was divided into N-terminal (1-102 amino acids, SEQ ID NO: 17) and C-terminal (103-303 amino acids) ) ( Fig. 4B ). Using the GST pull-down experiment, the N-terminal of CDK4 was found to be important for binding to MRS by confirming the binding of CDK4 fragment to the entire MRS ( FIG. 4C ). The binding of the fragment of MRS to the whole CDK4 was confirmed by GST pull-down experiment, and the catalytic domain or tRNA binding domain of MRS was found to be important for binding to CDK4 ( FIG. 4D ). On the other hand, F4 fragments containing both GST-like domain and catalytic domain of MRS fragments did not bind to CDK4 even though they had a catalytic domain, and GST-like domain was thought to inhibit binding of MRS and CDK4.

Since the catalytic domain of MRS was found to be important for the binding of CDK4, the catalytic domain of MRS was subdivided into a methionine binding site and a methionine activating site (Met activation site) to confirm binding affinity. Histamine (H560) at position 560 of the MRS amino acid and lysine (K596) at position 596 are known to be essential for the binding of methionine and activation of methionine, respectively (Fourmy et al., 1991; Mechulam et al, 1991). The mutation (H560A) in which H560 was replaced with alanine and the mutation (K596Q) in which K596 was replaced with glutamine was introduced into MRS and the result of binding with CDK4 using co-IP ( Fig. 4E ) ) Was still bound to CDK4, but the K596Q mutation (KQ) did not bind to CDK4, indicating that the methionine activation site in the catalytic domain of MRS is particularly important for the binding of CDK4. In addition, cycloheximide (CHX) was used to synthesize new proteins ( de In one situation inhibit novo protein synthesis), wild-type (WT) MRS, or H560A mutations (HA) is, K596Q mutation (KQ), while that more than four hours, retained by stabilizing the CDK4 were pre-existing inhibit protein synthesis in H460 cells Failed to stabilize CDK4, indicating that CDK4 was degraded to a level that was not detected by the immunoblot 4 hours before.

The above results show that MRS plays a role of stabilizing the CDK4 protein not to be degraded by directly binding to CDK4 specifically in the cells. It was found that the MRS catalytic domain and the N-terminal of CDK4 are important for the binding of MRS and CDK4, and that the methionine activation site, K596, is essential for binding to CDK4 in the MRS catalytic domain.

< Example 3>

Using methionine analogs MRS Wow CDK4 Inhibition of binding

<3-1> Screening of methionine analogs

To understand the effect of methionine depletion on the activity of MRS and consequently the mechanism of inhibiting the binding of MRS and CDK4, methionine mimetics were screened and the effect of MRS on the binding of CDK4 was investigated.

In purchase a methionine analog of 13 kinds of commercially available using each of the analog of firefly luciferase (firefly luciferase) The effects on the translation of the in vitro translation system and the effect of CDK4 and CDK4 on the protein levels of Cyclin D1, pRb (phosphorylated on the 780th amino acid serine (S780) of Rb) ( Fig. 5 ). The methionine analogues used in the experiment are as shown in Table 1 . Three of the 13 methionine analogues tested had no effect on protein translation and CDK4-related signal transduction proteins (3, 10, 13 analogs), while five of the analogs expressed protein translation and CDK4- (2, 4, 5, 6, 7 analogues), the other five analogues inhibited only protein translation and had no effect on CDK4 - related signaling proteins.

<3-2> Effects of methionine analogue FSMO on MRS activity

Among the five kinds of analogues that were found to reduce the levels of CDK4-related signaling proteins in <Example 3-1>, 6 similarities in which the level of CDK4-related signaling protein was decreased without changing the level of MRS Chain Fmoc-Sec (Mob) -OH (FSMO) were selected to investigate the effect of FSMO on the activity of MRS.

In the translation system using firefly luciferase, the protein translation was greatly reduced by FSMO (1 mM) ( Fig. 6A ). When methionine was added to the reaction (+ M), the protein translation reduced by FSMO was restored, S), cysteine (+ C), and leucine (+ L). Also in vitro aminoacylation reaction (Catalytic activity) measurements (Fig. 6B), FSMO (150μM) is in the inhibited the activity of MRS, CRS (cysteinyl-tRNA synthetase ), SRS (seryl-tRNA synthetase), KRS (lysyl-tRNA synthetase) There was no effect on activity. In other words, it was confirmed that FSMO is a methionine-specific response competitor and a specific inhibitor of MRS. In particular, as shown in the ATP-PPi exchange assay ( FIG. 6C ), FSMO inhibited the methionine activation reaction of MRS in a concentration-dependent manner. In Example 2-2, it was confirmed that a site where methionine activation reaction occurs in MRS, specifically K596, is particularly important for binding with CDK4. FSMO binds to the methionine-activated site of MRS and is likely to inhibit the response of MRS to CDK4.

Finally, the effects of FSMO on protein translation and CDK4 protein levels were compared with borrelidin, a potent inhibitor of threonyl-tRNA synthetase. FSMO and borrelidin were treated with H460 cells at different concentrations, respectively, and the level of CDK4-related protein was monitored by immunoblot ( Fig. 6D ). Separately, the concentration-dependent inhibition of protein translation of FSMO and borrelidin was measured using a firefly luciferase system ( Fig. 6E ). FSMO showed a concentration-dependent effect on MRS activity, generally at a concentration of 25 μM that did not inhibit total protein translation levels, a significant reduction in the levels of CDK4 and Cyclin D1 protein. This shows that the activity of MRS to regulate CDK4 and the catalytic activity required for normal protein translation can be somewhat distinguished. In addition, borrelidin did not significantly affect the protein level of CDK4 at 25 nM, which is a 70% reduction in total protein translation, because the modulation of CDK4 protein levels by MRS is not simply due to inhibition of normal protein translation CDK4-specific phenomenon.

<3-3> Effects of FSMO on CDK4 and MRS binding and cell cycle

We investigated whether FSMO, which is an MRS specific inhibitor and changes the protein level of CDK4, directly inhibits the binding of CDK4 and MRS and changes cell cycle progression. In the preceding <Example 3-2>, it has been observed that FSMO inhibits the regulatory reaction at the methionine activation site of MRS which is important for binding of CDK4.

In order to investigate the effect of FSMO on the binding of CDK4 and MRS, FSMO (25, 50, 100 μM) was treated with various concentrations of GST and pull-down test. As a result, the level of CDK4 protein bound to MRS decreased depending on FSMO concentration ( Fig. 7A ). The effect of FSMO directly inhibiting the reaction of MRS with CDK4 was also observed in co-IP experiments ( FIG. 7B ) and Venus BiFC experiments ( FIG. 7D ) performed by overexpressing MRS and CDK4 in H460 cells. Furthermore, the effect of FSMO on the binding of MRS and CDK4 implicitly expressed in H460 cells was confirmed by co-IP experiments ( Fig. 7C ). In particular, over the three hours of treatment with FSMO, the level of MRS already bound to CDK4 was significantly reduced before a decrease in CDK4 protein level, indicating that the dissociation of the binding of CDK4 and MRS preceded the decrease in CDK4 protein levels And that the combination of MRS and CDK4 is important for the stabilization of the CDK4 protein.

The effect of FSMO on cell cycle progression by inhibiting the binding of CDK4 and MRS was examined by analyzing the BrdU insertion rate ( FIG. 7E ) and the cell cycle distribution trend of cells ( FIG. 7F ). In the H460 cells treated with FSMO (50 μM), the BrdU insertion rate was significantly decreased compared to the control, and the proportion of cells suspended in the GO / G1 phase was significantly increased. It is understood that the FSMO inhibited the binding of MRS to CDK4 and decreased the protein level of CDK4 so that the cell cycle did not proceed normally.

Experimental results using the above-mentioned methionine analogue FSMO showed that the effect of MRS to regulate the protein level of CDK4 is not CDK4 protein specific but that it directly binds to CDK4 to stabilize the CDK4 protein . Thus, a substance capable of inhibiting the binding of MRS to CDK4, such as FSMO, may be able to inhibit CDK4-mediated cell cycle progression by destabilizing the CDK4 protein, resulting in reduced levels.

<Example 4>

MRS and p16 < RTI ID = 0.0 > ^INK4a Relationship

<4-1> p16 ^INK4a Expression and modulation of CDK4 by MRS

p16 ^INK4a is an inhibitor of CDK4 that is expressed intracellularly. p16 ^INK4a is known to competitively inhibit the activity of CDK4 and to increase the stability of CDK4 by binding to CDK4 competitively with cyclin D1 (Bockstaele et al., 2006). We ^investigated the functional relationship between MRS and p16 ^{INK4a in} the regulation of CDK4 stability.

In a variety of cell lines, siRNA (si-MRS) treatment of MRS and measurement of CDK4 levels ( Fig. 8A ) showed that CDK4 was destabilized only when the expression of MRS was suppressed in cells that did not express p16 ^INK4a , . Also FSMO has been observed to be effective for only reducing the level of CDK4 protein on criteria that inhibits the expression of p16 ^INK4a with siRNA (si-p16 ^INK4a) for p16 ^INK4a in the WI-26 lung-derived cell lines expressing the p16 ^INK4a ( Fig. 8B ).

To further confirm the relationship between the presence of p16 ^{INK4a in the} cells and the modulation effect of MRS on the level of CDK4 protein, FSMO (0, 25, 50, 100 μM) was treated with various cell lines expressing or not expressing p16 ^INK4a , The changes in protein levels of the cyclin-dependent kinases CDK2, MRS and the marker of protein translation inhibition, p-eIF2a, were analyzed by immunoblot and quantified using Image J software ( Fig. 8C ). The expression of p16 ^INK4a in the normal cell line (gray) and the cancer cell line (blue in the cervical cancer cell line, HELA; breast cancer cell line, HCC1937, MDA-MB-436, MDA-MB- A cancer cell line not expressing p16 ^INK4a (indicated in green; lung cancer cell line, A549, H460, H1299, breast cancer cell line MDA-MB-231) CDK4 protein levels were significantly decreased by FSMO in MDA-MB-231, MDA-MB-453, BT20, T47D and MCF7 colon cancer cell lines, HT-29, SW620 and HCT116. There was no significant change in the levels of CDK2, MRS, and p-eIF2α in all cell lines analyzed.

<4-2> p16 ^INK4a Wow MRS of CDK4 Bonded phase Comparison of features

Since p16 ^INK4a , an intrinsic inhibitor of CDK4, is known to bind to the N-terminus of CDK4 in the same manner as MRS, the functional relationship between p16 ^INK4a and MRS was analyzed by analyzing its binding to CDK4.

p16 ^INK4a The amount of protein is fixed and the amount of MRS protein is increased ( Figs. 9A , Left panel), the amount of MRS protein was fixed, and p16 ^INK4a Conditions that increase the amount of protein ( Fig. 9A , In the GST pull-down experiment in the right panel, binding of CDK4 to CDK4 decreased the amount of p16 ^INK4a bound to CDK4 and increased the amount of p16 ^INK4a protein , The amount of MRS bound to CDK4 decreased, ^{indicating that} p16 ^INK4a and MRS directly compete with binding to CDK4. On the other hand the co-IP experiments using the WI-26 cells expressing p16 ^INK4a was found that the amount of the MRS to bind to CDK4 when sikyeoteul inhibiting the expression of p16 ^INK4a with siRNA (si-p16 ^INK4a) increased (Fig. 9B ).

Mutations of CDK4 were examined to determine whether MRS and p16 ^INK4a competitively binding to CDK4 bind at exactly the same position as CDK4. CDK4 binds to p16 ^INK4a and the 24th arginine (R24) of the amino acid sequence is essential. The mutation (R24C) in which arginine is substituted by cysteine is not active with the activity inhibitor p16 ^INK4a and is always active (Sotillo et al. , 2001). The R24C replacement mutation of CDK4 was found in patients with inherited and non-inherited skin cancer. R24C mutant mice were found to be involved in skin cancer, endocrine tumors, sarcomas such as hemangiosarcoma, lung cancer, liver cancer, lymphoma ). &Lt; / RTI > As a result of co-IP analysis of the R24C mutation of CDK4 and MRS in 293T cells ( Fig. 9C ), it was observed that MRS binds to the R24C mutation of CDK4 at a level similar to wild-type CDK4. This suggests that the MRS in combination with CDK4 competitively with p16 ^INK4a, but not to the same amino acid and the interaction of the p16 ^INK4a binding to CDK4 not.

On the other hand, in contrast to p16 ^INK4a , MRS did not inhibit CDK4 binding to Cyclin D1 through GST pull-down experiments ( Fig. 9D ). This indicates that the binding of MRS to CDK4 and the binding of p16 ^INK4a to CDK4 are functionally different.

These results suggest that p16 ^INK4a and MRS do not bind at the same position of CDK4 but they competitively bind to each other and inhibit the binding of MRS and CDK4 to reduce CDK4 protein levels in cells that do not express p16 ^INK4a It shows that it is clearly demonstrated. Furthermore, since the MRS binds not only to the wild type CDK4 but also to the R24C mutation, the CDK4 R24C protein, which is not ^under the control of p16 ^INK4a , is destabilized by regulating the expression of MRS or inhibiting the binding of the R24C mutation of MRS and CDK4, It is possible to inhibit the phosphorylation activity of the mutant CDK4.

< Example 5>

MRS Of Cancer Disease and Cancer Disease

<5-1> Cancer Cells and Cancer Model MRS And Cancer Disease

It is well known that cyclin D and CDK4 do not affect healthy normal cells but are essential for tumor development and maintenance of cancer cells (Choi et al., 2012) ; Vogelstein and Kinzler, 2004). CDK4 is also a highly expressed protein in breast cancer and lung cancer (An et al., 1999; Kim et al., 1997; Peurala et al., 2013; Samady et al., 2004; Wu et al., 2011; Yu et al., 2006). As confirmed in the previous examples, the CDK4 protein was found to be stabilized by MRS. Therefore, the correlation between the level of MRS and cancer development or progression was examined in cell and animal models.

The expression level of MRS and CDK4 protein in various breast cancer cell lines was confirmed by immunoblotting and quantified ( Fig. 10A ) . As a result, when the level of MRS expression was high in cells that did not express p16 ^INK4a , the level of CDK4 was high and the expression of MRS CDK4 levels were also found to be low.

To investigate the effect of MRS and CDK4 protein levels and CDK4 modulation by MRS on cancer development, MDA-MB-1 cells stably expressing shRNA (sh-MRS) or control shRNA (sh-Cont) 231 breast cancer cells, anchorage independent soft agar assay was performed ( Fig. 10B ). Transformability to cancer cells was significantly lower in sh-MRS cells than in the control (sh-Cont) cells in which MRS expression was inhibited.

In addition, MDA-MB-231 breast cancer cells stably expressing sh-MRS or sh-Cont were injected into the lateral side of the mouse to confirm the effect of suppression of MRS expression on tumor development or cancer progression, And xenograft experiments were performed to observe growth results ( Fig. 10C ). There was no significant difference in body weight between the mice injected with breast cancer cells suppressed the expression of MRS (sh-MRS) and the mice injected with breast cancer cells (sh-Cont) in which MRS expression was not inhibited 10D ), the tumor growth rate was observed to be significantly reduced ( Fig. 10E ). In addition, the expression level of MRS and CDK4 and Ki-67, a marker of cell division, in tumor tissues derived from breast cancer cells in which MRS expression was inhibited by sh-MRS was also expressed in tumor tissues derived from breast cancer cells expressing sh-Cont Compared with the control group. That is, it was confirmed that suppressing the expression of MRS and decreasing the level of CDK4 protein had an excellent effect in inhibiting tumor growth.

<5-2> In cancer patients Observed MRS And Cancer Disease

The correlation between the level of MRS and cancer development or progression was examined in cancer patients.

The correlation between MRS expression level and survival rate in breast cancer patients ( Fig. 11A , survival rate without recurrence, RFS) and lung cancer patients ( Fig. 11B , overall survival rate, OS) The survival rate was decreased and the prognosis was poor compared with patients with low MRS expression level.

Tissue microarrays (TMA) containing 272 breast cancer tissues were ^transfected with MRS, CDK4, Cyclin D1, pRb and p16 ^INK4a in order to correlate MRS4 and CDK4 signaling proteins in cancer ^progression . And the degree of expression was analyzed by assigning a score indicating the expression level to each protein ( FIG. 11C ). Analysis of breast cancer in this tissue reaches 74.9% of the entire organization, which do not express p16 ^INK4a, it showed that in breast cancer, the expression is considerably higher percentage of p16 ^INK4a away from the normal state. In breast cancer tissues not expressing p16 ^INK4a , the expression levels of MRS and CDK4 were highly correlated ( FIG. 11D ), whereas no correlation was found between expression levels of MRS and CDK4 in breast cancer tissues expressing p16 ^INK4a ( Fig. 11E ).

When MRS stabilizes the CDK4 protein, a strengthening effect of lower signaling activated by CDK4 as well as CDK4 appears. Therefore, if there is a high correlation between MRS and CDK4 in the correlation of protein expression in the previously analyzed cancer tissues, it is likely that a protein that leads to CDK4-Cyclin D1-pRb leading to Cyclin D1 binding to CDK4 and Rb phosphorylated by CDK4 There is a strong possibility that there is a continuous correlation with the MRS. When CDK4-Cyclin D1-pRb shows a continuous correlation with MRS, the sum of the expression scores of each protein is added to the additional point (AP) to calculate the weighted sum of score (WSS) We analyzed the possibility of continuous correlation. The specific addition point criteria are as shown in Table 2 . In cancer tissues that do not express p16 ^INK4a although the WSS of a high tissue (MRSH) MRS levels increased significantly in comparison with the low-MRS-level organization (MRSL) (Fig. 11F), in the tumor tissue expressing p16 ^INK4a MRSH and There was no significant difference between WSS of MRSL ( Fig. 11G ).

These results suggest that MRS is associated with the development or progression of cancer through binding with CDK4 protein. In particular, MRS and MRS in cancer tissues, which do not express p16 ^INK4a , CDK4 was found to be highly correlated. This suggests that by inhibiting the expression of MRS or inhibiting the binding of MRS to CDK4, it is possible to reduce the level of CDK4 protein and effectively prevent cancer progression, particularly in cancers that do not express p16 ^INK4a .

As described above, the anticancer agent that selects a substance that decreases the binding level of MRS and CDK4 by using an MRS (methionyl-tRNA synthetase) or a fragment thereof and CDK4 (cyclin-dependent kinase 4) or a fragment thereof according to the present invention Screening method can be used to develop an anticancer agent having a completely new mechanism of action. The anticancer composition comprising the selected anticancer agent according to the present invention as an active ingredient inhibits the expression of MRS or inhibits the binding of MRS to CDK4, thereby lowering the level of CDK4 protein. Therefore, the ^composition for cancer diseases not specifically expressing p16 ^INK4a And can be useful for developing therapeutic agents.

<110> Medicinal Bioconvergence Research Center <120> Methods for screening anti-cancer agents inhibiting interactions between MRS and CDK4 <130> NP15-0109 <160> 25 <170> Kopatentin 2.0 <210> 1 <211> 900 <212> PRT <213> Homo sapiens MRS protein (NP_004981.2) <400> 1 Met Arg Leu Phe Val Ser Asp Gly Val Pro Gly Cys Leu Pro Val Leu 1 5 10 15 Ala Ala Ala Gly Arg Ala Arg Gly Arg Ala Glu Val Leu Ile Ser Thr 20 25 30 Val Gly Pro Glu Asp Cys Val Val Pro Phe Leu Thr Arg Pro Lys Val 35 40 45 Pro Val Leu Gln Leu Asp Ser Gly Asn Tyr Leu Phe Ser Thr Ser Ala 50 55 60 Ile Cys Arg Tyr Phe Leu Leu Ser Gly Trp Glu Gln Asp Asp Leu 65 70 75 80 Thr Asn Gln Trp Leu Glu Trp Glu Ala Thr Glu Leu Gln Pro Ala Leu 85 90 95 Ser Ala Leu Tyr Tyr Leu Val Val Gln Gly Lys Lys Gly Glu Asp 100 105 110 Val Leu Gly Ser Val Arg Arg Ala Leu Thr His Ile Asp His Ser Leu 115 120 125 Ser Arg Gln Asn Cys Pro Phe Leu Ala Gly Glu Thr Glu Ser Leu Ala 130 135 140 Asp Ile Val Leu Trp Gly Ala Leu Tyr Pro Leu Leu Gln Asp Pro Ala 145 150 155 160 Tyr Leu Pro Glu Glu Leu Ser Ala Leu His Ser Trp Phe Gln Thr Leu 165 170 175 Ser Thr Gln Glu Pro Cys Gln Arg Ala Ala Glu Thr Val Leu Lys Gln 180 185 190 Gln Gly Val Leu Ala Leu Arg Pro Tyr Leu Gln Lys Gln Pro Gln Pro 195 200 205 Ser Pro Ala Glu Gly Arg Ala Val Thr Asn Glu Pro Glu Glu Glu Glu 210 215 220 Leu Ala Thr Leu Ser Glu Glu Glu Ile Ala Met Ala Val Thr Ala Trp 225 230 235 240 Glu Lys Gly Leu Glu Ser Leu Pro Pro Leu Arg Pro Gln Gln Asn Pro 245 250 255 Val Leu Pro Val Ala Gly Glu Arg Asn Val Leu Ile Thr Ser Ala Leu 260 265 270 Pro Tyr Val Asn As Val Val Pro His Leu Gly Asn Ile Gly Cys Val 275 280 285 Leu Ser Ala Asp Val Phe Ala Arg Tyr Ser Arg Leu Arg Gln Trp Asn 290 295 300 Thr Leu Tyr Leu Cys Gly Thr Asp Glu Tyr Gly Thr Ala Thr Glu Thr 305 310 315 320 Lys Ala Leu Glu Glu Gly Leu Thr Pro Gln Glu Ile Cys Asp Lys Tyr 325 330 335 His Ile Ile His Ala Asp Ile Tyr Arg Trp Phe Asn Ile Ser Phe Asp 340 345 350 Ile Phe Gly Arg Thr Thr Thr Pro Gln Gln Thr Lys Ile Thr Gln Asp 355 360 365 Ile Phe Gln Gln Leu Leu Lys Arg Gly Phe Val Leu Gln Asp Thr Val 370 375 380 Glu Gln Leu Arg Cys Glu His Cys Ala Arg Phe Leu Ala Asp Arg Phe 385 390 395 400 Val Glu Gly Val Cys Pro Phe Cys Gly Tyr Glu Glu Ala Arg Gly Asp 405 410 415 Gln Cys Asp Lys Cys Gly Lys Leu Ile Asn Ala Val Glu Leu Lys Lys 420 425 430 Pro Gln Cys Lys Val Cys Arg Ser Cys Pro Val Val Gln Ser Ser Gln 435 440 445 His Leu Phe Leu Asp Leu Pro Lys Leu Glu Lys Arg Leu Glu Glu Trp 450 455 460 Leu Gly Arg Thr Leu Pro Gly Ser Asp Trp Thr Pro Asn Ala Gln Phe 465 470 475 480 Ile Thr Arg Ser Trp Leu Arg Asp Gly Leu Lys Pro Arg Cys Ile Thr 485 490 495 Arg Asp Leu Lys Trp Gly Thr Pro Val Leu Glu Gly Phe Glu Asp 500 505 510 Lys Val Phe Tyr Val Trp Phe Asp Ala Thr Ile Gly Tyr Leu Ser Ile 515 520 525 Thr Ala Asn Tyr Thr Asp Gln Trp Glu Arg Trp Trp Lys Asn Pro Glu 530 535 540 Gln Val Asp Leu Tyr Gln Phe Met Ala Lys Asp Asn Val Pro Phe His 545 550 555 560 Ser Leu Val Phe Pro Cys Ser Ala Leu Gly Ala Glu Asp Asn Tyr Thr 565 570 575 Leu Val Ser His Leu Ile Ala Thr Glu Tyr Leu Asn Tyr Glu Asp Gly 580 585 590 Lys Phe Ser Lys Ser Arg Gly Val Gly Val Phe Gly Asp Met Ala Gln 595 600 605 Asp Thr Gly Ile Pro Ala Asp Ile Trp Arg Phe Tyr Leu Leu Tyr Ile 610 615 620 Arg Pro Glu Gly Gln Asp Ser Ala Phe Ser Trp Thr Asp Leu Leu Leu 625 630 635 640 Lys Asn Asn Ser Glu Leu Leu Asn Asn Leu Gly Asn Phe Ile Asn Arg 645 650 655 Ala Gly Met Phe Val Ser Lys Phe Phe Gly Gly Tyr Val Pro Glu Met 660 665 670 Val Leu Thr Pro Asp Asp Gln Arg Leu Leu Ala His Val Thr Leu Glu 675 680 685 Leu Gln His Tyr His Gln Leu Leu Glu Lys Val Arg Ile Arg Asp Ala 690 695 700 Leu Arg Ser Ile Leu Thr Ile Ser Arg His Gly Asn Gln Tyr Ile Gln 705 710 715 720 Val Asn Glu Pro Trp Lys Arg Ile Lys Gly Ser Glu Ala Asp Arg Gln 725 730 735 Arg Ala Gly Thr Val Thr Gly Leu Ala Val Asn Ile Ala Ala Leu Leu 740 745 750 Ser Val Met Leu Gln Pro Tyr Met Pro Thr Val Ser Ala Thr Ile Gln 755 760 765 Ala Gln Leu Gln Leu Pro Pro Ala Cys Ser Ile Leu Leu Thr Asn 770 775 780 Phe Leu Cys Thr Leu Pro Ala Gly His Gln Ile Gly Thr Val Ser Pro 785 790 795 800 Leu Phe Gln Lys Leu Glu Asn Asp Gln Ile Glu Ser Leu Arg Gln Arg 805 810 815 Phe Gly Gly Gly Gln Ala Lys Thr Ser Pro Lys Pro Ala Val Val Glu 820 825 830 Thr Val Thr Thr Ala Lys Pro Gln Gln Ile Gln Ala Leu Met Asp Glu 835 840 845 Val Thr Lys Gln Gly Asn Ile Val Arg Glu Leu Lys Ala Gln Lys Ala 850 855 860 Asp Lys Asn Glu Val Ala Ala Glu Val Ala Lys Leu Leu Asp Leu Lys 865 870 875 880 Lys Gln Leu Ala Val Ala Glu Gly Lys Pro Pro Glu Ala Pro Lys Gly 885 890 895 Lys Lys Lys Lys 900 <210> 2 <211> 2703 <212> DNA <213> Homo sapiens MRS mRNA (NM_004990.3) <400> 2 atgagactgt tcgtgagtga tggcgtcccg ggttgcttgc cggtgctggc cgccgccggg 60 agagcccggg gcagagcaga ggtgctcatc agcactgtag gcccggaaga ttgtgtggtc 120 ccgttcctga cccggcctaa ggtccctgtc ttgcagctgg atagcggcaa ctacctcttc 180 tccactagtg caatctgccg atattttttt ttgttatctg gctgggagca agatgacctc 240 actaaccagt ggctggaatg ggaagcgaca gagctgcagc cagctttgtc tgctgccctg 300 tactatttag tggtccaagg caagaagggg gaagatgttc ttggttcagt gcggagagcc 360 ctgactcaca ttgaccacag cttgagtcgt cagaactgtc ctttcctggc tggggagaca 420 gaatctctag ccgacattgt tttgtgggga gccctatacc cattactgca agatcccgcc 480 tacctccctg aggagctgag tgccctgcac agctggttcc agacactgag tacccaggaa 540 ccatgtcagc gagctgcaga gactgtactg aaacagcaag gtgtcctggc tctccggcct 600 tacctccaaa agcagcccca gcccagcccc gctgagggaa gggctgtcac caatgagcct 660 gaggaggagg agctggctac cctatctgag gaggagattg ctatggctgt tactgcttgg 720 gagaagggcc tagaaagttt gcccccgctg cggccccagc agaatccagt gttgcctgtg 780 gctggagaaa ggaatgtgct catcaccagt gccctccctt acgtcaacaa tgtcccccac 840 cttgggaaca tcattggttg tgtgctcagt gccgatgtct ttgccaggta ctctcgcctc 900 cgccagtgga acaccctcta tctgtgtggg acagatgagt atggtacagc aacagagacc 960 aaggctctgg aggagggact aaccccccag gagatctgcg acaagtacca catcatccat 1020 gctgacatct accgctggtt taacatttcg tttgatattt ttggtcgcac caccactcca 1080 cagcagacca aaatcaccca ggacattttc cagcagttgc tgaaacgagg ttttgtgctg 1140 caagatactg tggagcaact gcgatgtgag cactgtgctc gcttcctggc tgaccgcttc 1200 gtggagggcg tgtgtccctt ctgtggctat gaggaggctc ggggtgacca gtgtgacaag 1260 tgtggcaagc tcatcaatgc tgtcgagctt aagaagcctc agtgtaaagt ctgccgatca 1320 tgccctgtgg tgcagtcgag ccagcacctg tttctggacc tgcctaagct ggagaagcga 1380 ctggaggagt ggttggggag gacattgcct ggcagtgact ggacacccaa tgcccagttt 1440 atcacccgtt cttggcttcg ggatggcctc aagccacgct gcataacccg agacctcaaa 1500 tggggaaccc ctgtaccctt agaaggtttt gaagacaagg tattctatgt ctggtttgat 1560 gccactattg gctatctgtc catcacagcc aactacacag accagtggga gagatggtgg 1620 aagaacccag agcaagtgga cctgtatcag ttcatggcca aagacaatgt tcctttccat 1680 agcttagtct ttccttgctc agccctagga gctgaggata actatacctt ggtcagccac 1740 ctcattgcta cagagtacct gaactatgag gatgggaaat tctctaagag ccgcggtgtg 1800 gggtgtttg gggacatggc ccaggacacg gggatccctg ctgacatctg gcgcttctat 1860 ctgctgtaca ttcggcctga gggccaggac agtgctttct cctggacgga cctgctgctg 1920 aagaataatt ctgagctgct taacaacctg ggcaacttca tcaacagagc tgggatgttt 1980 gtgtctaagt tctttggggg ctatgtgcct gagatggtgc tcacccctga tgatcagcgc 2040 ctgctggccc atgtcaccct ggagctccag cactatcacc agctacttga gaaggttcgg 2100 atccgggatg ccttgcgcag tatcctcacc atatctcgac atggcaacca atatattcag 2160 gtgaatgagc cctggaagcg gattaaaggc agtgaggctg acaggcaacg ggcaggaaca 2220 gtgactggct tggcagtgaa tatagctgcc ttgctctctg tcatgcttca gccttacatg 2280 cccacggtta gtgccacaat ccaggcccag ctgcagctcc cacctccagc ctgcagtatc 2340 ctgctgacaa acttcctgtg taccttacca gcaggacacc agattggcac agtcagtccc 2400 ttgttccaaa aattggaaaa tgaccagatt gaaagtttaa ggcagcgctt tggagggggc 2460 caggcaaaaa cgtccccgaa gccagcagtt gtagagactg ttacaacagc caagccacag 2520 cagatacaag cgctgatgga tgaagtgaca aaacaaggaa acattgtccg agaactgaaa 2580 gcacaaaagg cagacaagaa cgaggttgct gcggaggtgg cgaaactctt ggatctaaag 2640 aaacagttgg ctgtagctga ggggaaaccc cctgaagccc ctaaaggcaa gaagaaaaag 2700 taa 2703 <210> 3 <211> 266 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens MRS F1 fragment protein (1-266 amino acids) <400> 3 Met Arg Leu Phe Val Ser Asp Gly Val Pro Gly Cys Leu Pro Val Leu 1 5 10 15 Ala Ala Ala Gly Arg Ala Arg Gly Arg Ala Glu Val Leu Ile Ser Thr 20 25 30 Val Gly Pro Glu Asp Cys Val Val Pro Phe Leu Thr Arg Pro Lys Val 35 40 45 Pro Val Leu Gln Leu Asp Ser Gly Asn Tyr Leu Phe Ser Thr Ser Ala 50 55 60 Ile Cys Arg Tyr Phe Leu Leu Ser Gly Trp Glu Gln Asp Asp Leu 65 70 75 80 Thr Asn Gln Trp Leu Glu Trp Glu Ala Thr Glu Leu Gln Pro Ala Leu 85 90 95 Ser Ala Leu Tyr Tyr Leu Val Val Gln Gly Lys Lys Gly Glu Asp 100 105 110 Val Leu Gly Ser Val Arg Arg Ala Leu Thr His Ile Asp His Ser Leu 115 120 125 Ser Arg Gln Asn Cys Pro Phe Leu Ala Gly Glu Thr Glu Ser Leu Ala 130 135 140 Asp Ile Val Leu Trp Gly Ala Leu Tyr Pro Leu Leu Gln Asp Pro Ala 145 150 155 160 Tyr Leu Pro Glu Glu Leu Ser Ala Leu His Ser Trp Phe Gln Thr Leu 165 170 175 Ser Thr Gln Glu Pro Cys Gln Arg Ala Ala Glu Thr Val Leu Lys Gln 180 185 190 Gln Gly Val Leu Ala Leu Arg Pro Tyr Leu Gln Lys Gln Pro Gln Pro 195 200 205 Ser Pro Ala Glu Gly Arg Ala Val Thr Asn Glu Pro Glu Glu Glu Glu 210 215 220 Leu Ala Thr Leu Ser Glu Glu Glu Ile Ala Met Ala Val Thr Ala Trp 225 230 235 240 Glu Lys Gly Leu Glu Ser Leu Pro Pro Leu Arg Pro Gln Gln Asn Pro 245 250 255 Val Leu Pro Val Ala Gly Glu Arg Asn Val 260 265 <210> 4 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens MRS F1 fragment mRNA <400> 4 atgagactgt tcgtgagtga tggcgtcccg ggttgcttgc cggtgctggc cgccgccggg 60 agagcccggg gcagagcaga ggtgctcatc agcactgtag gcccggaaga ttgtgtggtc 120 ccgttcctga cccggcctaa ggtccctgtc ttgcagctgg atagcggcaa ctacctcttc 180 tccactagtg caatctgccg atattttttt ttgttatctg gctgggagca agatgacctc 240 actaaccagt ggctggaatg ggaagcgaca gagctgcagc cagctttgtc tgctgccctg 300 tactatttag tggtccaagg caagaagggg gaagatgttc ttggttcagt gcggagagcc 360 ctgactcaca ttgaccacag cttgagtcgt cagaactgtc ctttcctggc tggggagaca 420 gaatctctag ccgacattgt tttgtgggga gccctatacc cattactgca agatcccgcc 480 tacctccctg aggagctgag tgccctgcac agctggttcc agacactgag tacccaggaa 540 ccatgtcagc gagctgcaga gactgtactg aaacagcaag gtgtcctggc tctccggcct 600 tacctccaaa agcagcccca gcccagcccc gctgagggaa gggctgtcac caatgagcct 660 gaggaggagg agctggctac cctatctgag gaggagattg ctatggctgt tactgcttgg 720 gagaagggcc tagaaagttt gcccccgctg cggccccagc agaatccagt gttgcctgtg 780 gctggagaaa ggaatgtg 798 <210> 5 <211> 331 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens MRS F2 fragment protein (267-597 amino acids) <400> 5 Leu Ile Thr Ser Ala Leu Pro Tyr Val Asn Asn Val Pro His Leu Gly 1 5 10 15 Asn Ile Ile Gly Cys Val Leu Ser Ala Asp Val Phe Ala Arg Tyr Ser 20 25 30 Arg Leu Arg Gln Trp Asn Thr Leu Tyr Leu Cys Gly Thr Asp Glu Tyr 35 40 45 Gly Thr Ala Thr Glu Thr Lys Ala Leu Glu Glu Gly Leu Thr Pro Gln 50 55 60 Glu Ile Cys Asp Lys Tyr His Ile Ile His Ala Asp Ile Tyr Arg Trp 65 70 75 80 Phe Asn Ile Ser Phe Asp Ile Phe Gly Arg Thr Thr Thr Pro Gln Gln 85 90 95 Thr Lys Ile Thr Gln Asp Ile Phe Gln Gln Leu Leu Lys Arg Gly Phe 100 105 110 Val Leu Gln Asp Thr Val Glu Gln Leu Arg Cys Glu His Cys Ala Arg 115 120 125 Phe Leu Ala Asp Arg Phe Val Glu Gly Val Cys Pro Phe Cys Gly Tyr 130 135 140 Glu Glu Ala Arg Gly Asp Gln Cys Asp Lys Cys Gly Lys Leu Ile Asn 145 150 155 160 Ala Val Glu Leu Lys Lys Pro Gln Cys Lys Val Cys Arg Ser Cys Pro 165 170 175 Val Val Gln Ser Ser Gln His Leu Phe Leu Asp Leu Pro Lys Leu Glu 180 185 190 Lys Arg Leu Glu Glu Trp Leu Gly Arg Thr Leu Pro Gly Ser Asp Trp 195 200 205 Thr Pro Asn Gln Phe Ile Thr Arg Ser Trp Leu Arg Asp Gly Leu 210 215 220 Lys Pro Arg Cys Ile Thr Arg Asp Leu Lys Trp Gly Thr Pro Val Pro 225 230 235 240 Leu Glu Gly Phe Glu Asp Lys Val Phe Tyr Val Trp Phe Asp Ala Thr 245 250 255 Ile Gly Tyr Leu Ser Ile Thr Ala Asn Tyr Thr Asp Gln Trp Glu Arg 260 265 270 Trp Trp Lys Asn Pro Glu Gln Val Asp Leu Tyr Gln Phe Met Ala Lys 275 280 285 Asp Asn Val Pro Phe His Ser Leu Val Phe Pro Cys Ser Ala Leu Gly 290 295 300 Ala Glu Asp Asn Tyr Thr Leu Val Ser Ser Leu Ile Ala Thr Glu Tyr 305 310 315 320 Leu Asn Tyr Glu Asp Gly Lys Phe Ser Lys Ser 325 330 <210> 6 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens MRS F2 fragment mRNA <400> 6 ctcatcacca gtgccctccc ttacgtcaac aatgtccccc accttgggaa catcattggt 60 tgtgtgctca gtgccgatgt ctttgccagg tactctcgcc tccgccagtg gaacaccctc 120 tatctgtgtg ggacagatga gtatggtaca gcaacagaga ccaaggctct ggaggaggga 180 ctaacccccc aggagatctg cgacaagtac cacatcatcc atgctgacat ctaccgctgg 240 tttaacattt cgtttgatat ttttggtcgc accaccactc cacagcagac caaaatcacc 300 caggacattt tccagcagtt gctgaaacga ggttttgtgc tgcaagatac tgtggagcaa 360 ctgcgatgtg agcactgtgc tcgcttcctg gctgaccgct tcgtggaggg cgtgtgtccc 420 ttctgtggct atgaggaggc tcggggtgac cagtgtgaca agtgtggcaa gctcatcaat 480 gctgtcgagc ttaagaagcc tcagtgtaaa gtctgccgat catgccctgt ggtgcagtcg 540 agccagcacc tgtttctgga cctgcctaag ctggagaagc gactggagga gtggttgggg 600 aggacattgc ctggcagtga ctggacaccc aatgcccagt ttatcacccg ttcttggctt 660 cgggatggcc tcaagccacg ctgcataacc cgagacctca aatggggaac ccctgtaccc 720 ttagaaggtt ttgaagacaa ggtattctat gtctggtttg atgccactat tggctatctg 780 tccatcacag ccaactacac agaccagtgg gagagatggt ggaagaaccc agagcaagtg 840 gacctgtatc agttcatggc caaagacaat gttcctttcc atagcttagt ctttccttgc 900 tcagccctag gagctgagga taactatacc ttggtcagcc acctcattgc tacagagtac 960 ctgaactatg aggatgggaa attctctaag agc 993 <210> 7 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens MRS F3 fragment protein (598-900 amino acids) <400> 7 Arg Gly Val Gly Val Phe Gly Asp Met Ala Gln Asp Thr Gly Ile Pro 1 5 10 15 Ala Asp Ile Trp Arg Phe Tyr Leu Leu Tyr Ile Arg Pro Glu Gly Gln 20 25 30 Asp Ser Ala Phe Ser Trp Thr Asp Leu Leu Leu Lys Asn Asn Ser Glu 35 40 45 Leu Leu Asn Asn Leu Gly Asn Phe Ile Asn Arg Ala Gly Met Phe Val 50 55 60 Ser Lys Phe Phe Gly Gly Tyr Val Pro Glu Met Val Leu Thr Pro Asp 65 70 75 80 Asp Gln Arg Leu Leu Ala His Val Thr Leu Glu Leu Gln His Tyr His 85 90 95 Gln Leu Leu Glu Lys Val Arg Ile Arg Asp Ala Leu Arg Ser Ile Leu 100 105 110 Thr Ile Ser Arg His Gly Asn Gln Tyr Ile Gln Val Asn Glu Pro Trp 115 120 125 Lys Arg Ile Lys Gly Ser Glu Ala Asp Arg Gln Arg Ala Gly Thr Val 130 135 140 Thr Gly Leu Ala Val Asn Ile Ala Ala Leu 145 150 155 160 Pro Tyr Met Pro Thr Val Ser Ala Thr Ile Gln Ala Gln Leu Gln Leu 165 170 175 Pro Pro Ala Cys Ser Ile Leu Leu Thr Asn Phe Leu Cys Thr Leu 180 185 190 Pro Ala Gly His Gln Ile Gly Thr Val Ser Pro Leu Phe Gln Lys Leu 195 200 205 Glu Asn Gln Ile Glu Ser Leu Arg Gln Arg Phe Gly Gly Gly Gln 210 215 220 Ala Lys Thr Ser Pro Lys Pro Ala Val Val Glu Thr Val Thr Thr Ala 225 230 235 240 Lys Pro Gln Gln Ile Gln Ala Leu Met Asp Glu Val Thr Lys Gln Gly 245 250 255 Asn Ile Val Arg Glu Leu Lys Ala Gln Lys Ala Asp Lys Asn Glu Val 260 265 270 Ala Ala Glu Val Ala Lys Leu Leu Asp Leu Lys Lys Gln Leu Ala Val 275 280 285 Ala Glu Gly Lys Pro Pro Glu Ala Pro Lys Gly Lys Lys Lys Lys 290 295 300 <210> 8 <211> 909 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens MRS F3 fragment mRNA <400> 8 cgcggtgtgg gagtgtttgg ggacatggcc caggacacgg ggatccctgc tgacatctgg 60 cgcttctatc tgctgtacat tcggcctgag ggccaggaca gtgctttctc ctggacggac 120 ctgctgctga agaataattc tgagctgctt aacaacctgg gcaacttcat caacagagct 180 gggatgtttg tgtctaagtt ctttgggggc tatgtgcctg agatggtgct cacccctgat 240 gatcagcgcc tgctggccca tgtcaccctg gagctccagc actatcacca gctacttgag 300 aaggttcgga tccgggatgc cttgcgcagt atcctcacca tatctcgaca tggcaaccaa 360 tatattcagg tgaatgagcc ctggaagcgg attaaaggca gtgaggctga caggcaacgg 420 gcaggaacag tgactggctt ggcagtgaat atagctgcct tgctctctgt catgcttcag 480 ccttacatgc ccacggttag tgccacaatc caggcccagc tgcagctccc acctccagcc 540 tgcagtatcc tgctgacaaa cttcctgtgt accttaccag caggacacca gattggcaca 600 gtcagtccct tgttccaaaa attggaaaat gaccagattg aaagtttaag gcagcgcttt 660 ggagggggcc aggcaaaaac gtccccgaag ccagcagttg tagagactgt tacaacagcc 720 aagccacagc agatacaagc gctgatggat gaagtgacaa aacaaggaaa cattgtccga 780 gaactgaaag cacaaaaggc agacaagaac gaggttgctg cggaggtggc gaaactcttg 840 gatctaaaga aacagttggc tgtagctgag gggaaacccc ctgaagcccc taaaggcaag 900 aagaaaaag 909 <210> 9 <211> 597 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens MRS F4 fragment protein (1-597 amino acids) <400> 9 Met Arg Leu Phe Val Ser Asp Gly Val Pro Gly Cys Leu Pro Val Leu 1 5 10 15 Ala Ala Ala Gly Arg Ala Arg Gly Arg Ala Glu Val Leu Ile Ser Thr 20 25 30 Val Gly Pro Glu Asp Cys Val Val Pro Phe Leu Thr Arg Pro Lys Val 35 40 45 Pro Val Leu Gln Leu Asp Ser Gly Asn Tyr Leu Phe Ser Thr Ser Ala 50 55 60 Ile Cys Arg Tyr Phe Leu Leu Ser Gly Trp Glu Gln Asp Asp Leu 65 70 75 80 Thr Asn Gln Trp Leu Glu Trp Glu Ala Thr Glu Leu Gln Pro Ala Leu 85 90 95 Ser Ala Leu Tyr Tyr Leu Val Val Gln Gly Lys Lys Gly Glu Asp 100 105 110 Val Leu Gly Ser Val Arg Arg Ala Leu Thr His Ile Asp His Ser Leu 115 120 125 Ser Arg Gln Asn Cys Pro Phe Leu Ala Gly Glu Thr Glu Ser Leu Ala 130 135 140 Asp Ile Val Leu Trp Gly Ala Leu Tyr Pro Leu Leu Gln Asp Pro Ala 145 150 155 160 Tyr Leu Pro Glu Glu Leu Ser Ala Leu His Ser Trp Phe Gln Thr Leu 165 170 175 Ser Thr Gln Glu Pro Cys Gln Arg Ala Ala Glu Thr Val Leu Lys Gln 180 185 190 Gln Gly Val Leu Ala Leu Arg Pro Tyr Leu Gln Lys Gln Pro Gln Pro 195 200 205 Ser Pro Ala Glu Gly Arg Ala Val Thr Asn Glu Pro Glu Glu Glu Glu 210 215 220 Leu Ala Thr Leu Ser Glu Glu Glu Ile Ala Met Ala Val Thr Ala Trp 225 230 235 240 Glu Lys Gly Leu Glu Ser Leu Pro Pro Leu Arg Pro Gln Gln Asn Pro 245 250 255 Val Leu Pro Val Ala Gly Glu Arg Asn Val Leu Ile Thr Ser Ala Leu 260 265 270 Pro Tyr Val Asn As Val Val Pro His Leu Gly Asn Ile Gly Cys Val 275 280 285 Leu Ser Ala Asp Val Phe Ala Arg Tyr Ser Arg Leu Arg Gln Trp Asn 290 295 300 Thr Leu Tyr Leu Cys Gly Thr Asp Glu Tyr Gly Thr Ala Thr Glu Thr 305 310 315 320 Lys Ala Leu Glu Glu Gly Leu Thr Pro Gln Glu Ile Cys Asp Lys Tyr 325 330 335 His Ile Ile His Ala Asp Ile Tyr Arg Trp Phe Asn Ile Ser Phe Asp 340 345 350 Ile Phe Gly Arg Thr Thr Thr Pro Gln Gln Thr Lys Ile Thr Gln Asp 355 360 365 Ile Phe Gln Gln Leu Leu Lys Arg Gly Phe Val Leu Gln Asp Thr Val 370 375 380 Glu Gln Leu Arg Cys Glu His Cys Ala Arg Phe Leu Ala Asp Arg Phe 385 390 395 400 Val Glu Gly Val Cys Pro Phe Cys Gly Tyr Glu Glu Ala Arg Gly Asp 405 410 415 Gln Cys Asp Lys Cys Gly Lys Leu Ile Asn Ala Val Glu Leu Lys Lys 420 425 430 Pro Gln Cys Lys Val Cys Arg Ser Cys Pro Val Val Gln Ser Ser Gln 435 440 445 His Leu Phe Leu Asp Leu Pro Lys Leu Glu Lys Arg Leu Glu Glu Trp 450 455 460 Leu Gly Arg Thr Leu Pro Gly Ser Asp Trp Thr Pro Asn Ala Gln Phe 465 470 475 480 Ile Thr Arg Ser Trp Leu Arg Asp Gly Leu Lys Pro Arg Cys Ile Thr 485 490 495 Arg Asp Leu Lys Trp Gly Thr Pro Val Leu Glu Gly Phe Glu Asp 500 505 510 Lys Val Phe Tyr Val Trp Phe Asp Ala Thr Ile Gly Tyr Leu Ser Ile 515 520 525 Thr Ala Asn Tyr Thr Asp Gln Trp Glu Arg Trp Trp Lys Asn Pro Glu 530 535 540 Gln Val Asp Leu Tyr Gln Phe Met Ala Lys Asp Asn Val Pro Phe His 545 550 555 560 Ser Leu Val Phe Pro Cys Ser Ala Leu Gly Ala Glu Asp Asn Tyr Thr 565 570 575 Leu Val Ser His Leu Ile Ala Thr Glu Tyr Leu Asn Tyr Glu Asp Gly 580 585 590 Lys Phe Ser Lys Ser 595 <210> 10 <211> 1791 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens MRS F4 fragment mRNA <400> 10 atgagactgt tcgtgagtga tggcgtcccg ggttgcttgc cggtgctggc cgccgccggg 60 agagcccggg gcagagcaga ggtgctcatc agcactgtag gcccggaaga ttgtgtggtc 120 ccgttcctga cccggcctaa ggtccctgtc ttgcagctgg atagcggcaa ctacctcttc 180 tccactagtg caatctgccg atattttttt ttgttatctg gctgggagca agatgacctc 240 actaaccagt ggctggaatg ggaagcgaca gagctgcagc cagctttgtc tgctgccctg 300 tactatttag tggtccaagg caagaagggg gaagatgttc ttggttcagt gcggagagcc 360 ctgactcaca ttgaccacag cttgagtcgt cagaactgtc ctttcctggc tggggagaca 420 gaatctctag ccgacattgt tttgtgggga gccctatacc cattactgca agatcccgcc 480 tacctccctg aggagctgag tgccctgcac agctggttcc agacactgag tacccaggaa 540 ccatgtcagc gagctgcaga gactgtactg aaacagcaag gtgtcctggc tctccggcct 600 tacctccaaa agcagcccca gcccagcccc gctgagggaa gggctgtcac caatgagcct 660 gaggaggagg agctggctac cctatctgag gaggagattg ctatggctgt tactgcttgg 720 gagaagggcc tagaaagttt gcccccgctg cggccccagc agaatccagt gttgcctgtg 780 gctggagaaa ggaatgtgct catcaccagt gccctccctt acgtcaacaa tgtcccccac 840 cttgggaaca tcattggttg tgtgctcagt gccgatgtct ttgccaggta ctctcgcctc 900 cgccagtgga acaccctcta tctgtgtggg acagatgagt atggtacagc aacagagacc 960 aaggctctgg aggagggact aaccccccag gagatctgcg acaagtacca catcatccat 1020 gctgacatct accgctggtt taacatttcg tttgatattt ttggtcgcac caccactcca 1080 cagcagacca aaatcaccca ggacattttc cagcagttgc tgaaacgagg ttttgtgctg 1140 caagatactg tggagcaact gcgatgtgag cactgtgctc gcttcctggc tgaccgcttc 1200 gtggagggcg tgtgtccctt ctgtggctat gaggaggctc ggggtgacca gtgtgacaag 1260 tgtggcaagc tcatcaatgc tgtcgagctt aagaagcctc agtgtaaagt ctgccgatca 1320 tgccctgtgg tgcagtcgag ccagcacctg tttctggacc tgcctaagct ggagaagcga 1380 ctggaggagt ggttggggag gacattgcct ggcagtgact ggacacccaa tgcccagttt 1440 atcacccgtt cttggcttcg ggatggcctc aagccacgct gcataacccg agacctcaaa 1500 tggggaaccc ctgtaccctt agaaggtttt gaagacaagg tattctatgt ctggtttgat 1560 gccactattg gctatctgtc catcacagcc aactacacag accagtggga gagatggtgg 1620 aagaacccag agcaagtgga cctgtatcag ttcatggcca aagacaatgt tcctttccat 1680 agcttagtct ttccttgctc agccctagga gctgaggata actatacctt ggtcagccac 1740 ctcattgcta cagagtacct gaactatgag gatgggaaat tctctaagag c 1791 <210> 11 <211> 634 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens MRS F5 fragment protein (267-900 amino acids) <400> 11 Leu Ile Thr Ser Ala Leu Pro Tyr Val Asn Asn Val Pro His Leu Gly 1 5 10 15 Asn Ile Ile Gly Cys Val Leu Ser Ala Asp Val Phe Ala Arg Tyr Ser 20 25 30 Arg Leu Arg Gln Trp Asn Thr Leu Tyr Leu Cys Gly Thr Asp Glu Tyr 35 40 45 Gly Thr Ala Thr Glu Thr Lys Ala Leu Glu Glu Gly Leu Thr Pro Gln 50 55 60 Glu Ile Cys Asp Lys Tyr His Ile Ile His Ala Asp Ile Tyr Arg Trp 65 70 75 80 Phe Asn Ile Ser Phe Asp Ile Phe Gly Arg Thr Thr Thr Pro Gln Gln 85 90 95 Thr Lys Ile Thr Gln Asp Ile Phe Gln Gln Leu Leu Lys Arg Gly Phe 100 105 110 Val Leu Gln Asp Thr Val Glu Gln Leu Arg Cys Glu His Cys Ala Arg 115 120 125 Phe Leu Ala Asp Arg Phe Val Glu Gly Val Cys Pro Phe Cys Gly Tyr 130 135 140 Glu Glu Ala Arg Gly Asp Gln Cys Asp Lys Cys Gly Lys Leu Ile Asn 145 150 155 160 Ala Val Glu Leu Lys Lys Pro Gln Cys Lys Val Cys Arg Ser Cys Pro 165 170 175 Val Val Gln Ser Ser Gln His Leu Phe Leu Asp Leu Pro Lys Leu Glu 180 185 190 Lys Arg Leu Glu Glu Trp Leu Gly Arg Thr Leu Pro Gly Ser Asp Trp 195 200 205 Thr Pro Asn Gln Phe Ile Thr Arg Ser Trp Leu Arg Asp Gly Leu 210 215 220 Lys Pro Arg Cys Ile Thr Arg Asp Leu Lys Trp Gly Thr Pro Val Pro 225 230 235 240 Leu Glu Gly Phe Glu Asp Lys Val Phe Tyr Val Trp Phe Asp Ala Thr 245 250 255 Ile Gly Tyr Leu Ser Ile Thr Ala Asn Tyr Thr Asp Gln Trp Glu Arg 260 265 270 Trp Trp Lys Asn Pro Glu Gln Val Asp Leu Tyr Gln Phe Met Ala Lys 275 280 285 Asp Asn Val Pro Phe His Ser Leu Val Phe Pro Cys Ser Ala Leu Gly 290 295 300 Ala Glu Asp Asn Tyr Thr Leu Val Ser Ser Leu Ile Ala Thr Glu Tyr 305 310 315 320 Leu Asn Tyr Glu Asp Gly Lys Phe Ser Ser Ser Arg Gly Val Gly Val 325 330 335 Phe Gly Asp Met Ala Gln Asp Thr Gly Ile Pro Ala Asp Ile Trp Arg 340 345 350 Phe Tyr Leu Leu Tyr Ile Arg Pro Glu Gly Gln Asp Ser Ala Phe Ser 355 360 365 Trp Thr Asp Leu Leu Leu Lys Asn Asn Ser Glu Leu Leu Asn Asn Leu 370 375 380 Gly Asn Phe Ile Asn Arg Ala Gly Met Phe Val Ser Lys Phe Phe Gly 385 390 395 400 Gly Tyr Val Pro Glu Met Val Leu Thr Pro Asp Asp Gln Arg Leu Leu 405 410 415 Ala His Val Thr Leu Glu Leu Gln His Tyr His Gln Leu Leu Glu Lys 420 425 430 Val Arg Ile Arg Asp Ala Leu Arg Ser Ile Leu Thr Ile Ser Arg His 435 440 445 Gly Asn Gln Tyr Ile Gln Val Asn Glu Pro Trp Lys Arg Ile Lys Gly 450 455 460 Ser Glu Ala Asp Arg Gln Arg Ala Gly Thr Val Thr Gly Leu Ala Val 465 470 475 480 Asn Ile Ala Leu Leu Ser Val Met Leu Gln Pro Tyr Met Pro Thr 485 490 495 Val Ser Ala Thr Ile Gln Ala Gln Leu Gln Leu Pro Pro Ala Cys 500 505 510 Ser Ile Leu Leu Thr Asn Phe Leu Cys Thr Leu Pro Ala Gly His Gln 515 520 525 Ile Gly Thr Val Ser Pro Leu Phe Gln Lys Leu Glu Asn Asp Gln Ile 530 535 540 Glu Ser Leu Arg Gln Arg Phe Gly Gly Gly Gln Ala Lys Thr Ser Pro 545 550 555 560 Lys Pro Ala Val Val Glu Thr Val Thr Thr Ala Lys Pro Gln Gln Ile 565 570 575 Gln Ala Leu Met Asp Glu Val Thr Lys Gln Gly Asn Ile Val Arg Glu 580 585 590 Leu Lys Ala Gln Lys Ala Asp Lys Asn Glu Val Ala Ala Glu Val Ala 595 600 605 Lys Leu Leu Asp Leu Lys Lys Gln Leu Ala Val Ala Glu Gly Lys Pro 610 615 620 Pro Glu Ala Pro Lys Gly Lys Lys Lys Lys 625 630 <210> 12 <211> 1902 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens MRS F5 fragment mRNA <400> 12 ctcatcacca gtgccctccc ttacgtcaac aatgtccccc accttgggaa catcattggt 60 tgtgtgctca gtgccgatgt ctttgccagg tactctcgcc tccgccagtg gaacaccctc 120 tatctgtgtg ggacagatga gtatggtaca gcaacagaga ccaaggctct ggaggaggga 180 ctaacccccc aggagatctg cgacaagtac cacatcatcc atgctgacat ctaccgctgg 240 tttaacattt cgtttgatat ttttggtcgc accaccactc cacagcagac caaaatcacc 300 caggacattt tccagcagtt gctgaaacga ggttttgtgc tgcaagatac tgtggagcaa 360 ctgcgatgtg agcactgtgc tcgcttcctg gctgaccgct tcgtggaggg cgtgtgtccc 420 ttctgtggct atgaggaggc tcggggtgac cagtgtgaca agtgtggcaa gctcatcaat 480 gctgtcgagc ttaagaagcc tcagtgtaaa gtctgccgat catgccctgt ggtgcagtcg 540 agccagcacc tgtttctgga cctgcctaag ctggagaagc gactggagga gtggttgggg 600 aggacattgc ctggcagtga ctggacaccc aatgcccagt ttatcacccg ttcttggctt 660 cgggatggcc tcaagccacg ctgcataacc cgagacctca aatggggaac ccctgtaccc 720 ttagaaggtt ttgaagacaa ggtattctat gtctggtttg atgccactat tggctatctg 780 tccatcacag ccaactacac agaccagtgg gagagatggt ggaagaaccc agagcaagtg 840 gacctgtatc agttcatggc caaagacaat gttcctttcc atagcttagt ctttccttgc 900 tcagccctag gagctgagga taactatacc ttggtcagcc acctcattgc tacagagtac 960 ctgaactatg aggatgggaa attctctaag agccgcggtg tgggagtgtt tggggacatg 1020 gcccaggaca cggggatccc tgctgacatc tggcgcttct atctgctgta cattcggcct 1080 gagggccagg acagtgcttt ctcctggacg gacctgctgc tgaagaataa ttctgagctg 1140 cttaacaacc tgggcaactt catcaacaga gctgggatgt ttgtgtctaa gttctttggg 1200 ggctatgtgc ctgagatggt gctcacccct gatgatcagc gcctgctggc ccatgtcacc 1260 ctggagctcc agcactatca ccagctactt gagaaggttc ggatccggga tgccttgcgc 1320 agtatcctca ccatatctcg acatggcaac caatatattc aggtgaatga gccctggaag 1380 cggattaaag gcagtgaggc tgacaggcaa cgggcaggaa cagtgactgg cttggcagtg 1440 aatatagctg ccttgctctc tgtcatgctt cagccttaca tgcccacggt tagtgccaca 1500 atccaggccc agctgcagct cccacctcca gcctgcagta tcctgctgac aaacttcctg 1560 tgtaccttac cagcaggaca ccagattggc acagtcagtc ccttgttcca aaaattggaa 1620 aatgaccaga ttgaaagttt aaggcagcgc tttggagggg gccaggcaaa aacgtccccg 1680 aagccagcag ttgtagagac tgttacaaca gccaagccac agcagataca agcgctgatg 1740 gatgaagtga caaaacaagg aaacattgtc cgagaactga aagcacaaaa ggcagacaag 1800 aacgaggttg ctgcggaggt ggcgaaactc ttggatctaa agaaacagtt ggctgtagct 1860 gaggggaaac cccctgaagc ccctaaaggc aagaagaaaa ag 1902 <210> 13 <211> 303 <212> PRT <213> Homo sapiens CDK4 WT protein (NP_000066.1) <400> 13 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 <210> 14 <211> 912 <212> DNA <213> Homo sapiens CDK4 WT mRNA (NM_000075.3) <400> 14 atggctacct ctcgatatga gccagtggct gaaattggtg tcggtgccta tgggacagtg 60 tacaaggccc gtgatcccca cagtggccac tttgtggccc tcaagagtgt gagagtcccc 120 aatggaggag gaggtggagg aggccttccc atcagcacag ttcgtgaggt ggctttactg 180 aggcgactgg aggcttttga gcatcccaat gttgtccggc tgatggacgt ctgtgccaca 240 tcccgaactg accgggagat caaggtaacc ctggtgtttg agcatgtaga ccaggaccta 300 aggacatatc tggacaaggc acccccacca ggcttgccag ccgaaacgat caaggatctg 360 atgcgccagt ttctaagagg cctagatttc cttcatgcca attgcatcgt tcaccgagat 420 ctgaagccag agaacattct ggtgacaagt ggtggaacag tcaagctggc tgactttggc 480 ctggccagaa tctacagcta ccagatggca cttacacccg tggttgttac actctggtac 540 cgagctcccg aagttcttct gcagtccaca tatgcaacac ctgtggacat gtggagtgtt 600 ggctgtatct ttgcagagat gtttcgtcga aagcctctct tctgtggaaa ctctgaagcc 660 gaccagttgg gcaaaatctt tgacctgatt gggctgcctc cagaggatga ctggcctcga 720 gatgtatccc tgccccgtgg agcctttccc cccagagggc cccgcccagt gcagtcggtg 780 gtacctgaga tggaggagtc gggagcacag ctgctgctgg aaatgctgac ttttaaccca 840 cacaagcgaa tctctgcctt tcgagctctg cagcactctt atctacataa ggatgaaggt 900 aatccggagt ga 912 <210> 15 <211> 303 <212> PRT <213> Homo sapiens CDK4 R24C protein <400> 15 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Cys Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 <210> 16 <211> 912 <212> DNA <213> Homo sapiens CDK4 R24C mRNA <400> 16 atggctacct ctcgatatga gccagtggct gaaattggtg tcggtgccta tgggacagtg 60 tacaaggcct gtgatcccca cagtggccac tttgtggccc tcaagagtgt gagagtcccc 120 aatggaggag gaggtggagg aggccttccc atcagcacag ttcgtgaggt ggctttactg 180 aggcgactgg aggcttttga gcatcccaat gttgtccggc tgatggacgt ctgtgccaca 240 tcccgaactg accgggagat caaggtaacc ctggtgtttg agcatgtaga ccaggaccta 300 aggacatatc tggacaaggc acccccacca ggcttgccag ccgaaacgat caaggatctg 360 atgcgccagt ttctaagagg cctagatttc cttcatgcca attgcatcgt tcaccgagat 420 ctgaagccag agaacattct ggtgacaagt ggtggaacag tcaagctggc tgactttggc 480 ctggccagaa tctacagcta ccagatggca cttacacccg tggttgttac actctggtac 540 cgagctcccg aagttcttct gcagtccaca tatgcaacac ctgtggacat gtggagtgtt 600 ggctgtatct ttgcagagat gtttcgtcga aagcctctct tctgtggaaa ctctgaagcc 660 gaccagttgg gcaaaatctt tgacctgatt gggctgcctc cagaggatga ctggcctcga 720 gatgtatccc tgccccgtgg agcctttccc cccagagggc cccgcccagt gcagtcggtg 780 gtacctgaga tggaggagtc gggagcacag ctgctgctgg aaatgctgac ttttaaccca 840 cacaagcgaa tctctgcctt tcgagctctg cagcactctt atctacataa ggatgaaggt 900 aatccggagt ga 912 <210> 17 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens CDK4 N-terminal fragment protein (1-102 amino acids) <400> 17 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr 100 <210> 18 <211> 306 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens CDK4 N-terminal fragment mRNA <400> 18 atggctacct ctcgatatga gccagtggct gaaattggtg tcggtgccta tgggacagtg 60 tacaaggccc gtgatcccca cagtggccac tttgtggccc tcaagagtgt gagagtcccc 120 aatggaggag gaggtggagg aggccttccc atcagcacag ttcgtgaggt ggctttactg 180 aggcgactgg aggcttttga gcatcccaat gttgtccggc tgatggacgt ctgtgccaca 240 tcccgaactg accgggagat caaggtaacc ctggtgtttg agcatgtaga ccaggaccta 300 aggaca 306 <210> 19 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA against MRS <400> 19 cuaccgcugg uuuaacauuu cguuu 25 <210> 20 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA against p16INK4a <400> 20 cgcaccgaau aguuacggu 19 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > shRNA against MRS (sequence as DNA) <400> 21 ggcgaaactc ttggatcta 19 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> MRS forward primer <400> 22 gaggatggga aattctctaa gagccg 26 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> MRS reverse primer <400> 23 ttggttgcca tgtcgagata tggtgaggat act 33 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CDK4 forward primer <400> 24 atggctacct ctcgatatga gcca 24 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CDK4 reverse primer <400> 25 tcactccgga ttaccttcat cctt 24

Claims

(a) a fragment which does not contain the 1st to 266th amino acids of the MRS (methionyl-tRNA synthetase) sequence of SEQ ID NO: 1 and a fragment of CDK4 (cyclin-dependent kinase 4) amino acid sequence of SEQ ID NO: 13 or 15 under the presence or absence of the test substance Contacting a fragment comprising a 1 to 102 amino acid sequence of the sequence;
(b) measuring the binding of the MRS fragment to the CDK4 fragment in the presence or absence of the test substance;
(c) comparing the binding of the MRS fragment with the CDK4 fragment in the presence of the test substance and the binding of the MRS fragment and the CDK4 fragment under the absence of the test substance to determine the change in the binding level of the MRS fragment and the CDK4 fragment by the test substance;
(d) selecting a test substance that reduces the level of binding between the MRS fragment and the CDK4 fragment; And
(e) identifying an anticancer activity of the selected test substance in a cell or an animal.

The anticancer agent screening method according to claim 1, wherein the anticancer agent screening method comprises the steps of (d) and (e)
(1) contacting the test substance with cells expressing CDK4;
(2) measuring the CDK4 protein level in the control cells not in contact with the test substance; And
(3) selecting a test substance that reduces CDK4 protein levels compared to control cells.

2. The method of claim 1, wherein the cell does not express p16 ^INK4a .

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The method of claim 1, wherein the fragment of MRS is a fragment comprising the 596th lysine in the amino acid sequence of SEQ ID NO: 1.

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The method of claim 1, wherein the fragment of CDK4 comprises the amino acid sequence of SEQ ID NO: 17.

The anticancer agent according to any one of claims 1 to 3, 5, or 8, wherein the anticancer agent is selected from the group consisting of a methionine amino acid analogue, siRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acid (PNA), antisense oligonucleotide, Antibodies, aptamers, peptides, natural extracts, and chemicals.

The method of claim 1, wherein the cancer is a cancer that does not express p16 ^INK4a .

The method of claim 10, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, angioma, Osteosarcoma, prostate cancer, colon cancer, pancreatic cancer, renal cancer, stomach cancer, skin cancer, basal cell carcinoma, melanoma, squamous cell carcinoma, oral squamous cell carcinoma, colon cancer, rectum Wherein the cancer is selected from the group consisting of blastoma, endometrial cancer and malignant glioma.

(Boc-S-trityl-L-homocysteine, Fmoc-DL-selenomethionine, Fmoc-DL-ethionine and Fmoc-Sec (Mob) -OH) of any one of claims 1 to 3, FSMO), Fmoc-? -Methyl-DL-methionine, siRNA comprising the nucleotide sequence shown in SEQ ID NO: 19 or 21, shRNA and antisense nucleotides are screened for effective As an active ingredient.

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