WO2014142619A1 - Procédé de criblage d'agent thérapeutique pour des cancers par utilisation d'une interaction aimp2-dx2 et pl4/arf - Google Patents

Procédé de criblage d'agent thérapeutique pour des cancers par utilisation d'une interaction aimp2-dx2 et pl4/arf Download PDF

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WO2014142619A1
WO2014142619A1 PCT/KR2014/002196 KR2014002196W WO2014142619A1 WO 2014142619 A1 WO2014142619 A1 WO 2014142619A1 KR 2014002196 W KR2014002196 W KR 2014002196W WO 2014142619 A1 WO2014142619 A1 WO 2014142619A1
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aimp2
arf
protein
cancer
fragment
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Korean (ko)
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박범준
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부산대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to a method for screening a cancer therapeutic agent using the interaction of AIMP2-DX2 with pl4 / ARF, and more particularly, (a) isolated AIMP2-DX2 protein or fragment thereof and isolated P14 / ARF protein or its Contacting the fragment with the test substance; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, and the method for screening an anticancer agent that inhibits the interaction between the two proteins. It relates to a pharmaceutical composition for treating cancer comprising the substance screened by the screening method as an active ingredient.
  • ARF known as P14 / ARF in humans and pl9ARF in mice, is a tumor suppressor protein.
  • ARF proteins bind to E2F-1, -2 and -3 transcriptional activators to inhibit their transcriptional activity and degrade them via the 26S proteasome pathway. This binding results in a p53-independent ARF activity mechanism that is seen in cell cycle progression and tumor sensitivity.
  • AIMP2 (ARS-interacting multi-functional protein 2) is the aminoacyl-tRNA sum One of the proteins involved in the formation of aminoacyl-tRNA synthetase (ARSs), also called p38 / JTV-l or p38.
  • ARSs aminoacyl-tRNA synthetase
  • the present inventors have shown that AIMP2 is a novel cancer suppressor that enhances the signaling of TGF- ⁇ through direct interaction with Smad2 / 3, cancer cell lines and tissues. It was confirmed that AIMP2-DX2, a variant of AIMP2 lacking in AMP2, was specifically expressed in.
  • AIMP2 levels are dramatically reduced in AIMP2-DX2-type transfected cells irrespective of TGF- ⁇ , resulting in loss of AIMP2 activity in AIMP2-DX2 production (Patent 10-0762995).
  • AIMP2-DX2-knock down model has excellent anticancer effects in benzopyrine-induced lung cancer, the role of AIMP2-DX2 in lung cancer progression is not yet clear. Since AIMP2-DX2 alone does not reliably induce lung cancer, the tumorigenic properties of AIMP2-DX2 may be dependent on other genetic factors.
  • an object of the present invention is the step of (a) contacting a test substance with an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, as compared to the control group to which the test substance was not contacted.
  • Another object of the present invention is to contact a test substance with a cell or tissue expressing an isolated AIMP2-DX2 protein or fragment thereof and an isolated P14 / ARF protein or fragment thereof; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in the cell or tissue to which the test substance is contacted, wherein the test substance is not contacted.
  • the present invention comprises the steps of: (a) contacting the test substance with the isolated AIMP2-DX2 protein or fragment thereof and the isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2—DX2 protein or fragment thereof and the p / ARF protein or fragment thereof, the test as compared to the control group not contacted with the test substance.
  • An anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF when the interaction of AIMP2-DX2 with pl4 / ARF is reduced in contact with the substance; Provides a screening method.
  • the present invention comprises (a) testing the AIMP2-DX2 separating material protein or of the fragment mat separate pl4 / ARF protein or, cells to a fragment thereof string or tissue and on contact with the key in order to achieve all-object of the present invention Is a step; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in the cells or tissues to which the test substance is contacted, wherein the AIMP2- is in contact with the test substance when compared to the control group to which the test substance is not contacted.
  • the present invention provides a method for screening an anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF, including selecting the test substance as a primary candidate.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a substance screened by the screening method as an active ingredient.
  • a pharmaceutical composition for preventing or treating cancer which comprises a substance screened by the screening method as an active ingredient.
  • the present invention comprises the steps of (a) contacting a test substance with an isolated AIMP2-DX2 protein or fragment thereof and an isolated pl4 / ARF protein or fragment thereof; And (b) determining whether the test substance inhibits the interaction of the AIMP2-DX2 protein or fragment thereof and the pl4 / ARF protein or fragment thereof, when compared with the control group to which the test substance is not contacted.
  • AIMP2-DX2 protein and pl4 / ARF protein are characterized in that it is provided in the form of cells or tissues expressing it.
  • Step (b) is a two-hybrid method, a co-immunoprecipitation method (c immunoprecipitat ion assay), a co-localization assay, a scintillation proximity assay (SPA), UV or chemical crosslinking. Binding methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance, fluorescence polarization assays (FPA), and in vitro pull-down assays It is characterized in that it is carried out by any one method selected from the group consisting of in vitro pull-down assay.
  • c immunoprecipitat ion assay a co-localization assay
  • SPA scintillation proximity assay
  • UV or chemical crosslinking Binding methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance, fluorescence polarization assays (FPA), and in vitro pull-down assays It is characterized in that it is carried out by any
  • the present invention comprises the steps of (a) contacting a test substance with a cell or tissue expressing the isolated AIMP2-DX2 protein or fragment thereof and the isolated pM / ARF protein or fragment thereof; And (b) measuring the expression level of the AIMP2-DX2 protein or fragment thereof in cells or tissues to which the test substance is contacted, wherein the test substance is in contact with the test substance as compared to the control group not contacted with the test substance.
  • the present invention provides a method for screening an anticancer agent that inhibits the interaction of AIMP2-DX2 with pl4 / ARF, including selecting the test substance as a primary candidate.
  • Step (b) comprises co-immunoprecipitation, enzyme-linked unosor bent assay, radioimmunoassay (RI A), immunohistochemistry, Westton blotting ( Western Blotting) and flow cytometry (FACS) is characterized in that it is measured by a method selected from the group consisting of.
  • RI A radioimmunoassay
  • FACS flow cytometry
  • the AIMP2-DX2 is a variant in which the region of exon 2 is deleted in the AIMP2 protein sequence (312aa version: AAC50391.1 or GI: 1215669; 320aa version: AAH13630.1, GI: 15489023, BC013630.1), and AIMP2 equivalent (amino acid Functional derivatives having a substantially equivalent activity to AIMP2, or a derivative having a substantially equivalent activity to AIMP2 but having a modification that increases or decreases physicochemical properties with a modification by substitution, deletion, insertion, or combination thereof in the sequence) Contains a protein from which the region of axon 2 is deleted.
  • deleting the axon 2 region in the AIMP2 protein sequence means that the variant resulting from the partial or total loss of the amino acid sequence of the axon 2 region in the AIMP2 protein forms an AIMP2 protein and a heterodimer, thereby reducing the normal function of AIMP2. It means to disturb.
  • the AIMP2-DX2 protein may have deleted all of the amino acid sequences of axon 2 of the AIMP2 protein or the amino acid sequence of this region, including some of these regions in exon 1, exon 3, axon 4 or both of these regions, Only part of the amino acid sequence includes the deleted protein.
  • AIMP2-DX2 proteins and pl4 / ARF proteins of the present invention are also within the scope of the present invention, as well as proteins having their native amino acid sequences, as well as amino acid sequence variants thereof.
  • Variant means a protein in which the natural amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, RL Hill, The Proteins, Academic Press, New York, 1979).
  • the most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / GIy. In some cases, it is modified by phosphorylation, sulfation, acrylation, glycosylat ion, methylat ion and pansylation ( ⁇ 1 " 1 3 131 ⁇ 011). May be.
  • the AIMP2-DX2 protein or pl4 / ARF protein and variants thereof may be extracted or synthesized in nature (Merrifleld, J. Araer. Chem. Soc .. 85: 2149-2156, 1963) or recombinant based on DNA sequences. It may be prepared by the method (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989). In the case of gene recombination technology, the nucleic acid encoding the protein is inserted into an appropriate expression vector, the host cell is cultured to express the protein in a transformant transformed with the recombinant expression vector, and then Recovering the proteins It can be obtained by the process.
  • the AIMP2-DX2 protein of the present invention may be represented by the amino acid sequence of SEQ ID NO: 4 or 5, more preferably AIMP2-DX2 may be represented by the amino acid sequence of SEQ ID NO: 4, pl4 / ARF is It may be represented by the amino acid sequence of SEQ ID NO.
  • the AIMP2-DX2 protein fragment of the present invention may be composed of at least 5 to 100 amino acid sequences including exon 1-3 junction of AIMP2 protein.
  • fragments of the pM / A F protein of the present invention may be represented by the 1st to 29th or 2nd to 29th amino acid sequence of SEQ ID NO: 6.
  • the anticancer screening method comprising the steps (a) to (b) described above,
  • step (c) contacting the primary candidate selected in step (b) with a protein other than pl4 / ARF and AIMP2-DX2 binding thereto
  • the step (c) may further comprise the step of selecting a primary candidate material that does not affect the interaction of the protein other than pl4 / ARF and AIMP2-DX2 binding thereto as a secondary candidate material.
  • the proteins other than the AIMP2-DX2 are, but are not limited to, p53, MDM2 (Cell, Vol . 92, 713723, March 20, 1998), E2F1 (Oncogene (2001) 20, 1033 ⁇ 1041), • prOgerin (Ceir Cycle 12: 14, 22772290; July 15, 2013), and may preferably be p53.
  • Step (c) is a two-hybrid method, a coimmunoprecipitation method, a co-localizatkm assay, a scintillation proximity assay (SPA).
  • reaction means that AIMP2-DX2 and pl4 / ARF proteins directly bind AIMP2—DX2 to inhibit the function of pl4 / ARF. All.
  • anti-cancer agent that inhibits interaction means treating and preventing cancer by inhibiting AIMP2-DX2 from inhibiting the function of pl4 / ARF.
  • inhibit interaction includes both inhibiting, alleviating and eliminating the interaction between AIMP2-DX2 and pl4 / ARF. Inhibition of interaction may inhibit the interaction between AIMP2-DX2 and pl4 / ARF itself, but also inhibits the binding of each other by reducing the activity of AIMP2-DX2.
  • the term “anticancer” means inhibiting or preventing the growth of cancer.
  • “Inhibiting or preventing cancer growth” is a concept that includes reducing cancer growth and cancer metastasis as compared to when not treated or treated.
  • “Metastasis” means that tumor cells It refers to a process that spreads to distant parts of the body, as used herein, the term also encompasses cancer that occurs through metastasis.
  • the term “recombinant vector” refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell and comprises an essential regulatory element operably linked to express the gene insert.
  • P14 / ARF is present in the nucleus and is involved in cell cycle resting and apoptosis.
  • inactivation of pl4 / ARF can promote metastatic phenotype of lung cancer. Because the carcinogenic properties of AIMP2-DX2 were similar to those of pl4 / ARF inactivated cancers, we tested the functional association between AIMP2-DX2 and pl4 / ARF. First, the effect of AIMP2-DX2 on pl4 / ARF expression was observed.
  • AIMP2-DX2 reduced P14 / ARF expression.
  • RKIP negative control RKIP
  • AIMP2-DX2 selectively inhibits pl4 / ARF expression
  • the protein half-life was measured after each of the two proteins was removed using si-RNA acting on each protein. Removal of AIMP2-DX2 prolonged the half-life of pl4 / ARF and similarly removal of pl4 / ARF prolonged expression of DX-2 (see FIG. 2).
  • Si-AIMP2-DX2 also promoted intranuclear expression of pl4 / ARF. (See Figure 3).
  • Glutathione S-transferase (GST) -pul 1 down was performed.
  • GSP-pull down assay using p53 ol the interaction with AIMP2 as well as AIMP2-DX2 was observed. This result is in agreement with previous findings that AIMP2 and AIMP2-DX2 bind directly to p53.
  • GST-pl4 / ARF shows selective interaction with AIMP2—DX2 (see FIG. 7).
  • pRb deficiency is essential for small cell lung cancer (SCLC) progression, and pRb is known to be involved in cell differentiation and proliferation. Therefore, we examined how AIMP2-DX2 affects pRb function. When AIMP2-DX2 was transfected, the nuclear pRb was released into the cytoplasm. (See Figure ' 8)-. And this, again, check the connection through the cell fractions', AIMP2 DX2 has confirmed that interferes with pRb expression in the nucleus. (See FIG. 9)
  • the GST-pull down assay confirmed that the candidate selected as a pl4 / ARF-DX2 specific binding inhibitor inhibited the P 14 / ARF-DX2 binding, and actually inhibited the binding of pl4 / ARF-DX2. It could be confirmed (see FIG. 23).
  • the screening method of the present invention can effectively select pl4 / ARF-DX2 specific binding inhibitors.
  • compositions for preventing or treating cancer comprising the substance screened by the screening method of the present invention as an active ingredient.
  • composition of the present invention comprises an effective amount of the substance screened by the screening method of the present invention or a pharmaceutically acceptable carrier of the substance.
  • effective amount means an amount sufficient to exert the therapeutic efficacy of cancer.
  • a pharmaceutically pharmaceutical less contained in eu compositions of the balttang - as carrier duty is commonly used at the time o preparation ⁇ , carbohydrate-type compound (e.g., lactose, amylose, dextrose, sucrose, sorbitan Mold, mantle, starch, cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, salt solution, alcohol , Gum arabic, vegetable oils (e.g.,
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like.
  • Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response. Ordinarily, the skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prevention.
  • a suitable daily dosage is 0.0001-100 mg / kg body weight. Administration may be once a day or may be divided several times.
  • the pharmaceutical composition of the present invention may be formulated using a pharmaceutically acceptable carrier and / or excipient, in unit dosage form, according to a method which can be easily carried out by those skilled in the art. It can be made or prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or an aqueous medium, or may be in the form of axes, powders, granules, tablets, or tablets, and may further include a dispersant or stabilizer.
  • Inhibitors of interaction of P14 / ARF can be a variety of substances.
  • the active ingredient includes a single compound, a combination of compounds (eg, a natural extract or a cell or tissue culture), an antibody or a peptide.
  • the eu - ⁇ 2- DX2 protein and pl4 / ARF danbek query "method for cleaning a disc anti-cancer agent for inhibiting the interaction, it is effective in the manufacture of a cancer by screening for anti-cancer substances.
  • FIG. 1 shows the expression level of pl4 / ARF following AIMP2-DX2 transfection.
  • EV Vector not including AIMP2-DX2 gene / Myc-AIMP2-DX2: Contains AIMP2-DX2 gene tagged with Myc
  • Vector Vector not including AIMP2-DX2 gene / Myc-AIMP2-DX2: Contains AIMP2-DX2 gene tagged with Myc
  • Figure 2 is a result of measuring how the removal of AIMP2-DX2, pl4 / ARF affect each other half-life.
  • si-Count control group
  • si-AIMP2-DX2 experimental group inhibited AIMP2-DX2 expression
  • si-pl4 / ARF experimental group inhibited pl4 / ARF expression
  • CHX chlorhexidine,-, 2,7 (hr): CHX Time processed
  • Figure 3 looks at the positional shift of pl4 / ARF to the nucleus following AIMP2-DX2 removal. (Green: pl4 / ARF, blue: nucleus, 1.5, 2.5: concentration treated with si-AIMP2-DX2)
  • Figure 5 shows the presence of AIMP2-DX2 and pl4 / ARF in the nucleus by immunofluorescence staining.
  • Figure 6 is the result of observing the interaction between proteins made AIMP2-DX2 pl4 / ARF directly recombinant protein.
  • Pur Group separating sediment supernatant (Group not associated with AIMP2-DX2), IP: GST: Group precipitated with GST antibody)
  • FIG. 8 shows the results of observing with immunofluorescence that AIMP2—DX2 influences the migration of pRb.
  • EV HCT116 cells transfected with vector containing no AIMP2-DX2 gene
  • AIMP2-DX2 HCT116 cells transfected with vector containing AIMP2-DX2 gene
  • FIG. 9 shows Western pRb expression by cell fraction. This is the result confirmed by the blot.
  • Fig. 10 shows the result of confirming that pl4 / ARF and pRb bind to each other by Western blot. (Input: cell lysate)
  • FIG. 18 It is a schematic diagram which shows that AIMP2-DX2 binds to the N-terminal site of pl4 / ARF.
  • 18 is a schematic diagram showing a pl4 / ARF-DX2 specific inhibitor screening method.
  • 19 shows a method for finding a test substance that inhibits pl4 / ARF-DX2 binding using ELISA.
  • FIG. 21 is a GST pull down result confirming pl4 / ARF-DX2 binding inhibition of primary candidates selected by ELISA-based screening.
  • FIG. 22 shows specificity test results confirming that primary candidates selected through ELISA-based screening inhibit protein binding other than P14 / ARF—DX2.
  • Cells HEK293 and HCT116 were purchased from American Type Culture Collection (ATCC, Manassas, VA). NCI-H23 cells were obtained from Prof. Kim Sung-hoon of Seoul National University. The medium contains RPMI— 1640 or DMEM containing 10% FBS and antibiotics. Used. Common reagents were purchased from Sigma (St Louis, MO, USA) and chemical inhibitors were purchased from Calbiochem (San Die, CA, USA).
  • Protein extraction from cells was performed using a radioimmunoprecipitation assay (RIPA; 150 mM NaCl, 25 mM Tris-Cl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture). After cell lysate in the buffer solution, it was stored at 95 ° C for 7 minutes. Samples were subjected to Western blot after SDS-PAGE. The membrane to which the protein was transferred was incubated with the primary antibodies. The antibodies used are shown in Table 1 below. The membrane was was washed for 5 minutes and then incubated with HRP-conjugated 2nd antibody for 1 hour. stomach The positive membrane was washed three times for 5 minutes.
  • RIPA radioimmunoprecipitation assay
  • the pl4 / A F fragment (Full-length) was ligated to the Hind II site of the pGEX-TEV vector prepared by adding the TEV protease cleavage site to the pGEX-4TL site. Recombinant protein is expressed in GST binding form in E. coli strain BL2KDE3). Proteins are separated by glutathione affinity chromatography. The amino acid sequence of the isolated pl4 / ARF is shown by SEQ ID NO: 6.
  • Mammalian expression plasmid encoding GFP-pl4 / ARF attach GFP to the N terminus of pl4 / ARF
  • GFP-pl4 / ARF-N N-terminal region
  • His-AIMP2 His ⁇ AIMP2-DX2 vector
  • pl4 / ARF protein was expressed from the second amino acid sequence using the start codon of the GFP protein.
  • Wild type and two variant pRb (567L, 661W; point mutant; Sellers, 1998) were purchased from Addgene (Cambridge, USA).
  • Si-RNAs that interfere with each gene are listed in Table 2 below. Transfection was performed for 24 hours using Jet-pei (Polypi us Trans feet ion, New York, NY) reagent It became. In short, the cells sown a day ago were washed with PBS and incubated with DNA / Jet-pei mixture for 4 hours under serum-free conditions. The control is a sequence without a target.
  • Jet-pei Polypi us Trans feet ion, New York, NY
  • Test materials used for screening are described in Synthetic Compounds and Natural Products Libraries (Lee, SH et al., Oncogene 29, 4576-4587, 2010; Lee, SH et al., P53, Oncogene 28, 2005-2014, 2009).
  • the 8000 chemicals provided by the Korean Chemical Society were used.
  • His-DX2 recombinant protein was immobilized with 1% paraformaldehyde in a 96-well plate. After the plates were dried and washed, the final concentration of the O.lmM chemicals and the GST-pl4 / ARF protein were incubated. After 1 hour, the plate was washed with Tris-buf fered saline-Tween20 and incubated with anti-GST antibody (1: 10,000) for 30 minutes and anti-mouse IgG-HRP (1: 50,000) for 1 hour.
  • pl4 / ARF-DX2 inhibitors were selected as primary candidates. Among them, other known chemicals were excluded by screening. Among the primary candidates, pl4 / ARF-DX2 specific binding inhibitors were selected by excluding non-specific inhibitors that inhibit the binding of p53-pl4 / ARF through the GST pull down assay.
  • AIMP2-DX2 regulates the expression of pl4 / ARF.
  • FIG. 1 After dividing AIMP2-DX2 into the group transfected and the group not transfected, P 14 / ARF expression was measured by Western blot, and the results are shown in FIG. 1.
  • FIG. 1 it can be seen that transfection of AIMP2-DX2 reduced pl4 / ARF expression.
  • RKIKRaf kinase inhibitory protein / cancer inhibitory function used as a negative control was expressed regardless of AIMP2-DX2 type vaginal injection. Therefore, AIMP2-DX2 appears to selectively inhibit pl4 / ARF.
  • si- ⁇ which acts on AIMP2 3X2 and pl4 / ARF, respectively, inhibited the expression of proteins, and then treated with CHX (Cycloheximide) every 0, 2 and 7 hours to observe the half-life of the expression level of the remaining proteins.
  • CHX Cycloheximide
  • AIMP2-DX2 and pl4 / ARF directly bind and interact.
  • HEK293 cell lysates expressed by GFP-pl4 / ARF were incubated with Histidine-labeled AIMP2-DX2 (His-AIMP2-DX2).
  • Histidine antibody After the culture was immunoprecipitated with Histidine antibody, it was confirmed by GFP antibody whether the precipitated protein contained pl4, pRb (cancer inhibitor protein), nucleoline (cancer inhibitor inhibitor protein).
  • pl4 was found in the precipitated protein. All.
  • all of pl4, pRb and nucleolin were found in the supernatant except the precipitate (see Fig. 4).
  • AIMP2-DX2 binds to pl4.
  • AIMP2-DX2 was labeled with His (His-AIMP2-DX2) and pl4 / ARF was labeled with GST (GST-pW).
  • His-AIMP2-DX2 was not detected when added alone, but when GST-pl4 was added.
  • AIMP2-DX2 binds to pl4 / ARF (see FIG. 6).
  • GST pulldown was performed using p53.
  • p53 has been shown to interact with AIMP2-DX2, AIMP2.
  • protein was detected after pull-down and His immunoprecipitation, regardless of AIMP2-DX2 and AIMP2.
  • His immunoprecipitation was performed. Protein was detected.
  • AIMP2-DX2 binds to the N-terminus of pl4 / ARF, and AIMP2 does not bind (see FIG. 16).
  • AIMP2-DX2 is the deletion of the exon 2 region of AIMP2, the AIMP2-DX2 domain corresponding to the site where the axon 1 and 3 regions of AIMP2 are linked to the pl4 / ARF N-terminal region (see Figure 17).
  • pRb is inhibited by AIMP2-DX2 via pl4 / ARF.
  • AIMP2-DX2 affects pRb function.
  • AIMP2-DX2 and pRb vectors were transfected into HCT116 cells for 24 hours. After fixation, the cells were anti-pRb (green) and DAPI stained (blue). As a result, the position of pRb moved from the nucleus to the cytoplasm by AIMP2-DX2 (see FIG. 8).
  • the transfected HCT116 cells were fractionated and subjected to Western blot in each fraction.
  • AIMP2-DX2 reduced the expression of pRb in the nucleus (see FIG. 9).
  • pRb and pl4 overexpressed HEK293 cell lysates were incubated with His labeled AIMP2-DX2 recombinant protein. Thereafter, after co-immunoprecipitation with anti-pRb antibody, Western blot was performed. As a result, pRb did not bind to AIMP2-DX2 but directly to pl4 / ARF (see FIG. 10).
  • GFP-pW / ARF was transfected into NCI-H23 cells (human lung cancer cell line expressing endogenous AIMP2-DX2) and treated with si-AIMP2-DX2, followed by IF. It was confirmed that the deficiency promoted the migration of pRb and pl4 / ARF into the nucleus (see FIG. 15).
  • pl4 / ARF-DX2 binding specific inhibitors were selected.
  • ELISA-based screening was performed to select inhibitors specific for P14 / ARF-DX2 binding. After screening chemicals that inhibited more than 703 ⁇ 4> of pl4 / ARF-DX2 binding by 9200 ELISAs, the screened material was removed from the candidates (see 18-20). Fourteen candidates obtained through the above process were confirmed to inhibit P 53-pl4 / ARF pair binding or DX2-pl4 / ARF pair binding by GST pull down assay. As a result, it was confirmed that only five candidates (D12, F6, F9, 889-A4, SLC36) are pl4 / ARF-DX2 specific binding inhibitors that do not affect p53-pl4 / ARF binding (FIG. 21 and 22).
  • GST-pull down assay confirmed that SLC36, which was selected as a pl4 / ARF-DX2 specific binding inhibitor, inhibited pl4 / ARF-DX2 binding. It was confirmed that the inhibition of the binding (see Fig. 23).
  • an anticancer agent that inhibits the interaction of AIMP2-DX2 protein and pl4 / ARF protein, there is an industrial applicability since an anticancer agent can be selected to produce an anticancer agent.

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Abstract

La présente invention concerne un procédé de criblage d'un agent thérapeutique pour des cancers par utilisation d'une interaction AIMP2-DX2 et pl4/ARF, et concerne, plus particulierement, un procédé de criblage d'un médicament anticancéreux qui supprime l'interaction AIMP2-DX2 et pl4/ARF, qui comprend les étapes consistant : (a) à amener une substance d'essai en contact avec une protéine AIMP2-DX2 séparée ou un fragment de cette dernière et une protéine pl4/ARF séparée ou un fragment de cette dernière ; et (b) pour une mesure servant à vérifier si la substance d'essai supprimel'interaction d'une protéine AIMP2-DX2 ou d'un fragment de ceete dernière et d'une protéine pl4/ARF ou d'un fragment de cette dernière, à choisir la substance d'essai comme premier candidat lorsque la substance fournit une diminution de l'interaction AIMP2-DX2 et pl4/ARF en raison d'un contact avec la substance d'essai par comparaison avec un groupe témoin qui n'a pas été amené en contact avec la substance d'essai. L'invention concerne également une composition pharmaceutique pour traiter des cancers, qui comprend, en tant qu'ingrédient actif, une substance qui a été criblée par le procédé de criblage. Le procédé est destiné au criblage d'un médicament anticancéreux qui supprime l'interaction de protéine AIMP2-DX2 et de protéine pw/ARF et est efficace dans la fabrication d'un agent anticancéreux par criblage de substances anticancéreuses.
PCT/KR2014/002196 2013-03-14 2014-03-14 Procédé de criblage d'agent thérapeutique pour des cancers par utilisation d'une interaction aimp2-dx2 et pl4/arf WO2014142619A1 (fr)

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KR20180117529A (ko) * 2017-04-19 2018-10-29 주식회사 프로티나 단백질-단백질 상호작용 분석에 의한 약물 반응성 예측 방법
WO2018194406A1 (fr) * 2017-04-19 2018-10-25 주식회사 프로티나 Procédé et appareil d'analyse d'interaction protéine-protéine
WO2019132517A1 (fr) * 2017-12-26 2019-07-04 주식회사 프로티나 Méthode et appareil d'analyse d'interaction protéine-protéine intracellulaire ou intercellulaire

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CN109073638A (zh) * 2016-03-07 2018-12-21 医药生命融合研究团 抑制aimp2-dx2和hsp70结合的抗癌剂的筛选方法
CN109073638B (zh) * 2016-03-07 2022-04-26 医药生命融合研究团 抑制aimp2-dx2和hsp70结合的抗癌剂的筛选方法
WO2018026236A1 (fr) * 2016-08-04 2018-02-08 재단법인 의약바이오컨버젼스연구단 Méthode de criblage d'agents anticancéreux inhibant la liaison entre aimp2-dx2 et k-ras
KR20180015847A (ko) * 2016-08-04 2018-02-14 재단법인 의약바이오컨버젼스연구단 AIMP2-DX2와 K-Ras의 결합을 저해하는 항암제 스크리닝 방법
KR102290511B1 (ko) 2016-08-04 2021-08-17 주식회사 자이메디 AIMP2-DX2와 K-Ras의 결합을 저해하는 항암제 스크리닝 방법

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