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WO2019024518A1 - Médicament pour le traitement d'une maladie impliquant la protéine chimioattractive des monocytes de type 1 (mcp-1) par ajustement de la phosphorylation de yb-1 - Google Patents

Médicament pour le traitement d'une maladie impliquant la protéine chimioattractive des monocytes de type 1 (mcp-1) par ajustement de la phosphorylation de yb-1 Download PDF

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WO2019024518A1
WO2019024518A1 PCT/CN2018/080675 CN2018080675W WO2019024518A1 WO 2019024518 A1 WO2019024518 A1 WO 2019024518A1 CN 2018080675 W CN2018080675 W CN 2018080675W WO 2019024518 A1 WO2019024518 A1 WO 2019024518A1
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substance
phosphorylation
protein
regulates
drug
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刘斌
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吉林众泰生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a kind of medicine and a screening method thereof for treating diseases involving monocyte chemoattractant protein-1 by regulating YB-1 phosphorylation.
  • Y-box-binding protein-1 (YB-1) is a type of Y-Box sequence (a highly conserved cis-DNA sequence) that specifically binds to the promoter of the gene of interest and the enhancer.
  • a transcription factor a member of the Cold shock proteins superfamily, contains a highly conserved Cold Shock domain (CSD), which binds to nucleic acids.
  • the protein code of YB-1 is: Protein Symbol: P67809-YBOX1_HUMAN, which is composed of 324 amino acids and has a molecular weight of 35924 Daltons.
  • YB-1 is widely found in microorganisms, plants, animals and humans.
  • YB-1 is at the cellular level.
  • Cell proliferation and differentiation, cellular stress response, and transformation of tumor cells play an important role; in clinical pathology, YB-1 has been confirmed to be associated with the occurrence of various diseases, such as tumors, including liver cancer, breast cancer, colon Adenocarcinoma, lung cancer; liver fibrosis; inflammation; atherosclerosis; organ transplant rejection; reperfusion injury; YB-1 can also be used as a marker for tumor diagnosis, and / or prognosis.
  • the chemotactic cytokine chemokine is a family of proteins composed of more than ten proteins with large homology and a molecular weight of 8-10 kD. These proteins contain one or two cysteines at the amino terminus. Chemokine cytokines are classified into subfamilies according to the arrangement of cysteine. Two cysteines are arranged in the Cys-X-Cys (cysteine-any amino acid-cysteine) manner.
  • the chemotactic cytokine is a subfamily of ⁇ , also known as CXC chemotactic cytokine;
  • the chemotactic cytokines arranged in the Cys-Cys manner belong to the ⁇ subfamily, also known as CC chemotactic cytokines.
  • a chemotactic cytokine with only one cysteine at the amino terminus is called a gamma subfamily chemotactic cytokine, also known as a C-chemokine cytokine.
  • Chemotactic cytokines are mainly secreted by stromal cells in leukocytes and hematopoietic microenvironments, acting on any cell with a chemotactic cytokine receptor and chemotaxis and activation of their target cells.
  • IL-8 is a representative of the alpha subfamily and has chemotactic or enrichment effects on neutrophils.
  • Monocyte chemoattractant protein-1 (MCP-1 or CCL-2) is a representative of the ⁇ subfamily, which can chemoatize or enrich monocytes, basophils, and memory T cells. , and dendritic cells.
  • Lymphotactin is a representative of the gamma subfamily and has chemotactic or enrichment effects on lymphocytes.
  • MCP-1 is mainly involved in the development of inflammation, including atherosclerosis, systemic lupus erythematosus, and rheumatoid arthritis. So far, human treatment of these diseases is still very limited
  • the present invention provides a class of drugs for treating diseases involving monocyte chemoattractant protein-1 and the use of such drugs for the treatment of monocyte chemotactic proteins in the art based on the above-mentioned deficiencies and deficiencies in the art. 1
  • the above-mentioned various diseases involved provide a more effective drug and a new drug selection.
  • a medicament for treating a disease in which monocyte chemoattractant protein-1 is involved characterized in that YB-1 is a drug target; and the active ingredient of the drug includes a substance capable of directly or indirectly regulating YB-1 phosphorylation .
  • monocyte chemoattractant protein 1 The diseases in which monocyte chemoattractant protein 1 is involved include inflammation, atherosclerosis, type 2 diabetes, tumors, autoimmune diseases, obesity, and encephalopathy (dementia, epilepsy, etc.).
  • the active ingredient of the drug includes a substance that is directly or indirectly down-regulated, and/or directly or indirectly inhibits YB-1 phosphorylation;
  • the substance that directly or indirectly downregulates, and/or directly or indirectly inhibits YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor.
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of any one or any of the following: a substance that promotes dephosphorylation of YB-1, a substance that blocks or delays phosphorylation of YB-1, and a down regulation Or a substance that inhibits the protein kinase activity of YB-1, a substance that up-regulates or stimulates the activity of YB-1 phosphorylase.
  • Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer
  • the YB-1 phosphorylation enhancer is selected from the group consisting of any one or any of the following: a substance that blocks or delays YB-1 dephosphorylation, a substance that up-regulates or stimulates YB-1 phosphorylation. A substance that up-regulates or stimulates the protein kinase activity of YB-1, a substance that inhibits or down-regulates the activity of YB-1 phosphorylase.
  • the YB-1 phosphorylation inhibitor comprises: a substance as shown in Formula I:
  • the YB-1 mutein refers to a mutant protein obtained by mutation of the serine at position 102 of the amino acid sequence of the wild type YB-1 protein.
  • the active ingredient of the drug further comprises a polymer of the YB-1 phosphorylation inhibitor, and/or the YB-1 phosphorylation inhibitor is a pharmaceutically acceptable salt compound; and/or an ester a compound; and/or a synergistic compound.
  • the medicament also includes pharmaceutically acceptable excipients and/or carriers.
  • a screening method for a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved characterized in that a substance which can directly or indirectly regulate YB-1 phosphorylation is used as a drug candidate for pathological experiments, and/or a clinical trial And/or, treating, screening for a substance having an effect, and/or producing a therapeutic effect, for use as an active ingredient of the drug.
  • the substance which can directly or indirectly regulate YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is preferably selected from: a substance that promotes dephosphorylation of YB-1, and/or a substance that blocks or delays phosphorylation of YB-1, and/or downregulates or inhibits YB-1.
  • a substance which can directly or indirectly regulate YB-1 phosphorylation in the preparation of a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved characterized in that the phosphorylation of YB-1 can be directly or indirectly regulated
  • the substance is placed in a package of goods labeled for the therapeutic use of the disease in which monocyte chemoattractant protein 1 is involved;
  • the substance that directly or indirectly regulates YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of: a substance that promotes dephosphorylation of YB-1, and/or a substance that blocks or delays phosphorylation of YB-1, and/or, A substance that down-regulates or inhibits the protein kinase activity of YB-1, and/or a substance that up-regulates or stimulates YB-1 phosphorylase activity.
  • the present invention first provides a class of drugs for treating a disease in which monocyte chemoattractant protein 1 is involved, characterized in that YB-1 is used as a drug target; and the active ingredient of the drug includes direct or indirect regulation of YB- 1 phosphorylated substance.
  • the present invention pioneered the discovery of a novel disease resistance mechanism of the above-mentioned drugs for the treatment of diseases in which monocyte chemoattractant protein 1 is involved: YB-1 is directly involved in the regulation of MCP-1 mRNA stability. Non-phosphorylated YB-1 down-regulates MCP-1 levels by promoting degradation of MCP-1 mRNA, thereby reducing monocyte aggregation.
  • Non-phosphorylated YB-1 facilitates binding to UK114 (nuclease active protein) to form a complex containing at least YB-1, UK114, and GR (glucocorticoid receptor), which selectively reduces cells by
  • MCP-1 monocyte chemoattractant protein-1
  • Increased mildness of intracellular non-phosphorylated YB-1 (increased by dephosphorylation of YB-1 or decreased phosphorylation, or a synergistic effect of both) can significantly inhibit the expression of MCP-1, thereby inhibiting monocytes Agglomeration in local tissues ultimately leads to inhibition, alleviation, or prevention of diseases involving monocyte chemoattractant protein 1.
  • regulation of YB-1 phosphorylation can effectively regulate MCP-1 and thereby control diseases caused by MCP-1 dysregulation.
  • the present invention demonstrates the role of this mechanism of disease resistance using an atherosclerotic model of transgenic mice.
  • the diseases in which the monocyte chemoattractant protein 1 is involved include inflammation, atherosclerosis, type 2 diabetes, tumor, autoimmune disease, obesity, and encephalopathy (dementia, epilepsy, etc.) .
  • the active ingredient of the drug comprises a substance that is directly or indirectly downregulated, and/or that directly or indirectly inhibits YB-1 phosphorylation;
  • the substance that directly or indirectly downregulates, and/or directly or indirectly inhibits YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor.
  • the present invention originally discovered the role of YB-1 phosphorylation in regulating MCP-1 expression and the inhibition of MCP-1 expression by YB-1 phosphorylation inhibitors in the process of studying the regulation of MCP-1 expression. effect.
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of any one or any of the following: a substance that promotes dephosphorylation of YB-1, blocks or delays A substance phosphorylated by YB-1, a substance which down-regulates or inhibits the protein kinase activity of YB-1, a substance which up-regulates or activates YB-1 phosphorylase activity.
  • those substances which are highly selective and directly cause YB-1 dephosphorylation or high selectivity to directly block YB-1 phosphorylation, and substances which highly selectively down-regulate the protein kinase activity of YB-1 and their high selection A substance that upregulates the phosphorylase activity of YB-1.
  • protein kinase The role of protein kinase is to phosphorylate the target protein, and a significant decrease in protein kinase can lead to a decrease in the total activity of the protein kinase. Down-regulation or inhibition of protein kinase activity can inhibit or down-regulate the phosphorylation of the target protein;
  • phosphorylase acts as a dephosphorylation (dephosphorylation), upregulates or stimulates the activity of the target protein phosphorylase, thereby enhancing the dephosphorylation of phosphorylase, thereby enhancing the dephosphorylation of the target protein. In turn, it aims to inhibit or down-regulate target protein phosphorylation.
  • Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer
  • the YB-1 phosphorylation enhancer is selected from the group consisting of any one or any of the following: a substance that blocks or delays YB-1 dephosphorylation, a substance that up-regulates or stimulates YB-1 phosphorylation. A substance that up-regulates or stimulates the protein kinase activity of YB-1, a substance that inhibits or down-regulates the activity of YB-1 phosphorylase.
  • the YB-1 phosphorylation inhibitor may also be: a substance which has an opposite effect to the above YB-1 phosphorylation enhancer, and/or a YB-1 dephosphorylation enhancer.
  • the YB-1 phosphorylation inhibitor is a substance of formula I:
  • the YB-1 phosphorylation inhibitor is dexamethasone, or a YB-1 mutein; the YB-1 mutein refers to the 102-position serine of the amino acid sequence of the wild-type YB-1 protein Mutant protein obtained after mutation.
  • the sequence of the wild type YB-1 protein is shown in SEQ ID NO.
  • the active ingredient of the drug further comprises a polymer of the YB-1 phosphorylation inhibitor, and/or the YB-1 phosphorylation inhibitor is a pharmaceutically acceptable salt compound And/or an ester compound; and/or a synergistic compound.
  • the medicament further comprises a pharmaceutically acceptable adjuvant and/or carrier.
  • Another aspect of the present invention provides a method for screening a drug for treating a disease in which monocyte chemoattractant protein 1 is involved, characterized in that a substance which can directly or indirectly regulate YB-1 phosphorylation is used as a drug candidate for pathology Experiments, and/or, clinical trials, and/or treatments, screening for substances that have an effect, and/or produce a therapeutic effect, are used as active ingredients of the drug.
  • the substance that directly or indirectly regulates YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is preferably selected from: a substance which promotes dephosphorylation of YB-1, and/or a substance which blocks or delays phosphorylation of YB-1, and/or down-regulates or inhibits YB a substance of -1 protein kinase activity, and/or a substance that up-regulates or stimulates YB-1 phosphorylase activity.
  • the invention also claims the use of a substance which directly or indirectly regulates YB-1 phosphorylation in the preparation of a medicament for the treatment of a disease in which monocyte chemoattractant protein 1 is involved, characterized in that said direct or indirect regulation
  • the YB-1 phosphorylated substance is placed in a commercial packaging box for the therapeutic use of the disease in which monocyte chemoattractant protein 1 is involved;
  • the substance which can directly or indirectly regulate YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of: a substance that promotes dephosphorylation of YB-1, and/or a substance that blocks or delays phosphorylation of YB-1, and/or, down-regulates or A substance that inhibits the protein kinase activity of YB-1, and/or a substance that up-regulates or stimulates YB-1 phosphorylase activity.
  • the invention also claims to directly or indirectly regulate the phosphorylation of Y-box-binding protein-1 (YB-1) in clinical practice, as permitted by patent laws in some countries or regions. Indications and methods of use;
  • the clinical use of the YB-1 phosphorylation inhibitor of the present invention includes the use of such substances in the above-mentioned indications to treat, alleviate, and prevent the above indications.
  • the clinical application of the invention to the substances referred to in the invention includes the clinically used dosage forms and dosages of the substances referred to in the invention.
  • Certain compounds of the invention may exist in a particular geometric or steric structure.
  • the invention covers all such compounds, including cis, cis, R and S isomers, diastereomers, (d) and (l) isomers, racemic mixtures and others covered by the invention mixture. Also included are substitutions of asymmetric carbon atoms, such as substitutions in alkyl groups.
  • the isomeric mixture includes mixtures in any ratio containing the isomers of the invention.
  • the "effective amount of active ingredient" referred to in the present invention means a dose sufficient to produce the desired biological effect.
  • the effective dose referred to herein may vary depending on the circumstances (e.g., route of administration, individual differences, pharmacokinetics of the different compounds, type of disease, and therapeutic target).
  • treatment of the disease referred to in the invention includes methods of curing, alleviating, delaying, or ameliorating the condition, including prevention of the condition.
  • Treatment may be one or more symptoms of the disease or pathological mechanisms that cause the symptoms.
  • the reduction or prevention referred to herein represents at least a 10% difference (measured by any standard technique) relative to an equally untreated control group.
  • Prevention includes methods of preventing, delaying, avoiding, or stopping the onset, exacerbation, or recurrence of a disease or condition.
  • “Pharmaceutically acceptable adjuvant” as used herein includes excipients, carriers, solvents, diluents, and encapsulating materials for carrying and transporting the drug in the body.
  • examples of such materials are: sugar, cellulose and its derivatives, scutellite powder, talc, gelatin, oil, alcohols, agar, buffer, diethyl ester, emulsion, lubricant, non-pyrogenic water, Alginic acid, toning and flavoring agents, preservatives, emulsifiers, aerosols, lubricants, antioxidants, sustained release agents, and other substances used in pharmaceutical formulations.
  • isolated or purified refers to a material that is substantially free of natural concomitants in the ordinary circumstances. Purity or homogeneity is determined by chemical methods such as polyacrylamide condensation electrophoresis or high performance liquid chromatography.
  • “Individual” as used herein includes, but is not limited to, humans, primates, rodents, and the like that receive the treatment. “Individual” and “patient” are used interchangeably for humans.
  • dephosphorylation ie, “dephosphorylation” generally refers to the removal of a phosphate group, and the meaning herein is consistent with the ordinary meaning as understood by those skilled in the art.
  • autoimmune disease refers to an autoimmune disease, specifically a disease caused by an organism's immune response to an autoantigen and causing damage to its own tissue. Many diseases have been classified as autoimmune diseases. It is worth mentioning that the existence of autoantibodies is not the same concept as autoimmune diseases. Autoantibodies can exist in normal people without autoimmune diseases, especially in the elderly. Such as anti-thyroglobulin antibodies, thyroid epithelial cell antibodies, gastric parietal cell antibodies, nuclear DNA antibodies, and the like. Sometimes, tissues with altered or antigenic changes can trigger the production of autoantibodies. For example, when myocardial ischemia, necrotic myocardium can lead to the formation of anti-myocardial autoantibodies, but this antibody has no pathogenic effect and is a secondary immune response.
  • the present invention has been confirmed by animal experiments that the drug claimed in the present invention can significantly alleviate the disease condition in which monocyte chemoattractant protein 1 is involved. Specifically, the drug of the present invention can effectively reduce the diseased arterial tissue in an animal. The interstitial volume (area and thickness) of fat, inflammatory cells and atherosclerosis, which can effectively inhibit or reduce the formation of atherosclerosis. The results of the effectiveness test of the present invention against animals demonstrate that the drug of the present invention can be as effective as 100% for animals.
  • FIG. 1 Human arterial immunohistochemical staining, showing high expression of YB-1 and phosphorylated YB-1 in human atherosclerotic tissue (AS), but very low expression in normal arterial wall (Normal) .
  • FIG. 1 Mouse arterial oil red-European staining. Compared with the negative control group (DMSO), it was shown that MK2206 significantly inhibited the formation of atherosclerosis. The red area indicates the fat in the atherosclerotic plaque.
  • DMSO negative control group
  • FIG. 3 Panel A: Western blot hybridization (IB) and reverse transcription-polymerase chain reaction (RT-PCR), showing that MK2206 (MK, YB-1 protein kinase specific inhibitor) can efficiently inhibit vascular smooth muscle cells YB-1 phosphorylation and MCP-1 mRNA levels.
  • MK2206 MK, YB-1 protein kinase specific inhibitor
  • RT-PCR reverse transcription-polymerase chain reaction
  • Panel B model male Aorta oil red (European Red O) staining, showed that MK2206 effectively inhibited the formation of atherosclerosis in model males.
  • DMSO was a drug-free group, 6-week-old male C57BL/6, N 9, ApoE -/- mice were fed with high fat for 10 weeks; MK was administered group: 6 weeks old male C57BL/6, ApoE -/- mice After 10 weeks of simultaneous administration with high fat, the red area in the figure shows the fat in the atherosclerotic lesion of the arterial wall.
  • Panel C aortic arch frozen section oil red European staining, showing that MK significantly reduced the degree of atherosclerosis at the aortic arch.
  • Panel D HE staining of aortic arch frozen sections, showed that MK significantly reduced the interstitial layer area of atherosclerosis.
  • EH map immunohistochemical staining, respectively showed that MK significantly reduced MCP-1 (E), macrophages (F), and foam cells (G) in atherosclerotic plaques, and significantly reduced phosphorylation YB-1 (H). (Brown red is a positive staining area).
  • FIG. 4 shows that V5 antibody (purchased from Invitrogen/Life Technologies, Grand Island, NY) selectively precipitates YB-1 mutant protein with V5 oligopeptide label in immunoprecipitation experiments;
  • V5 antibody purchased from Invitrogen/Life Technologies, Grand Island, NY
  • YBX is wild type Synonymous mutation of YB-1
  • 3dS is a deletion of 102 serine and two adjacent amino acids upstream thereof
  • WT wild type
  • IP immunoprecipitation
  • IB immunoblot hybridization
  • YB YB-1
  • protein extracts for immunoblot hybridization were derived from wild-type arterial wall smooth muscle cells and arterial wall stably transfected with YBX and 3dS, respectively. Smooth muscle cell line.
  • Panel B The top four images are shown in Figures i, ii, iii, and iv; wherein, Figure i shows that UK114 protein (UK) can only be combined with YB-1 (YB) from the V5 antibody in the A map.
  • the extract was purified (Fig. i, IB: UK immunoblot hybridization).
  • the precipitate containing UK and YB purified in immunoprecipitation has an activity of degrading MCP-1 mRNA.
  • Reverse transcription-polymerase chain reaction (RT-PCR) results showed that the UK content obtained by immunoprecipitation was directly proportional to the biological activity of the immunoprecipitate (the function of degrading MCP-1 mRNA) (Fig. ii).
  • Addition of the UK antibody to the immunoprecipitated complex blocked the above-described biological activity of degrading MCP-1 mRNA (Fig. iii), but the control PKC ⁇ antibody showed no blocking effect (Fig. iv).
  • Extract protein extract
  • rhUK recombinant source UK114
  • Panel A is a schematic representation of the base changes of the YB-1 mutant.
  • YB mut YB-1 mutant.
  • Figure B shows the results of reverse transcription-polymerase chain reaction (RT-PCR): RNA from a stably transfected YB-1 mutant cell line (stable transfected cell line that failed to obtain S/A mutant) and wild type Arterial smooth muscle cells. Wild and YB-1 mutant-containing cells in culture were incubated with 10 ng/ml PDGF (platelet-derived growth factor) for 2 hours, followed by selective addition of 1 micromolar Dex (dexamethasone) for 2 hours as shown in Figure B. RNA derived from the above cells was used for RT-PCR.
  • PDGF platelet-derived growth factor
  • dexamethasone also down-regulates MCP-1 by down-regulating YB-1 phosphorylation (see Figure 6), it was used as a positive control for this regulatory system in this experiment. Although dexamethasone down-regulates MCP-1, Clinical application shows that it has serious side effects and cannot be used for a long time). This experimental result demonstrates the important role that YB-1 plays in phosphorylation at its 100/102 position in regulating MCP-1 levels in cells.
  • FIG. 6 Screening of signaling pathways for the down-regulation of MCP-1 by dexamethasone.
  • RT-PCR results of panel A dexamethasone (Dex) significantly down-regulated MCP-1 mRNA (see lane 2), and this effect is insensitive to the six protein kinase inhibitors used (see Table 1) (see 3). To 8 lanes).
  • RT-PCR results of panel B dexamethasone (Dex) significantly down-regulates MCP-1 mRNA (see lane 2), and this effect is effectively blocked by a phosphorylase inhibitor (Cal) (lane 6) However, it is not sensitive to the other five phosphorylase inhibitors used (see Table 1 for details) (see lanes 3, 4, 5, 7, 8). This experiment predicts that dexamethasone down-regulates MCP-1 by phosphorylase.
  • the human arterial tissue used in the examples and/or experimental examples of the present invention is derived from a clinical sample of Peking Union Medical College Hospital; ApoE knockout mice (C57BL/6, ApoE-/-), 6-week-old male C57BL/6, ApoE- /- mice were purchased from Vital River, China (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.); arterial wall smooth muscle cell strains are commercially available.
  • OCT complex (Tissue-Tek, IL1-9302) was purchased from Tissue-Tek;
  • YB-1 antibody (Y0396) was purchased from sigma company; secondary antibody (PV-6001) was purchased from Zhongshan Jinqiao Company; AEC (AEC-0037) was purchased from Maixin Bio Company; Oil Red O staining kit (ab150678) was purchased from the United States Abcam (www.abcam.com).
  • the total RNA extraction kit (RNeasy kit) used in Experimental Example 3 was purchased from Qiagen Inc, Valencia, CA.
  • the PKC ⁇ antibody used in Experimental Example 4 was purchased from Sigma Aldrich; the anti-YB1 antibody (Y0396) was purchased from Sigma Aldrich; the human UK recombinant protein (rhUK, H00010247-P01) and its antibody (00010247-M01) were purchased from Abnova (Littleton, CO); V5 antibody was purchased from Invitrogen/Life Technologies, Grand Island, NY.
  • the present group of embodiments provides a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved.
  • the drug has the following common feature: the drug uses YB-1 as a drug target; and the active ingredient of the drug includes a substance that directly or indirectly regulates YB-1 phosphorylation .
  • the novel anti-disease mechanism of the medicament provided by the present invention in the treatment of diseases in which monocyte chemoattractant protein 1 is involved is as follows: YB-1 selectively regulates single-core by reducing phosphorylation (mRNA) stability of the cell by its own phosphorylation Expression of monocyte chemotactic protein-1 (MCP-1). YB-1 plays a major role in regulating the mRNA stability of MCP-1.
  • Increased mildness of intracellular non-phosphorylated YB-1 (increased by dephosphorylation of YB-1 or decreased phosphorylation, or a synergistic effect of both) can significantly inhibit the expression of MCP-1, thereby inhibiting monocytes Agglomeration in local tissues ultimately leads to inhibition, alleviation, or prevention of diseases involving monocyte chemoattractant protein 1.
  • regulation of YB-1 phosphorylation is effective in regulating MCP-1 and thereby controlling diseases caused by MCP-1 dysregulation.
  • the present invention demonstrates the role of this mechanism of disease resistance using an atherosclerotic model of transgenic mice.
  • the term "disease involved in monocyte chemoattractant protein 1" as used herein means: prior art published prior to the filing date (priority date) of the present inventors according to the present invention.
  • diseases in which monocyte chemoattractant protein 1 is involved specifically: inflammation, atherosclerosis, type 2 diabetes, obesity, autoimmune disease, tumor, and encephalopathy. (dementia, epilepsy, etc.);
  • the active ingredient of the drug comprises a substance that is directly or indirectly downregulated, and/or that directly or indirectly inhibits YB-1 phosphorylation;
  • the substance that directly or indirectly downregulates, and/or directly or indirectly inhibits YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor.
  • the present invention originally discovered the role of YB-1 phosphorylation in regulating MCP-1 expression and the inhibition of MCP-1 expression by YB-1 phosphorylation inhibitors in the process of studying the regulation of MCP-1 expression. effect.
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of any one or any of the following: a substance that promotes dephosphorylation of YB-1, blocks or delays YB-1 A phosphorylated substance, a substance that down-regulates or inhibits YB-1 protein kinase, a substance that up-regulates or stimulates YB-1 phosphorylase.
  • a substance that promotes dephosphorylation of YB-1 blocks or delays YB-1 A phosphorylated substance
  • a substance that down-regulates or inhibits YB-1 protein kinase a substance that up-regulates or stimulates YB-1 phosphorylase.
  • those substances which are highly selective and directly cause YB-1 dephosphorylation or high selectivity to directly block YB-1 phosphorylation and substances which highly selectively down-regulate the protein kinase activity of YB-1 and their high selection A substance that upregulates the phosphorylase activity of YB
  • Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer
  • the YB-1 phosphorylation enhancer is selected from the group consisting of any one or any of the following: a substance that blocks or delays YB-1 dephosphorylation, a substance that up-regulates or stimulates YB-1 phosphorylation. A substance that up-regulates or stimulates the protein kinase activity of YB-1, a substance that inhibits or down-regulates the activity of YB-1 phosphorylase.
  • the YB-1 phosphorylation inhibitor may also be: a substance which has an opposite effect to the above YB-1 phosphorylation enhancer, and/or a YB-1 dephosphorylation enhancer.
  • one specific example of the YB-1 phosphorylation inhibitor is a substance as shown in Formula I:
  • the YB-1 phosphorylation inhibitor may also be a currently clinically applied drug: dexamethasone; dexamethasone may dephosphorylate YB-1 by increasing the activity of a phosphorylase that has not yet been determined.
  • dexamethasone may dephosphorylate YB-1 by increasing the activity of a phosphorylase that has not yet been determined.
  • YB-1 phosphorylation inhibitor is a YB-1 mutein
  • the YB-1 mutein refers to a mutation obtained by mutating a serine at position 102 of the amino acid sequence of the wild type YB-1 protein. protein. Since serine contains hydroxyl groups, it is a common phosphorylation site. Removal or replacement of the serine at this site will cause YB-1 to lose the molecular basis at which it is phosphorylated, so this site can no longer be phosphorylated.
  • the above-mentioned 102-site serine-based YB-1 mutant protein has a function of down-regulating MCP-1 in cells.
  • mutant YB-1 is also assigned to the YB-1 phosphorylation inhibitor and falls within the scope of the YB-1 phosphorylation inhibitors of the present invention.
  • the active ingredient of the drug further comprises a polymer of the YB-1 phosphorylation inhibitor, and/or the YB-1 phosphorylation inhibitor is a pharmaceutically acceptable salt compound; And/or an ester compound; and/or a synergistic compound.
  • a polymer of the YB-1 phosphorylation inhibitor and/or the YB-1 phosphorylation inhibitor is a pharmaceutically acceptable salt compound; And/or an ester compound; and/or a synergistic compound.
  • Those skilled in the art may be based on actual needs, for example, the cost of drug production, the dosage form of the drug, the convenience of obtaining the raw material, and other properties (such as stability, disintegration in the body). Performance, etc.), selecting an appropriate derivative of a substance of formula I that achieves an equivalent or similar potency.
  • the medicament further comprises a pharmaceutically acceptable adjuvant and/or carrier.
  • a pharmaceutically acceptable adjuvant and/or carrier may be based on actual needs, for example, the cost of drug production, the dosage form of the drug, the convenience of obtaining the raw material, and other properties (such as stability, disintegration in the body). The performance, the shelf life of the drug, etc.), the selection of appropriate excipients, specifically, excipients, carriers, solvents, diluents, and encapsulating materials for carrying and transporting the drug in the body.
  • Such materials are: sugar, cellulose and its derivatives, scutellite powder, talc, gelatin, oil, alcohols, agar, buffer, diethyl ester, emulsion, lubricant, non-pyrogenic water, Alginic acid, toning and flavoring agents, preservatives, emulsifiers, aerosols, lubricants, antioxidants, sustained release agents, and other substances used in pharmaceutical formulations.
  • the present group of embodiments provides a screening method for a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved as provided in the first group of examples. All the examples in this group have the following common features: use of substances that can directly or indirectly regulate YB-1 phosphorylation as a drug candidate for pathological experiments, and / or, clinical trials, and / or treatment, screening and effect And/or a substance that produces a therapeutic effect is used as an active ingredient of the drug.
  • the substance that modulates YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is preferably selected from: a substance that promotes dephosphorylation of YB-1, and/or a substance that blocks or delays phosphorylation of YB-1, and/or a substance that down-regulates or inhibits YB-1 protein kinase, and/or a substance that up-regulates or stimulates YB-1 phosphorylase.
  • Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer
  • the YB-1 phosphorylation enhancer is selected from the group consisting of any one or any of the following: a substance that blocks or delays YB-1 dephosphorylation, a substance that up-regulates or stimulates YB-1 phosphorylation. A substance that up-regulates or stimulates the protein kinase activity of YB-1, a substance that inhibits or down-regulates the activity of YB-1 phosphorylase.
  • the YB-1 phosphorylation inhibitor may also be: a substance which has an opposite effect to the above YB-1 phosphorylation enhancer, and/or a YB-1 dephosphorylation enhancer.
  • the specific operation of the screening method can be carried out as follows: treating a atherosclerotic model mouse with a specific substance A, and performing pathological section observation (for example, oil red euro staining, or HE staining); Immunoblot hybridization (IB) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the effect of this substance A on MCP-1 at the molecular and protein levels.
  • pathological section observation for example, oil red euro staining, or HE staining
  • IB Immunoblot hybridization
  • RT-PCR reverse transcription-polymerase chain reaction
  • the present embodiments provide the use of a substance that can down-regulate YB-1 phosphorylation in the preparation of a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved; or, for use in a treatment list as described in the first group of examples A method for preparing a drug for a disease in which nuclear cell chemoattractant protein 1 is involved. All of the examples in this group have the common feature of placing the down-regulated YB-1 phosphorylated material in a commercial package containing the disease therapeutic use in which monocyte chemoattractant protein 1 is involved;
  • the substance that binds, activates, and/or upregulates YB-1 comprises a YB-1 phosphorylation inhibitor
  • the YB-1 phosphorylation inhibitor is selected from the group consisting of: a substance that dephosphorylates YB-1, and/or a substance that blocks YB-1 phosphorylation, and/or downregulates A substance of YB-1 protein kinase, and/or a substance that up-regulates YB-1 phosphorylase.
  • the present invention provides a method of using the drug of the first group of examples, and/or the drug selected by the screening method of the second group of examples, and/or the method for preparing the drug prepared by the preparation method of the third group of examples. All of the examples in this group have the feature that the substance which phosphorylates YB-1 can be directly or indirectly regulated in the prevention and/or treatment of diseases involving MCP-1.
  • the method of use comprises the clinical use of the drug, i.e., the use of the substance in an individual for the above indications to treat, alleviate, prevent the above indications.
  • the clinical use of the medicament comprises the clinically used dosage form and dosage of the substance to which the invention refers.
  • the primary antibodies were anti-YB-1 antibody (sigma, Y0396) and anti-phospho-YB-1 antibody (Cell Signaling, C34A2), respectively, diluted with PBS (1:100) and incubated with the sectioned tissue at 4 degrees overnight.
  • the second antibody was purchased from Zhongshan Jinqiao (PV-6001).
  • AEC Alignin reagent AEC-0037
  • red is a positive region.
  • YB-1 and phosphorylated YB-1 are highly expressed in human atherosclerotic tissue (AS), while the expression level in normal arterial wall (Normal) is extremely low.
  • the atherosclerotic model mice commonly used in the art are treated with the drug according to any one of the first group of the present invention, the administration mode is: intraperitoneal injection of 85 ⁇ g/day; the control mice are treated with the same amount of DMSO per day; After 70 days of administration, the arterial tissues of the mice in the treated group and the mice in the control group were stained with the Oil Red O staining kit, respectively, and the staining method is the most commonly used standard method for displaying fat in frozen section tissues.
  • the specific procedure is in accordance with the product specification of the Oil Red O staining kit (ab150678) available from the American abcam company (www.abcam.com).
  • Atherosclerosis model mouse and “model mouse” herein refer to C57BL/6, N9, ApoE -/- mice after 10 weeks of high fat feeding.
  • Immunoblot hybridization (IB) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the effect of MK on MCP-1 at the molecular and protein levels; the specific operation was: mouse and human vascular smooth muscle cells (mSMC, hSMC) was incubated for 4 hours in 0.5 ⁇ mol of MK2206 medium, and cells incubated in MK2206-free medium were used as control (C). Protein and ribonucleic acid are extracted from the above cells for IB and RT-PCR to detect YB-1 and MCP-1 mRNA contents therein; and the methods of operation of IB and RT-PCR are carried out according to conventional procedures well known to those skilled in the art.
  • the specific operation of reverse transcription-polymerase chain reaction (RT-PCR) is as follows:
  • the primers used were 145-595 nucleic acid coding segments spanning MCP-1 and 340-625 nucleic acid coding segments of GAPDH; primers were 20 bases in length (the nucleic acid coding region sequences of rat MCP-1 and GAPDH were available). Log in to Genebank for a query).
  • the PCR products were 145-595 for the monocyte chemoattractant cDNA sequence, 340-625 for GAPDH, 420-988 for YB-1, and 441-988 for the YB-1 mutant.
  • the forward primer of the YB-1 mutant is TGTGGAATTCGACGTCGTC, which corresponds to the wild type TGTGAGTTTGATGTTGTT.
  • the above primers are suitable for all RT-PCRs herein unless otherwise stated.
  • Total RNA was extracted from rat cells using the RNeasy kit (Qiagen Inc, Valencia, CA). Rat smooth muscle cells were isolated and cultured as described in Brock, 1985, Hypertension. The operation was as follows: 200-300 g of smooth muscle cells in the middle thoracic artery of male Sprague-Dawley rats were obtained by enzymatic hydrolysis. Cultured cells of 5-14 passages were used in all experiments. The cells were cultured in Dulbecco modified Eagle medium (DMEM; Gibco Laboratories, Gaithersburg, Md.) containing 10% calf serum and incubated in a 37 ° carbon dioxide incubator. When the cells in the culture dish reached approximately 60% full, the cells were used to prepare RNA.
  • DMEM Dulbecco modified Eagle medium
  • RNA transfection was performed according to the method of Lipofectamine 2000 reagent manufacturer Ivitrogen, and Lipofectamine 2000 was purchased from Invitrogen. The cell density at the time of transfection was approximately 30%.
  • Each reverse transcription-polymerase chain reaction uses 200 nanograms of RNA.
  • the reaction system was carried out using a Masterscript RT-PCR system (5 PRIME Inc., Gaithersbueg, MD).
  • the RT-PCR reaction procedure was: 54 ° C for 30 minutes; 94 ° C for 2 minutes; "94 ° C for 22 seconds, 55 ° C for 22 seconds, 73 ° C for 44 seconds" for one cycle, a total of 27 cycles; 73 ° C for 6 minutes.
  • the above parameters apply to all RT-PCR reactions herein unless otherwise stated.
  • YB-1 mutant herein generally refers to a nucleotide sequence corresponding to the "YB-1 mutein”.
  • FIG. 3 A: Western blot hybridization (IB) and reverse transcription-polymerase chain reaction (RT-PCR), showing that MK2206 (MK, YB-1 protein kinase specific inhibitor) can Highly inhibits YB-1 phosphorylation and MCP-1 mRNA levels in vascular smooth muscle and monocytes.
  • IB Western blot hybridization
  • RT-PCR reverse transcription-polymerase chain reaction
  • the atherosclerotic model mice are treated with the medicament according to any one of the first group of the present invention, and the administration method is: intramuscular injection of 85 ⁇ g/day for the experimental rats with high fat feeding (about a mouse model) 1/13 of the therapeutic tumor dose. See Mol Cancer Ther.
  • mice used an equal amount of DMSO instead of the drug of the present invention every day;
  • the arterial tissues of the mice in the treatment group and the mice in the control group were stained with oil red O and arterial HE, respectively, and the specific operation of the oil red O staining was as described in Experimental Example 2;
  • HE hematoxylin-eosin staining method, the specific operation refers to the "Haematoxylin Eosin (H&E) staining
  • HE staining is the most commonly used standard histological staining method in the field to show the morphology and structure of tissues (hematoxylin is dark blue or purple, combined with nucleic acids. Eosin is pink, combined with amino acids); the therapeutic effects of this group of experiments are as follows:
  • Figure B is a B-D diagram of the model male aorta oil red O (Oil Red O) staining, showing that MK2206 efficiently inhibits the formation of atherosclerosis in model males.
  • Oil Red O Oil Red O
  • DMSO is a drug-free group, 6-week-old male C57BL/6, ApoE -/- mice are fed with high fat for 10 weeks; MK is a drug-administered group: 6-week-old male C57BL/6, ApoE -/- mice with high fat
  • Panel C aortic arch oil red European staining, showed that MK significantly reduced the degree of atherosclerosis at the aortic arch.
  • Panel D arterial HE staining, showed that MK significantly reduced the interstitial layer area of atherosclerosis.
  • this experimental example also tests the drug of the present invention for reducing inflammation and reducing inflammatory cells, and the therapeutic administration process of the control group and the administration group is as described in the above paragraph, and the mice of the control group and the administration group after the treatment are completed.
  • the arterial tissues were subjected to immunohistochemical staining, respectively.
  • the method was the same as in Experimental Example 1.
  • the staining results were as shown in the EH diagram of Figure 3, immunohistochemical staining, respectively, showing that MK significantly reduced MCP-1(E) in atherosclerotic plaques. , macrophages (F), and foam cells (G), and significantly reduced phosphorylated YB-1 (H). (Brown red is a positive staining area).
  • Atherosclerosis is inflammation of the arterial wall mediated by MCP-1.
  • the above experimental example uses atherosclerosis as a model to verify the role of YB-1 in inflammatory pathology and the anti-inflammatory effect produced by regulating YB-1 with the drug of the present invention to regulate MCP-1 levels in vivo.
  • the medicament of the present invention can also be used for treating type 2 diabetes, tumor, autoimmune disease, obesity, and encephalopathy (dementia, epilepsy, etc.) mediated by MCP-1, and can A therapeutic effect similar to Experimental Examples 2 and 3 herein is expected.
  • the YB-1 mutein with the V5 oligopeptide tag was selectively precipitated by the V5 antibody by immunoprecipitation assay.
  • the specific operations are as follows:
  • the YB-1 mutein was obtained by the specific procedure of RT-PCR as described in Experimental Example 3 (including the use of YB-1 mutant primers, reaction procedure, etc.) to thereby make rat YB-1 (GEnBank accession no) .NM031563) cDNA spanning the initiation and termination cryptodomains was obtained by reverse transcription-polymerase chain reaction and cloned between HindIII and XbaI of the pBluescript II KS(+) plasmid. All YB-1 mutant proteins were synthesized by polymerase chain reaction (PCR) or other artificial sequences.
  • PCR polymerase chain reaction
  • YB-1 mutants were subcloned between the Hind III and XbaI sites of the pcDNA3.1/V5-His A plasmid.
  • the Kozak sequence, GCCACC, was introduced upstream of the initiation codon.
  • the termination code of the YB-1 3' terminus was replaced by the Xba I site.
  • the V5-His tag peptide and the stop codon contained in the plasmid follow the Xba I site in coding order.
  • the cloned PCR final products were confirmed by DNA sequencing.
  • the YB-1 mutein with the V5 oligopeptide marker was obtained by the pcDNA3.1/V5-His A vector plasmid carrying the V5-labeled peptide DNA sequence, and the YB-1 mutant without the stop codon was inserted into the box. A hybrid plasmid was obtained after cloning into the upstream of V5. This hybrid plasmid expresses a YB-1 mutein with V5 in the cell. This heterozygous mutant protein binds specifically to the V5 antibody.
  • the procedures for immunoprecipitation and immunoblot hybridization herein are as follows: 30 microliters of G protein bead suspension (Roche, Germany) was used for each sample. G protein beads were washed 3 times with pre-chilled S100 buffer (10 mM Tris [pH 7.4], 1.5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 0.5 mM PMSF). Another 3 ⁇ l of V5-antibody was incubated with G-protein beads for 1 hour at 4 ° C, followed by incubation with 1 ⁇ l of 20% bovine serum albumin (BSA Fraction V from Sigma Aldrich) for 1 hour.
  • BSA Fraction V bovine serum albumin
  • V5 antibody coated G protein beads were washed once with pre-chilled S100 buffer and then resuspended in 0.5 ml Eppendorf tubes in 150 microliters of cold S100 buffer in an ice bath.
  • Add 30-50 ⁇ l (10 ⁇ g protein) cytoplasmic extract (for extraction method, refer to Poon M, Liu B, Taubman MB. 1999. Identification of a novel dexamethasone-sensitive RNA-destabilizing region on rat monocyte chemoattractant protein 1 mRNA.
  • the test results are shown in Figures 4 and 5, respectively.
  • the control PKC ⁇ antibody used in Figure iv of Figure 4 is a protein kinase that specifically binds to human, mouse, and rat protein kinases, which have no inhibitory effect on MCP-1 mRNA degradation.
  • Broken action; serine at position 102 of YB-1 corresponds to serine at 100 sites in rats.
  • rat YB-1 this deletion of serine resulted in YB-1 not being phosphorylated at this site.
  • This site of non-phosphorylated YB-1 binds to UK114 (a ribonuclease) and GR (glucocorticoid receptor) to form an active complex that selectively degrades MCP-1 mRNA.
  • Phosphorylated YB-1 at this site is not conducive to binding to UK114.
  • concentrations in the above table refer to the final concentration of each inhibitor in the cell culture medium; +: complete inhibition; +/-: partial inhibition; -: no inhibition
  • mice Twenty-four model mice, 12 rats in the control group and the drug-administered group were administered to the drug-administered group and the control group according to the administration mode and the control method as shown in Experimental Example 3, respectively. 50 mice of the administration group were intraperitoneally injected with the drug described in any one of the first group of the present invention; after 10 weeks, the oil red O (Oil Red O) staining and immunization as shown in Experimental Example 3 was used. Histochemical tissues of the two groups of mice were stained by histochemical staining, and the results of the treatment of Experimental Example 2-3 were finally obtained.
  • Oil Red O Oil Red O

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Abstract

L'invention concerne un médicament pour le traitement d'une maladie impliquant la MCP-1. Les ensembles médicamenteux YB-1 à titre de cibles médicamenteuses, et leurs principes actifs, comprennent une substance qui peut directement ou indirectement ajuster la phosphorylation de YB-1.
PCT/CN2018/080675 2017-08-03 2018-03-27 Médicament pour le traitement d'une maladie impliquant la protéine chimioattractive des monocytes de type 1 (mcp-1) par ajustement de la phosphorylation de yb-1 WO2019024518A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007109908A1 (fr) * 2006-03-29 2007-10-04 The University Of British Columbia Leures thérapeutiques de la phosphorylation par le yb-1
WO2017043779A1 (fr) * 2015-09-07 2017-03-16 동국대학교 산학협력단 Gène stk32c lié au cancer du sein et utilisation de ce dernier

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007109908A1 (fr) * 2006-03-29 2007-10-04 The University Of British Columbia Leures thérapeutiques de la phosphorylation par le yb-1
WO2017043779A1 (fr) * 2015-09-07 2017-03-16 동국대학교 산학협력단 Gène stk32c lié au cancer du sein et utilisation de ce dernier

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LATIKA, D. ET AL.: "Y-Box Binding Protein 1 and RNase UK 114 Mediate Monocyte Chemoattractant Protein 1 mRNA Stability in Vascular Smooth Muscle Cells", MOLECULAR AND CELLULAR BIOLOGY, vol. 32, no. 18, 30 September 2012 (2012-09-30), pages 3768 - 3775, XP055570140 *

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