WO2019024518A1 - Drug for treating monocyte chemoattractant protein-1 (mcp-1) involved disease by adjusting phosphorylation of yb-1 - Google Patents

Drug for treating monocyte chemoattractant protein-1 (mcp-1) involved disease by adjusting phosphorylation of 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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Chinese (zh)
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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
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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

Abstract

Provided is a drug for treating a MCP-1 involved disease. The drug sets YB-1 as a drug target, and the active components thereof comprise a substance that can directly or indirectly adjust phosphorylation of YB-1.

Description

[根据细则37.2由ISA制定的发明名称]　通过调控YB-1磷酸化治疗MCP-1参与的疾病的药物[Name of invention established by ISA according to Rule 37.2] Drugs for the treatment of diseases in which MCP-1 is involved by regulating YB-1 phosphorylation 技术领域Technical field
本发明属于生物医药领域，具体涉及一类通过调控YB-1磷酸化治疗由单核细胞趋化蛋白-1参与的疾病的药物及其筛选方法。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.
背景技术Background technique
Y区结合蛋白-1(Y-box-binding protein 1,YB-1)是一类特异性结合目的基因启动子和增强子内部Y-Box序列(一种高度保守的顺式的DNA序列)的转录因子，属于冷休克蛋白(Cold shock proteins)超家族中的一员，它含有的一段高度保守的冷休克区域(Cold shock domain,CSD)，就是其与核酸的结合部位。YB-1的蛋白代号为：Protein Symbol:P67809-YBOX1_HUMAN，由324个氨基酸组成，分子量为35924道尔顿。YB-1广泛存在于微生物、植物、动物和人类中，在分子水平上，参与DNA修复，mRNA转录、剪接，调节mRNA的稳定性、翻译等生物学过程；在细胞水平上，YB-1在细胞增殖与分化、细胞应激反应、肿瘤细胞的转化上起重要作用；在临床病理方面，YB-1被证实，与多种疾病的发生有关联，例如，肿瘤，包括肝癌、乳腺癌、结肠腺癌、肺癌；肝纤维化；炎症；动脉粥样硬化；器官移植排异反应；再灌注损伤；YB-1也可作为肿瘤诊断，和/或预后的标志物。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. At the molecular level, it participates in DNA repair, mRNA transcription, splicing, regulation of mRNA stability, translation and other biological processes. At the cellular level, 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.
趋化性细胞因子chemokine是一个蛋白质家族，由十余种结构有较大同源性、分子量多为8～10kD的蛋白组成。这些蛋白在氨基端多含有一个或两个半胱氨酸。根据半胱氨酸的排列方式，将趋化性细胞因子分为亚家族。两个半胱氨酸按Cys-X-Cys(半胱氨酸-任一氨基酸-半胱氨酸)方式排列的趋化性细胞因子属α亚家族，也称CXC趋化性细胞因子；以Cys-Cys方式排列的趋化性细胞因子属β亚家族，也称CC趋化性细胞因子。氨基端只有一个半胱氨酸的趋化性细胞因子称γ亚家族趋化性细胞因子，也称C趋化性细胞因子。趋化性细胞因子主要由白细胞和造血微环境中的基质细胞分泌，可作用于任何具有趋化性细胞因子受体的细胞，并对他们的靶细胞具有趋化和激活作用。IL-8是α亚家族系的代表，对中性粒细胞有趋化或富集作用。单核细胞趋化蛋白-1(monocyte chemoattractant protein-1，MCP-1或CCL-2)是β亚家族的代表，可趋化或富集单核细胞,嗜碱性粒细胞，记忆性T细胞，及树突状细胞。淋巴细胞趋化蛋白(lymphotactin)是γ亚家族的代表，对淋巴细胞有趋化或富集作用。MCP-1主要参与炎症的发生发展过程，包括动脉粥样硬化,***性红斑狼疮，风湿性关节炎。目前为止，人类对上述疾病的治疗方法仍然十分有限且效果欠佳。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 α, also known as CXC chemotactic cytokine; The chemotactic cytokines arranged in the Cys-Cys manner belong to the β 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 β 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 and the effect is not good.
发明内容Summary of the invention
本发明基于本领域存在的上述缺陷和不足，提供一类治疗由单核细胞趋化蛋白-1参与的疾病的药物及该类药物的应用，用于为本领域治疗单核细胞趋化蛋白-1参与的上述各类疾病提供一种效果更好的药物以 及新的用药选择。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.
本发明的技术方案如下：The technical solution of the present invention is as follows:
用于治疗单核细胞趋化蛋白-1参与的疾病的药物，其特征在于，以YB-1为药物靶点；且所述药物的活性成分包括能直接或间接调控YB-1磷酸化的物质。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 .
所述单核细胞趋化蛋白1参与的疾病包括：炎症、动脉粥样硬化、2型糖尿病，肿瘤，自身免疫病，肥胖、及脑病(痴呆，癫痫等)。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.).
所述药物的活性成分包括直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质；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;
进一步地，所述直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质包括YB-1磷酸化抑制剂。Further, the substance that directly or indirectly downregulates, and/or directly or indirectly inhibits YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor.
所述YB-1磷酸化抑制剂选自由下述物质中的任一种或任几种组成的组：促使YB-1脱磷酸化的物质、阻断或延缓YB-1磷酸化的物质、下调或抑制YB－1的蛋白激酶活性的物质、上调或激发YB－1磷酸化酶活性的物质。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.
与所述YB-1磷酸化抑制剂相对的是YB-1磷酸化增强剂；Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer;
所述YB-1磷酸化增强剂选自由下述物质中的任一种或任几种组成的组：阻断或延缓YB-1脱磷酸化的物质、上调或激发YB-1磷酸化的物质、上调或激发YB－1的蛋白激酶活性的物质、抑制或下调YB－1磷酸化酶活性的物质。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.
所述YB-1磷酸化抑制剂包括：如式I所示的物质：The YB-1 phosphorylation inhibitor comprises: a substance as shown in Formula I:
Figure PCTCN2018080675-appb-000001
Figure PCTCN2018080675-appb-000001
***；和/或，Dexamethasone; and/or,
YB-1突变蛋白；YB-1 mutant protein;
所述YB-1突变蛋白指，野生型YB-1蛋白的氨基酸序列的102位点丝氨酸突变后得到的突变蛋白。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.
所述药物的活性成分还包括所述YB-1磷酸化抑制剂的聚合物，和/或，所述YB-1磷酸化抑制剂在药学上可接受的盐类化合物；和/或，酯类化合物；和/或；增效类化合物。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.
所述药物还包括药学上可接受的辅料和/或载体。The medicament also includes pharmaceutically acceptable excipients and/or carriers.
用于治疗单核细胞趋化蛋白1参与的疾病的药物的筛选方法，其特征在于，采用可直接或间接调控 YB-1磷酸化的物质做为候选药物进行病理实验，和/或，临床试验，和/或，治疗，筛选出具有效果，和/或产生疗效的物质用作所述药物的活性成分。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.
所述可直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；The substance which can directly or indirectly regulate YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor;
所述YB-1磷酸化抑制剂优选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。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 protein kinase active substance, and/or a substance that up-regulates or stimulates YB-1 phosphorylase activity.
可直接或间接调控YB-1磷酸化的物质在制备用于治疗单核细胞趋化蛋白1参与的疾病的药物方面的用途，其特征在于，将所述可直接或间接调控YB-1磷酸化的物质放置在标有单核细胞趋化蛋白1参与的疾病治疗用途的商品包装盒内；Use of 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;
在优选范围内，所述可直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；Within the preferred range, the substance that directly or indirectly regulates YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor;
在更进一步优选范围内，所述YB-1磷酸化抑制剂选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。In a still further preferred range, 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.
本发明首先提供一类用于治疗单核细胞趋化蛋白1参与的疾病的药物，其特征在于，以YB-1为药物靶点；且所述药物的活性成分包括可直接或间接调控YB-1磷酸化的物质。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.
本发明开拓性地发现了上述药物治疗单核细胞趋化蛋白1参与的疾病的新的抗病机理：YB-1直接参与MCP-1mRNA稳定性的调控。非磷酸化的YB-1通过促进MCP-1mRNA的降解而下调MCP-1水平，从而减少单核细胞的聚集。非磷酸化的YB-1有利于结合UK114(核酸酶活性蛋白)而形成一种至少含有YB－1，UK114，和GR(糖皮质激素受体)的复合物，该复合物通过选择性降低细胞内单核细胞趋化蛋白-1(monocyte chemoattractant protein-1:MCP-1)的mRNA稳定性而下调MCP-1的表达。有关分子生物学实验结果见图4。YB－1对调控MCP-1的mRNA稳定性起主要作用。细胞内非磷酸化YB－1的温和性增加(产生于YB－1的脱磷酸化增强或磷酸化减弱，或二者的协同作用)即可显著抑制MCP－1的表达，进而抑制单核细胞在局部组织中的集聚，最终达到抑制，减轻，或预防由单核细胞趋化蛋白1参与的疾病。因而，调控YB－1磷酸化能有效调控MCP－1进而控制由MCP－1失调引起的疾病。本发明用转基因小鼠的动脉粥样硬化模型证实了该抗病机理的作用。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 The mRNA stability of monocyte chemoattractant protein-1 (MCP-1) down-regulates the expression of MCP-1. The results of molecular biology experiments are shown in Figure 4. 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. Thus, 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.
在本发明具体的实施例中，所述单核细胞趋化蛋白1参与的疾病包括：炎症、动脉粥样硬化、2型糖尿病，肿瘤，自身免疫病，肥胖、及脑病(痴呆，癫痫等)。In a specific embodiment of the present invention, 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.) .
在本发明的一些实施例中，所述药物的活性成分包括直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质；In some embodiments of the invention, 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;
在进一步的实施例中，所述直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质包括YB-1磷酸化抑制剂。本发明在研究调控MCP－1的表达机理的过程中原创性地发现了YB－1磷酸化在调控 MCP-1表达中的作用，以及YB－1磷酸化的抑制剂对MCP－1表达的抑制作用。In a further embodiment, 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.
本文中的“单核细胞趋化因子1”、“单核细胞趋化蛋白1”、“单核细胞趋化蛋白-1”、“MCP-1”具有相同的本领域技术人员可常规理解的含义。"Monocyte chemotactic factor 1", "monocyte chemotactic protein 1", "monocyte chemotactic protein-1", "MCP-1" herein have the same conventionally understood by those skilled in the art. meaning.
在一些具体的实施例中，所述YB-1磷酸化抑制剂选自由下述物质中的任一种或任几种组成的组：可促使YB-1脱磷酸化的物质、阻断或延缓YB-1磷酸化的物质、下调或抑制YB－1的蛋白激酶活性的物质、上调或激发YB－1磷酸化酶活性的物质。In some specific embodiments, 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.
尤其是那些高选择性直接导致YB－1脱磷酸化或高选择性直接阻断YB－1磷酸化的物质，以及高选择性下调YB－1的蛋白激酶(kinase)活性的物质及其高选择性上调YB－1的磷酸化酶(phosphatase)活性的物质。In particular, 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.
蛋白激酶的作用是使目标蛋白磷酸化，而蛋白激酶的显著下降可导致蛋白激酶的总活性下降，下调或抑制蛋白激酶活性即可达到抑制或下调靶标蛋白磷酸化的目的；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;
相反，磷酸化酶的作用是脱磷酸化(去磷酸化)，上调或激发靶标蛋白磷酸化酶的活性，即可使磷酸化酶的脱磷酸化作用强化，从而增强靶标蛋白的去磷酸化作用，进而起到抑制或下调靶标蛋白磷酸化的目的。In contrast, 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.
与所述YB-1磷酸化抑制剂相对的是YB-1磷酸化增强剂；Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer;
所述YB-1磷酸化增强剂选自由下述物质中的任一种或任几种组成的组：阻断或延缓YB-1脱磷酸化的物质、上调或激发YB-1磷酸化的物质、上调或激发YB－1的蛋白激酶活性的物质、抑制或下调YB－1磷酸化酶活性的物质。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.
因此，所述YB-1磷酸化抑制剂还可以是：与上述YB-1磷酸化增强剂起相反作用的物质，和/或，YB-1去磷酸化增强剂。Therefore, 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.
在进一步具体的实施例中，所述YB-1磷酸化抑制剂为如式I所示的物质：In a further specific embodiment, the YB-1 phosphorylation inhibitor is a substance of formula I:
Figure PCTCN2018080675-appb-000002
Figure PCTCN2018080675-appb-000002
在另一些实施例中，所述YB-1磷酸化抑制剂为***、或YB－1突变蛋白；所述YB-1突变蛋白指，野生型YB-1蛋白氨基酸序列的102位点丝氨酸经突变后得到的突变蛋白。所述野生型YB-1蛋白的序列如SEQ ID NO.1所示。In other embodiments, 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.
在进一步的实施例中，所述药物的活性成分还包括所述YB-1磷酸化抑制剂的聚合物，和/或，所述YB-1磷酸化抑制剂在药学上可接受的盐类化合物；和/或，酯类化合物；和/或；增效类化合物。In a further embodiment, 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.
在更进一步的实施例中，所述的药物还包括药学上可接受的辅料和/或载体。In still further embodiments, the medicament further comprises a pharmaceutically acceptable adjuvant and/or carrier.
本发明的另一个方面提供了用于治疗单核细胞趋化蛋白1参与的疾病的药物的筛选方法，其特征在于，采用可直接或间接调控YB-1磷酸化的物质做为候选药物进行病理实验，和/或，临床试验，和/或，治疗，筛选出具有效果，和/或产生疗效的物质用作所述药物的活性成分。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.
在筛选方法的具体实施方案中，所述直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；In a specific embodiment of the screening method, the substance that directly or indirectly regulates YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor;
进一步地，所述YB-1磷酸化抑制剂优选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。Further, 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.
本发明还请求保护可直接或间接调控YB-1磷酸化的物质在制备用于治疗单核细胞趋化蛋白1参与的疾病的药物方面的用途，其特征在于，将所述可直接或间接调控YB-1磷酸化的物质放置在标有单核细胞趋化蛋白1参与的疾病治疗用途的商品包装盒内；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;
优选地，所述可直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；Preferably, the substance which can directly or indirectly regulate YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor;
更进一步优选地，所述YB-1磷酸化抑制剂选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。Still more preferably, 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.
在一些国家或地区的专利法允许的前提下，本发明还请求保护可直接或间接调控Y区结合蛋白－1(Y-box-binding protein 1:YB-1)磷酸化的物质在临床医疗中的适应症及其使用方法；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;
该发明关于YB－1磷酸化抑制剂的临床使用方法包括对上述适应症的个体使用该类物质以治疗，减轻，预防上述适应症。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.
该发明关于该发明所指物质的临床应用包括该发明所指物质的临床使用剂型和剂量。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.
除非另做说明，此处所有科技术语与该专利所涉及领域中科技常用术语具有相同意义。有机化学基本原理，及特殊官能团和反应参照Thomas Sorrell著《有机化学》,University Science Books,Sausalito:2006年版.Unless otherwise stated, all technical terms used herein have the same meaning as commonly used terms of technology in the field to which the patent relates. The basic principles of organic chemistry, and special functional groups and reactions refer to Thomas Sorrell, "Organic Chemistry", University Science Books, Sausalito: 2006 edition.
该发明的某些化合物或许以特定的几何或立体结构形式存在。该发明覆盖所有诸如此类的化合物，包括顺式，返式，R及S对应异构体，非对应异构体，(d)和(l)异构体，消旋混合物及该发明所覆盖的其他混合物。也包括非对称碳原子的替换，例如烷基中的替换。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.
异构混合物包括含有该发明异构体的任何比例的混合物。The isomeric mixture includes mixtures in any ratio containing the isomers of the invention.
该发明所指的“活性成份有效量”是指能足以产生所期待的生物学效应的剂量。此处所指的有效剂量可根据不同情况(例如给药途径，个体差异，不同化合物的药代，疾病的种类及治疗目标)而有所不同。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).
该发明所指的疾病的“治疗”包括治愈，减轻，延缓，或改善病情的方法，包括对病情的预防。治疗可能是针对疾病的一个或多个症状，或导致症状的病理学机制。相对于同等未接受治疗的对照组，此处所说的减轻或预防至少表现为10％的差异(用任何标准技术衡量)。The "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.
此处所指的“预防”包括防止，延缓，避免，或制止疾病或病情的发生，加重，或复发的方法。"Prevention" as used herein includes methods of preventing, delaying, avoiding, or stopping the onset, exacerbation, or recurrence of a disease or condition.
此处所指的“药学上可接受的辅料”包括赋形剂，载体，溶剂，稀释剂，及用以在体内携带，转运该药的包裹材料。这些材料的实例诸如：糖，纤维素及其衍生物,西黄蓍蚀粉，滑石粉，明胶,油，醇类，琼脂，缓冲剂，二乙酯，乳化物，润滑剂，无热源水，藻酸，调色及调味剂，防腐剂，乳化剂，湿剂，润滑剂，抗氧化剂，缓释剂，及其他制药配方中所使用的相应物质。"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.
本发明涉及的“分离的或纯化的”是指基本不含通常情况下的天然并存物的物质。纯度或均一性由诸如聚丙烯酰胺凝聚电泳或高效液相层析分析化学方法确定。As used herein, "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.
本发明所指的“个体”包括但不仅限于接受该治疗的人类，灵长类，啮齿类等。“个体”和“患者”可以互换用于人类。"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.
本文所提及的“脱磷酸化”即“去磷酸化”，一般指磷酸基团的除去，在本文中的含义与本领域技术人员所理解的通常含义一致。As used herein, "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.
除非另有定义，本文使用的全部科技术语具有与本发明所属领域技术人员通常理解相同的含义。比如，所述“自身免疫病”指自身免疫性疾病，具体指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病。许多疾病相继被列为自身免疫性疾病，值得提出的是，自身抗体的存在与自身免疫性疾病并非两个等同的概念，自身抗体可存在于无自身免疫性疾病的正常人特别是老年人，如抗甲状腺球蛋白抗体、甲状腺上皮细胞抗体、胃壁细胞抗体、细胞核DNA抗体等。有时，受损或抗原性发生变化的组织可激发自身抗体的产生，如心肌缺血时，坏死的心肌可导致抗心肌自身抗体形成，但此抗体并无致病作用，是一种继发性免疫反应。Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning For example, the "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.
本发明经动物实验证实，本发明请求保护的药物能十分显著地缓解单核细胞趋化蛋白1参与的疾病病症，具体地，本发明的所述药物能有效减少动物体内的病变动脉组织中的脂肪、炎性细胞及粥样硬化处间质层体积(面积及厚度)，所述药物可高效抑制或减少动脉粥样硬化的形成。本发明针对动物的有效性试验结果证实本发明的药物对动物有效性可高达百分之百。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.
附图说明DRAWINGS
图1.人动脉免疫组化染色，显示YB-1和磷酸化YB-1在人体动脉粥样硬化组织(AS)内的高表达，而在正常动脉管壁(Normal)中的表达量极低。Figure 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) .
图2.小鼠动脉油红欧染色。与阴性对照组(DMSO)相比，显示MK2206显著抑制了动脉粥样硬化的形成。红色区域表示粥样硬化斑块中的脂肪。Figure 2. 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.
图3.A图:免疫印迹杂交(IB)及反转录-聚合酶链式反应(RT-PCR)，显示MK2206(MK，YB-1的蛋白激酶特异性抑制剂)能高效抑制血管平滑肌细胞内的YB-1磷酸化及MCP-1mRNA水平。(小鼠及人血管平滑肌细胞-SMC在含0.5微摩尔的MK2206培养液中孵育4小时，在无MK2206培养液中孵育的细胞做为对照-C。从上述细胞中提取蛋白及核糖核酸，用于IB及RT-PCR以检测其中的YB-1和MCP-1mRNA含量)。B图，模型雄鼠主动脉油红欧(Oil Red O)染色，显示MK2206高效抑制了模型雄鼠动脉粥样硬化的形成。(DMSO为无药组，6周龄雄性C57BL/6,N 9,ApoE -/-小鼠经高脂饲养10周；MK为给药组：6周龄雄C57BL/6,ApoE -/-鼠经高脂饲养同时给药10周，图中出现红色的区域为显示动脉血管壁粥样硬化病变中的脂肪)。C图，主动脉弓冰冻切片油红欧染色，显示了MK显著减小了在主动脉弓处的粥样硬化程度。D图，主动脉弓冰冻切片HE染色，显示了MK显著减小了粥样硬化处间质层面积。 Figure 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. (Mouse and human vascular smooth muscle cells-SMC were incubated in 0.5 μmol of MK2206 medium for 4 hours, and cells incubated in MK2206-free medium were used as control-C. Protein and ribonucleic acid were extracted from the above cells. In IB and RT-PCR to detect YB-1 and MCP-1 mRNA content). 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.
E-H图，免疫组化染色，分别显示了MK显著减少了粥样硬化斑块中的MCP－1(E)，巨噬细胞(F)，及泡沫细胞(G)，并且显著减少了磷酸化的YB-1(H)。(棕红色为阳性染色区)。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).
图4.A图：显示V5抗体(购于Invitrogen/Life Technologies，Grand Island,NY)在免疫沉淀实验中能选择性沉淀带有V5寡肽标记物的YB－1突变蛋白；(YBX是野生型YB-1的同义突变；3dS是102丝氨酸及其上游两个相邻氨基酸的缺失型)，而野生型(WT)YB－1不含V5寡肽标记物，因此不能被V5抗体沉淀。Figure 4. Panel A: 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; (YBX is wild type Synonymous mutation of YB-1; 3dS is a deletion of 102 serine and two adjacent amino acids upstream thereof), while wild type (WT) YB-1 does not contain a V5 oligopeptide label and therefore cannot be precipitated by V5 antibody.
注：IP＝免疫沉淀，IB＝免疫印迹杂交，YB＝YB－1，用于免疫印迹杂交的蛋白提取物分别源于野生型动脉管壁平滑肌细胞及稳定转染了YBX和3dS的动脉管壁平滑肌细胞株。Note: 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.
B图：从上至下4幅图片分别为图i、ii、iii、iv；其中，图i显示UK114蛋白(UK)只能与YB－1(YB)一起被A图中的V5抗体从蛋白提取物中纯化出来(图i，IB：UK免疫印迹杂交)。而且，在免疫沉淀中被纯化出的含有UK和YB的沉淀物具有降解MCP－1mRNA的活性。逆转录－聚合酶链式反应(RT－PCR)结果显示免疫沉淀所得到的UK含量与免疫沉淀物的生物活性(降解MCP－1mRNA的功能)成正比(图ii)。向该免疫沉淀复合中加入UK抗体能阻断上述降解MCP－1mRNA的这一生物活性(图iii)，但对照PKCδ抗体则无阻断作用(图iv)。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). Moreover, 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 PKCδ antibody showed no blocking effect (Fig. iv).
注：Extract＝蛋白提取物，rhUK＝重组人源UK114Note: Extract=protein extract, rhUK=recombinant source UK114
图5.A图为YB－1突变子的碱基变化示意图。YB mut＝YB-1突变子。B图为逆转录－聚合酶链式(RT-PCR)反应结果：RNA来自YB－1突变子稳定转染的细胞株(未能获得S/A突变子的稳定转染细胞株)及野生型动脉平滑肌细胞。培养中的野生及含YB－1突变的细胞经10纳克/毫升PDGF(血小板源生长因子)孵育2小时，随后如图B选择性加入1微摩尔Dex(***)孵育2小时。源于以上细胞的RNA被用于RT－PCR。如前所述的YB－1，MCP－1，及YB－1突变子的特异性引物被用于RT－PCR。实验结果显示三种YB－1突变子均可被RT－PCR用特异性引物检出。三种突变子中只有3dS(丝氨酸缺失突变子)的MCP－1mRNA含量最低，而其他两个突变子的MCP－1mRNA与野生型没有显著差异。 经***处理的野生型细胞与3dS一样只含有痕迹量MCP－1mRNA。***能活化细胞中的磷酸化酶，后者使YB－1脱磷酸化。因为***也是通过下调YB－1磷酸化来下调MCP－1(见图6)，所以在该实验中被用作这一调控***的阳性对照(***虽能下调MCP－1，但临床应用表明它有严重的副作用而不能长期使用)。该实验结果证明了YB－1在其100/102位点的磷酸化在调控细胞中的MCP－1水平过程中所扮演的重要角色。 Figure 5. 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. Specific primers for the YB-1, MCP-1, and YB-1 mutants as described above were used for RT-PCR. The experimental results show that all three YB-1 mutants can be detected by RT-PCR with specific primers. Among the three mutants, only the 3dS (serine deletion mutant) had the lowest MCP-1 mRNA content, while the other two mutants had no significant difference in MCP-1 mRNA from the wild type. Dexamethasone-treated wild-type cells contained only trace amounts of MCP-1 mRNA as did 3dS. Dexamethasone activates phosphorylase in cells, which dephosphorylate YB-1. Because 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.
图6.筛选***下调MCP－1作用的信号传导路。A图的RT–PCR结果：显示***(Dex)显著下调MCP－1mRNA(见第2泳道)，而且该作用对所用的6种蛋白激酶抑制剂(详见表1)不敏感(见3至8泳道)。Figure 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).
B图的RT–PCR结果：显示***(Dex)显著下调MCP－1mRNA(见第2泳道)，而且该作用能被一种磷酸化酶抑制剂(Cal)所有效阻断(6泳道)，但对所用的其他5种磷酸化酶抑制剂(详见表1)不敏感(见3，4，5，7，8泳道)。该实验预示***通过磷酸化酶下调MCP－1。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.
C图的免疫印迹杂交结果：用YB－1和磷酸化YB－1的特异性抗体，通过免疫印迹杂交发现***显著下调磷酸化的YB－1(p-YB)水平(第2泳道)。Immunoblot hybridization results of panel C: Dexamethasone significantly down-regulated phosphorylated YB-1 (p-YB) levels by immunoblot hybridization with YB-1 and phosphorylated YB-1 specific antibodies (lane 2) .
具体实施方式Detailed ways
下面通过具体实施例进一步对本发明的内容进行详细描述，但并不以此限制本发明的保护范围。如无特殊说明，下述实施例中使用的耗材均可商购获得；操作步骤均为常规操作。The content of the present invention is further described in detail below by way of specific examples, but does not limit the scope of the present invention. The consumables used in the following examples are commercially available unless otherwise stated; the procedures are routine.
生物材料的来源Source of biological materials
本发明实施例和/或实验例中所使用的人体动脉组织源于北京协和医院的临床样本；ApoE敲除鼠(C57BL/6,ApoE-/-)、6周龄雄性C57BL/6,ApoE-/-小鼠均购自Vital River,China(北京维通利华实验动物技术有限公司)；动脉管壁平滑肌细胞株可商购获得。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.
试剂与耗材Reagents and consumables
OCT复合物(Tissue-Tek,IL1-9302)购自Tissue-Tek公司；OCT complex (Tissue-Tek, IL1-9302) was purchased from Tissue-Tek;
YB-1抗体(Y0396)购自sigma公司；二抗(PV-6001)购于中杉金桥公司；AEC(AEC-0037)购自迈新生物公司；油红O染色试剂盒(ab150678)购自美国abcam公司(www.abcam.com)。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).
实验例3使用的总RNA提取试剂盒(RNeasy试剂盒)购自Qiagen Inc,Valencia,CA。The total RNA extraction kit (RNeasy kit) used in Experimental Example 3 was purchased from Qiagen Inc, Valencia, CA.
实验例4中使用的PKCδ抗体购于SigmaAldrich公司；抗YB1抗体(Y0396)购于Sigma Aldrich公司；人UK重组蛋白(rhUK,H00010247-P01)及其抗体(00010247-M01)购于Abnova(Littleton,CO)；V5抗体购于Invitrogen/Life Technologies，Grand Island,NY。The PKCδ 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.
实验例5中使用的***可商购获得。Dexamethasone used in Experimental Example 5 is commercially available.
第1组实施例：本发明的药物 Group 1 Example: Drugs of the Invention
本组实施例提供一种用于治疗单核细胞趋化蛋白1参与的疾病的药物。在本组所有的实施例中，所述药物都具有如下共同特征：所述药物以YB-1为药物靶点；且所述药物的活性成分包括可直接或间接调控YB-1磷酸化的物质。The present group of embodiments provides a medicament for treating a disease in which monocyte chemoattractant protein 1 is involved. In all of the embodiments of the present group, 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 .
本发明所提供的药物在治疗单核细胞趋化蛋白1参与的疾病的新的抗病机理如下：YB-1通过自身的磷酸化选择性降低细胞内核糖核酸(mRNA)稳定性来调控单核细胞趋化蛋白－1(monocyte chemotacticprotein-1:MCP-1)的表达。YB－1对调控MCP-1的mRNA稳定性起主要作用。细胞内非磷酸化YB－1的温和性增加(产生于YB－1的脱磷酸化增强或磷酸化减弱，或二者的协同作用)即可显著抑制MCP－1的表达，进而抑制单核细胞在局部组织中的集聚，最终达到抑制，减轻，或预防由单核细胞趋化蛋白1参与的疾病。因而，调控YB－1磷酸化能有效调控MCP－1进而控制以由MCP－1失调引起的疾病。本发明用转基因小鼠的动脉粥样硬化模型证实了该抗病机理的作用。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. Thus, 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.
在具体的一些实施例中，本文所提及的“单核细胞趋化蛋白1参与的疾病”的含义为：本领域技术人员根据本发明的申请日(优先权日)之前公布的现有技术中所记载的发病机理和发病过程中有单核细胞趋化蛋白1参与的各类疾病，具体地，包括：炎症、动脉粥样硬化、2型糖尿病，肥胖，自身免疫病，肿瘤，及脑病(痴呆，癫痫等)；In some specific embodiments, 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. Among the pathogenesis and pathogenesis described in the disease, there are various diseases in which monocyte chemoattractant protein 1 is involved, specifically: inflammation, atherosclerosis, type 2 diabetes, obesity, autoimmune disease, tumor, and encephalopathy. (dementia, epilepsy, etc.);
而本文记载的上述各疾病类型“炎症、动脉粥样硬化、2型糖尿病，肥胖，自身免疫病，肿瘤，及脑病(痴呆，癫痫等)”，它们各自的含义与上述各疾病领域的普通技术人员所理解的通常含义和范围一致。The above-mentioned various types of diseases described herein are "inflammation, atherosclerosis, type 2 diabetes, obesity, autoimmune diseases, tumors, and encephalopathy (dementia, epilepsy, etc.)", and their respective meanings are common to the above-mentioned various diseases. The usual meaning and scope understood by the person is the same.
在本组实施例的另一些方案中，具体地，所述药物的活性成分包括直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质；In other aspects of this group of embodiments, in particular, 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;
在进一步的实施例中，所述直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质包括YB-1磷酸化抑制剂。本发明在研究调控MCP－1的表达机理的过程中原创性地发现了YB－1磷酸化在调控MCP-1表达中的作用，以及YB－1磷酸化的抑制剂对MCP－1表达的抑制作用。In a further embodiment, 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.
一些实施例中，所述YB-1磷酸化抑制剂选自由下述物质中的任一种或任几种组成的组：可促使YB-1脱磷酸化的物质、阻断或延缓YB-1磷酸化的物质、下调或抑制YB－1的蛋白激酶的物质、上调或激发YB－1磷酸化酶的物质。尤其是那些高选择性直接导致YB－1脱磷酸化或高选择性直接阻断YB－1磷酸化的物质，以及高选择性下调YB－1的蛋白激酶(kinase)活性的物质及其高选择性上调YB－1的磷酸化酶(phosphatase)活性的物质。In some embodiments, 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. In particular, 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.
与所述YB-1磷酸化抑制剂相对的是YB-1磷酸化增强剂；Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer;
所述YB-1磷酸化增强剂选自由下述物质中的任一种或任几种组成的组：阻断或延缓YB-1脱磷酸化 的物质、上调或激发YB-1磷酸化的物质、上调或激发YB－1的蛋白激酶活性的物质、抑制或下调YB－1磷酸化酶活性的物质。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.
因此，所述YB-1磷酸化抑制剂还可以是：与上述YB-1磷酸化增强剂起相反作用的物质，和/或，YB-1去磷酸化增强剂。Therefore, 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.
在上述实施例进一步的方案中，所述YB-1磷酸化抑制剂其中一个具体的实例为如式I所示的物质：In a further aspect of the above embodiment, one specific example of the YB-1 phosphorylation inhibitor is a substance as shown in Formula I:
Figure PCTCN2018080675-appb-000003
Figure PCTCN2018080675-appb-000003
上述物质是本领域技术人员根据本发明的记载及上述式I可以通过人工合成得到的，或者，可以通过商购获得；上述式I所述物质在本文中也被称为MK或MK2206。The above substances are known to those skilled in the art according to the present invention and the above formula I can be obtained by artificial synthesis, or can be obtained commercially; the substance of the above formula I is also referred to herein as MK or MK2206.
除了上述式I以外，所述YB-1磷酸化抑制剂还可以是目前临床应用的药物：***；***可以通过提高尚未被确定的磷酸化酶的活性来使YB－1脱磷酸化，从而减少细胞中的MCP－1。局部组织的MCP－1浓度是决定局部组织中单核/巨噬细胞数量的主要因素。小鼠动脉粥样硬化模型显示MCP－1缺陷可使动脉粥样硬化程度下降67％左右。In addition to the above 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. To reduce MCP-1 in cells. The local tissue MCP-1 concentration is the main factor determining the number of mononuclear/macrophage cells in local tissues. The mouse atherosclerosis model showed that MCP-1 deficiency reduced the degree of atherosclerosis by about 67%.
所述YB-1磷酸化抑制剂的另一个具体实例为YB-1突变蛋白；所述YB-1突变蛋白指，将野生型YB-1蛋白的氨基酸序列102位点的丝氨酸突变后所得的突变蛋白。由于丝氨酸含羟基，是常见的磷酸化位点。去除或替换该位点的丝氨酸会使YB－1失去在该位点被磷酸化的分子基础，所以该位点不再能被磷酸化。上述基于102位点丝氨酸的YB-1突变蛋白在细胞内有下调MCP－1的功能。此外，含102丝氨酸的YB－1多肽失去天然YB－1的功能，但能与天然YB－1竞争蛋白激酶，进而减少天然YB－1被磷酸化的概率。因此，本文中，这类突变的YB－1也被归属于YB－1磷酸化抑制剂，落入本发明YB－1磷酸化抑制剂的范畴。Another specific example of the 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. In addition, the 102-serine-containing YB-1 polypeptide loses the function of native YB-1, but competes with native YB-1 for protein kinases, thereby reducing the probability of natural YB-1 being phosphorylated. Thus, herein, such 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.
在一些实施例中，所述药物的活性成分还包括所述YB-1磷酸化抑制剂的聚合物，和/或，所述YB-1磷酸化抑制剂在药学上可接受的盐类化合物；和/或，酯类化合物；和/或；增效类化合物。本领域技术人员，基于本发明记载的内容，可根据实际需求，例如，药物制作成本、药物剂型、原料获取便捷程度、药物除药效以为的其它性质(比如，稳定性、在体内的崩解性能等)，选择适当的、能达到等同或相似药效的式I物质的衍生类物质。In some embodiments, 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. Those skilled in the art, based on the content described in the present invention, 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.
在另一些实施例中，所述的药物还包括药学上可接受的辅料和/或载体。本领域技术人员，基于本发明 记载的内容，可根据实际需求，例如，药物制作成本、药物剂型、原料获取便捷程度、药物除药效以为的其它性质(比如，稳定性、在体内的崩解性能、药物的保质期限等)，选择适当的辅料，具体地，包括赋形剂，载体，溶剂，稀释剂，及用以在体内携带，转运该药的包裹材料。这些材料的实例诸如：糖，纤维素及其衍生物,西黄蓍蚀粉，滑石粉，明胶,油，醇类，琼脂，缓冲剂，二乙酯，乳化物，润滑剂，无热源水，藻酸，调色及调味剂，防腐剂，乳化剂，湿剂，润滑剂，抗氧化剂，缓释剂，及其他制药配方中所使用的相应物质。In other embodiments, the medicament further comprises a pharmaceutically acceptable adjuvant and/or carrier. Those skilled in the art, based on the content described in the present invention, 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. 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.
第2组实施例：本发明药物的筛选方法Group 2 Example: Screening method of the drug of the present invention
本组实施例提供了第1组实施例所提供的用于治疗单核细胞趋化蛋白1参与的疾病的药物的筛选方法。本组所有的实施例都具有如下共同特征：采用可直接或间接调控YB-1磷酸化的物质做为候选药物进行病理实验，和/或，临床试验，和/或，治疗，筛选出具有效果，和/或产生疗效的物质用作所述药物的活性成分。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.
本组其中的一些实施例中，所述可调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；In some of the embodiments of the present invention, the substance that modulates YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor;
本组另一些实施例中，所述YB-1磷酸化抑制剂优选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶的物质，和/或，上调或激发YB－1磷酸化酶的物质。In other embodiments of the invention, 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.
与所述YB-1磷酸化抑制剂相对的是YB-1磷酸化增强剂；Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer;
所述YB-1磷酸化增强剂选自由下述物质中的任一种或任几种组成的组：阻断或延缓YB-1脱磷酸化的物质、上调或激发YB-1磷酸化的物质、上调或激发YB－1的蛋白激酶活性的物质、抑制或下调YB－1磷酸化酶活性的物质。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.
因此，所述YB-1磷酸化抑制剂还可以是：与上述YB-1磷酸化增强剂起相反作用的物质，和/或，YB-1去磷酸化增强剂。Therefore, 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.
具体地，筛选方法的具体操作可以按如下步骤进行：用某一具体A物质去处理动脉粥样硬化模型小鼠，并进行病理切片观察(例如，油红欧染色，或HE染色)；同时采用免疫印迹杂交(IB)及反转录-聚合酶链式反应(RT-PCR)检测该A物质在分子水平和蛋白水平对MCP-1的影响。Specifically, 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.
如果A物质处理动脉粥样硬化模型小鼠的治疗结果的病理切片与对照小鼠的切片相比较，出现与图3的B图-H图类似的结果；同时，A物质对MCP-1与YB-1磷酸化的IB与RT-PCR结果出现与图3的A图、B图类似的结果，即可确定A物质可作为本发明的候选药物之一。If the pathological section of the treatment results of the A substance-treated atherosclerotic model mouse is compared with the section of the control mouse, a result similar to the B-H diagram of FIG. 3 appears; at the same time, substance A pairs MCP-1 and YB. The results of -1 phosphorylated IB and RT-PCR appear similar to those of Fig. 3 and Fig. 3, and it can be confirmed that substance A can be one of the drug candidates of the present invention.
更具体的操作步骤详见实验例1-6的详细记载。More specific procedures are detailed in the description of Experimental Examples 1-6.
第3组实施例：本发明药物的制备方法Group 3 Example: Method for preparing the drug of the present invention
本组实施例提供了可下调YB-1磷酸化的物质在制备用于治疗单核细胞趋化蛋白1参与的疾病的药物方面的用途；或，第1组实施例所述的用于治疗单核细胞趋化蛋白1参与的疾病的药物的制备方法。本组所有的实施例都具有如下共同特征：将所述可下调YB-1磷酸化的物质放置在标有单核细胞趋化蛋白1参与的疾病治疗用途的商品包装盒内；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;
在本组优选的实施例中，所述可结合、激活、和/或上调YB-1的物质包括YB-1磷酸化抑制剂；In a preferred embodiment of the invention, the substance that binds, activates, and/or upregulates YB-1 comprises a YB-1 phosphorylation inhibitor;
在更进一步的实施例中，所述YB-1磷酸化抑制剂选自：可使YB-1脱磷酸化的物质，和/或，阻断YB-1磷酸化的物质，和/或，下调YB－1的蛋白激酶的物质，和/或上调YB－1磷酸化酶的物质。In still further embodiments, 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.
第4组实施例：本发明药物的使用方法Group 4 Example: Method of using the drug of the present invention
本组实施例提供第1组实施例所述药物，和/或第2组实施例筛选方法筛选得到的药物，和/或第3组实施例的制备方法制备得到的药物的使用方法。本组所有的实施例都具有如下特征：可直接或间接调控YB-1磷酸化的物质在预防和/或治疗由MCP－1参与的疾病方面的用途。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.
一些实施例中，具体提供YB-1磷酸化的抑制剂在临床医疗中的适应症及其使用方法；In some embodiments, specific indications for the inhibition of YB-1 phosphorylation in clinical practice and methods of use thereof are provided;
另一些实施例中，所述使用方法包括，所述药物的临床应用，即，包括对上述适应症的个体使用该类物质以治疗，减轻，预防上述适应症。In other embodiments, 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.
在进一步的实施例中，所述药物的临床应用包括该发明所指物质的临床使用剂型和剂量。In a further embodiment, the clinical use of the medicament comprises the clinically used dosage form and dosage of the substance to which the invention refers.
实验例1、人动脉免疫组化染色试验Experimental Example 1. Human arterial immunohistochemical staining test
将人新鲜动脉组织包埋于OCT复合物中(Tissue-Tek,IL1-9302),液氮速冷后的冰冻切片保存于－80度。对6微米厚的切片进行标准的免疫组化染色。所述免疫组化染色方法的操作参考“cellsignal公司”官网公布的“Immunohistochemistry Protocol(Frozen)”操作方法(网址：https://www.cst-c.com.cn/contents/resources-protocols/immunohistochemistry-protocol-(frozen)/ihc-frozen)。另外，本实验采用羊血清(中杉金桥，ZLI-9056)封闭非特异性背景。一抗分别为抗YB－1抗体(sigma,Y0396)和抗磷酸化YB－1抗体(Cell Signaling,C34A2)，用PBS稀释(1:100)后与切片组织在4度孵育过夜。二抗购于中杉金桥(PV-6001)。AEC(迈新试剂AEC-0037)用于显色，红色即为阳性区。Human fresh arterial tissue was embedded in OCT complex (Tissue-Tek, IL1-9302), and frozen sections after liquid nitrogen rapid cooling were stored at -80 degrees. Standard immunohistochemical staining was performed on 6 micron thick sections. The operation of the immunohistochemical staining method refers to the "Immunohistochemistry Protocol (Frozen)" operation method published on the official website of "cellsignal" (website: https://www.cst-c.com.cn/contents/resources-protocols/immunohistochemistry -protocol-(frozen)/ihc-frozen). In addition, this experiment used sheep serum (Zhushan Jinqiao, ZLI-9056) to block the non-specific background. 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 (Axin reagent AEC-0037) is used for color development, and red is a positive region.
实验结果显示YB-1和磷酸化YB-1在人体动脉粥样硬化组织(AS)内的高表达，而在正常动脉管壁(Normal)中的表达量极低。The experimental results show that 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.
实验例2、小鼠动脉油红O染色试验Experimental Example 2: Mouse arterial oil red O staining test
对本领域常用的动脉粥样硬化模型小鼠采用本发明第1组实施例任一所述的药物进行治疗，给药方式：腹腔注射85微克/天；对照组小鼠每天采用等量的DMSO；给药70天后，对治疗组的小鼠和对照组的小 鼠的动脉组织分别采用油红O染色试剂盒进行染色，其染色方法为本领域显示冷冻切片组织中脂肪的最常用的标准方法，具体操作步骤按照购自美国abcam公司(www.abcam.com)的油红O染色试剂盒(ab150678)的产品说明书。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 μg/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).
试验结果显示：如图2所示，与对照组(DMSO)相比，显示MK显著抑制了动脉粥样硬化的形成。红色区域表示粥样硬化斑块中的脂肪。The test results showed that, as shown in Fig. 2, MK was significantly inhibited from the formation of atherosclerosis as compared with the control group (DMSO). The red area indicates the fat in the atherosclerotic plaque.
本文中的“动脉粥样硬化模型小鼠”、“模型小鼠”指经高脂饲养10周后的C57BL/6,N9，ApoE -/-小鼠。 "Atherosclerosis model mouse" and "model mouse" herein refer to C57BL/6, N9, ApoE -/- mice after 10 weeks of high fat feeding.
实验例3、本发明药物防治动脉粥样硬化的效果Experimental Example 3, Effect of the medicament of the present invention for preventing and treating atherosclerosis
首先采用免疫印迹杂交(IB)及反转录-聚合酶链式反应(RT-PCR)检测MK在分子水平和蛋白水平对MCP-1的影响；具体操作为；小鼠及人血管平滑肌细胞(mSMC,hSMC)在含0.5微摩尔的MK2206培养液中孵育4小时，在无MK2206培养液中孵育的细胞做为对照(C)。从上述细胞中提取蛋白及核糖核酸，用于IB及RT-PCR以检测其中的YB-1和MCP-1mRNA含量；IB及RT-PCR的操作方法按本领域技术人员熟知的常规操作进行。其中逆转录－聚合酶链式反应(RT-PCR)的具体操作如下：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 μmol 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:
采用的引物分别为跨越MCP－1的145-595核酸编码区段及GAPDH的340-625核酸编码区段；引物长度均为20个碱基(大鼠MCP-1和GAPDH的核酸编码区序列可登录Genebank查询获知)。PCR产物分别是单核细胞趋化蛋白cDNA序列的145-595；GAPDH的340-625；YB-1的420-988；YB－1突变子的441-988。其中，YB－1突变子的正向引物为TGTGGAATTCGACGTCGTC,对应于野生型序TGTGAGTTTGATGTTGTT。除非另做说明，上述引物适用于本文的所有RT－PCR。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. Among them, 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.
总RNA(Total RNA)用RNeasy试剂盒(Qiagen Inc,Valencia,CA)从大鼠细胞提取。大鼠平滑肌细胞分离及培养见Brock,1985,Hypertension，操作大体如下：200-300克雄性Sprague-Dawley大鼠的胸中动脉中的平滑肌细胞经酶解法分离获得。所有实验采用5-14代的培养细胞。细胞培养于含10％小牛血清的Dulbecco modified Eagle medium(DMEM；Gibco Laboratories,Gaithersburg,Md.)培养液中，于37度二氧化碳孵箱孵育。当培养皿中的细胞达到大约60％满时，细胞被用于制备RNA。细胞转染按Lipofectamine2000试剂制造商Ivitrogen的操作方法,Lipofectamine2000购于Invitrogen。转染时的细胞密度约为30％。每个逆转录－聚合酶链式反应使用200纳克RNA。反应体系采用Masterscript RT-PCR system(5 PRIME Inc.,Gaithersbueg,MD)。RT-PCR反应程序为:54℃30分钟；94℃2分钟；以“94℃22秒,55℃22秒,73℃44秒”为1个循环，共进行27个循环；73℃6分钟。除非另做说明，以上参数适用于本文所有RT－PCR反应。Total RNA (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. Cell 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.
如无特殊说明，本文中的“YB－1突变子”一般指：所述“YB－1突变蛋白”对应的核苷酸序列。Unless otherwise specified, "YB-1 mutant" herein generally refers to a nucleotide sequence corresponding to the "YB-1 mutein".
检测结果如图3的A图所示:免疫印迹杂交(IB)及反转录-聚合酶链式反应(RT-PCR)，显示MK2206(MK，YB-1的蛋白激酶特异性抑制剂)能高效抑制血管平滑肌和单核细胞内的YB-1磷酸化及 MCP-1mRNA水平。The results are shown in Figure 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.
对动脉粥样硬化模型小鼠采用本发明第1组实施例任一所述的药物进行治疗，给药方式：对实验鼠高脂喂养的同时，腹腔注射85微克/天(约为小鼠模型中***剂量的1/13。见Hirai等人发表的 Mol Cancer Ther.2010Jul；9(7):1956-67.)；对照组小鼠每天采用等量的DMSO代替本发明的药物；给药10周后，对治疗组的小鼠和对照组的小鼠的动脉组织分别采用油红O染色和动脉HE染色，所述油红O染色的具体操作如实验例2所述；动脉HE染色指HE(苏木精－伊红)染色方法，其具体操作参照“protocolsonline”官网公布的“Haematoxylin Eosin(H&E)staining|Protocols Online”操作方法(官方网址： 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 μg/day for the experimental rats with high fat feeding (about a mouse model) 1/13 of the therapeutic tumor dose. See Mol Cancer Ther. 2010 Jul; 9(7): 1956-67.) by Hirai et al.; control mice used an equal amount of DMSO instead of the drug of the present invention every day; After 10 weeks, 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|Protocols Online" operation method published on the "protocolsonline" official website (official website:
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi？ID＝2503)。HE染色是本领域最常用的标准组织学染色方法以显示组织的形态和结构(苏木精为深蓝或紫色，与核酸结合。伊红呈粉色，与氨基酸结合)；本组试验的治疗效果如图3的B－D图所示：模型雄鼠主动脉油红O(Oil Red O)染色，显示MK2206高效抑制了模型雄鼠动脉粥样硬化的形成。(DMSO为无药组，6周龄雄性C57BL/6,ApoE -/-小鼠经高脂饲养10周；MK为给药组：6周龄雄C57BL/6,ApoE -/-鼠经高脂饲养同时给药10周，图中出现红色的区域为显示动脉血管壁粥样硬化病变中的脂肪)。C图，主动脉弓油红欧染色，显示了MK显著减小了在主动脉弓处的粥样硬化程度。D图，动脉HE染色，显示了MK显著减小了粥样硬化处间质层面积。 Http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi? ID=2503). 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. (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 The animals were given 10 weeks of simultaneous administration, and the red areas in the figure showed the fat in the atherosclerotic lesions of the arterial wall. 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.
同时，本实验例还对本发明的药物降低炎症、减少炎性细胞方面的试验，对照组和给药组动物的治疗给药过程如上一段所描述，治疗结束后对对照组和给药组小鼠的动脉组织分别进行免疫组化染色，方法同实验例1；染色结果如图3的E-H图，免疫组化染色，分别显示了MK显著减少了粥样硬化斑块中的MCP－1(E)，巨噬细胞(F)，及泡沫细胞(G)，并且显著减少了磷酸化的YB-1(H)。(棕红色为阳性染色区)。Meanwhile, 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).
动脉粥样硬化是由MCP－1介导的动脉血管壁的炎症。上述实验例以动脉粥样硬化为模型，验证了YB－1在炎症病理过程中的作用，以及通过用本发明的药物调控YB－1进而调控体内MCP－1水平所产生的抗炎效果。根据本发明药物的作用机理，本发明药物同样也能用于治疗由MCP-1介导发生的2型糖尿病，肿瘤，自身免疫病，肥胖、及脑病(痴呆，癫痫等)等病症，并能预期得到与本文实验例2和3类似的治疗效果。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. According to the mechanism of action of the medicament of the present invention, 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.
实验例4、YB-1突变蛋白抑制MCP-1活性验证Experimental Example 4, YB-1 mutein inhibits MCP-1 activity verification
首先通过免疫沉淀试验，采用V5抗体对带有V5寡肽标记物的YB-1突变蛋白进行选择性沉淀。具体操作如下：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:
YB-1突变蛋白通过如下方式获得：采用如实验例3所述的RT-PCR的具体操作(包括采用YB-1突变子引物、反应程序等)，从而使大鼠YB-1(GEnBank accession no.NM031563)跨越起始和终止密码区的cDNA经逆转录－聚合酶链式反应获得，并克隆于pBluescript II KS(+)质粒的HindIII和XbaI之间。所 有YB－1突变蛋白都采用聚合酶链式反应(PCR)生成或其它人工序列合成得到。这些YB－1突变子被亚克隆于pcDNA3.1/V5-His A质粒的Hind III和XbaI位点之间.起始密码上游都引入了Kozak sequence,GCCACC。YB－1 3‘端的终止密码被Xba I位点取代。质粒中所含的V5-His标记肽及终止密码按编码顺序紧随Xba I位点之后。克隆的PCR终产物都经DNA测序证实。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. These 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.
带有V5寡肽标记物的YB－1突变蛋白通过如下方式获得：pcDNA3.1/V5-His A载体质粒带有V5标记肽的DNA序列，将不带终止密码的YB-1突变体入框(in frame)克隆到V5的上游之后就得到杂交质粒。这种杂交质粒在细胞内能表达带有V5的YB-1突变蛋白。这种杂合的突变蛋白能与V5抗体特异性结合。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.
如实验例3所述的YB－1，MCP－1，及YB－1突变子的特异性引物以及RT-PCR反应程序被用于获得对应图5的B图所示的RT－PCR结果的RT-PCR试验中。The specific primers for the YB-1, MCP-1, and YB-1 mutants as described in Experimental Example 3 and the RT-PCR reaction program were used to obtain RT corresponding to the RT-PCR results shown in Figure B of Figure 5. - PCR test.
除非另做说明，本文中免疫沉淀、免疫印迹杂交的操作步骤均如下所示：每个样品使用30微升G蛋白珠悬液(Roche,Germany)。用预冷的S100缓冲液(10mM Tris[pH 7.4],1.5mM MgCl2,150mM KCl,0.5mM DTT,0.5mM PMSF)洗G蛋白珠3次。再用3微升V5-抗体与G蛋白珠在4℃孵育1小时,随后加1微升20％牛血清白蛋白(BSA Fraction V from Sigma Aldrich)继续孵育1小时。用预冷的S100缓冲液洗V5抗体包被好的G蛋白珠一次，然后用150微升冷S100缓冲液重悬于0.5毫升Eppendorf管中冰浴。加入30-50微升(10微克蛋白)胞浆抽提物(抽提方法参考Poon M,Liu B,Taubman MB.1999.Identification of a novel dexamethasone-sensitive RNA-destabilizing region on rat monocyte chemoattractantprotein 1mRNA.Molecular and cellular biology 19:6471-6478)于4℃共孵育1小时(含有重组人类UK蛋白的样品，需要将50微升胞浆抽提物于1微升重组人UK蛋白于室温孵育5分钟，再与V5抗体包被的G蛋白珠孵育)。用110xg离心免疫沉淀反应物1分钟以收集G蛋白珠,用S100缓冲液洗所得G蛋白珠2次,重悬于30微升S100缓冲液以备用于RNA降解实验(5微升＝1微克RNA与5微升G蛋白珠重悬液在室温共孵育5分钟，取2微升上清液用于20微升的逆转录－聚合酶链式反应中)，或30微升上样缓冲液(22μl RIPA buffer plus 8μl 4×loading dye:160mM Tris pH 6.8,4％SDS,40％Glycerol,575mM β-mecapital ethanol,20μl 2％Xylene Cyanol),继而用于免疫印迹杂交实验：95℃5分钟，洗脱液用于4-20％ SDS-PAGE(聚丙烯酰胺凝胶电泳)，电泳完成之后将凝胶中的蛋白转移到PVDF膜上，再用特异性抗体检测目的蛋白。Unless otherwise stated, 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 μl of V5-antibody was incubated with G-protein beads for 1 hour at 4 ° C, followed by incubation with 1 μl of 20% bovine serum albumin (BSA Fraction V from Sigma Aldrich) for 1 hour. The 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 μl (10 μg 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. Molecular And cellular biology 19:6471-6478) Incubate for 1 hour at 4 °C (samples containing recombinant human UK protein, 50 μl of cytosolic extract was incubated in 1 μl of recombinant human UK protein for 5 minutes at room temperature, then Incubate with V5 antibody-coated G protein beads). The immunoprecipitated reaction was centrifuged at 110 xg for 1 minute to collect G protein beads, and the resulting G protein beads were washed twice with S100 buffer and resuspended in 30 μl of S100 buffer for RNA degradation experiments (5 μL = 1 μg RNA). Incubate with 5 μl of G protein bead suspension for 5 minutes at room temperature, 2 μl of supernatant for 20 μl of reverse transcription-polymerase chain reaction, or 30 μl of loading buffer ( 22 μl RIPA buffer plus 8 μl 4×loading dye: 160 mM Tris pH 6.8, 4% SDS, 40% Glycerol, 575 mM β-mecapital ethanol, 20 μl 2% Xylene Cyanol), followed by immunoblot hybridization experiments: 95 ° C for 5 minutes, wash The deliquoring is used for 4-20% SDS-PAGE (polyacrylamide gel electrophoresis). After electrophoresis, the protein in the gel is transferred to the PVDF membrane, and the target protein is detected by a specific antibody.
试验结果分别如图4和图5所示。图4的B图中的图iv中使用的对照PKCδ抗体是一种蛋白激酶，该抗体特异性结合人、小鼠、和大鼠的这种蛋白激酶，该抗体对MCP-1mRNA降解活性无阻断作用；YB－1的102位点的丝氨酸相当于大鼠100位点的丝氨酸。在大鼠YB－1中，该丝氨酸的缺失导致YB－1不能在该位点被磷酸化。该位点无磷酸化的YB－1与UK114(一种核糖核酸酶)和GR(糖皮质激素受体)结合成活性复合物，该活性复合物选择性降解MCP－1mRNA。在该位点磷酸化了的YB－1不利于结合 UK114。The test results are shown in Figures 4 and 5, respectively. The control PKCδ 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. In 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.
图5中的三种突变子中只有3dS(丝氨酸缺失突变子)的MCP－1mRNA含量最低，而其他两个突变子的MCP－1mRNA与野生型没有显著差异，该实验结果证明了YB－1在其100/102位点的磷酸化在调控细胞中的MCP－1水平过程中所扮演的重要角色。Among the three mutants in Figure 5, only 3dS (serine-deficient mutant) had the lowest MCP-1 mRNA content, while the other two mutants had no significant difference in MCP-1 mRNA from the wild type. The experimental results confirmed that YB-1 was Phosphorylation at its 100/102 site plays an important role in regulating MCP-1 levels in cells.
实验例5、***抑制MCP-1活性以及下调YB-1磷酸化的验证Experimental Example 5: Verification of inhibition of MCP-1 activity by dexamethasone and down-regulation of YB-1 phosphorylation
本实验例也是通过RT-PCR试验验证了***也是通过下调YB－1磷酸化来下调MCP－1。This experimental example also verified by RT-PCR that dexamethasone also down-regulated MCP-1 by down-regulating YB-1 phosphorylation.
RT-PCR试验中使用的引物和具体实验操作步骤如实验例3所述。而试验中使用的其他6种磷酸化酶抑制剂的信息详见下表1：The primers used in the RT-PCR assay and the specific experimental procedures were as described in Experimental Example 3. The information on the other six phosphorylase inhibitors used in the test is shown in Table 1 below:
表1.用于筛选介导***下调MCP－1作用的信号传导路所使用的蛋白激酶和磷酸化酶抑制剂，以及这些试剂的使用浓度，靶分子，和抑制效果Table 1. Protein kinases and phosphorylase inhibitors used to screen signaling pathways that mediate the down-regulation of MCP-1 by dexamethasone, and the concentrations, target molecules, and inhibitory effects of these agents
Figure PCTCN2018080675-appb-000004
Figure PCTCN2018080675-appb-000004
注：上表中的浓度指各抑制剂在细胞培养基中的终浓度；+:完全抑制；+/-:部分抑制；-:不抑制Note: The concentrations in the above table refer to the final concentration of each inhibitor in the cell culture medium; +: complete inhibition; +/-: partial inhibition; -: no inhibition
试验结果如图6所示，结果表明：***(Dex)显著下调MCP－1mRNA，且***通过磷酸化酶下调MCP－1。The results of the experiment are shown in Figure 6. The results showed that dexamethasone (Dex) significantly down-regulated MCP-1 mRNA, and dexamethasone down-regulated MCP-1 by phosphorylase.
实验例6、本发明药物的动物实验有效性统计Experimental Example 6, Quantitative Statistics of Animal Experiments of the Drugs of the Invention
采用24只模型小鼠，对照组和给药组各12只，按照如实验例3所示的给药方式和对照方式分别对给药组和对照组进行给药或等量DMSO，给药指对给药组的50只小鼠腹腔注射本发明第1组实施例任一实施例所述的药物；10周后，采用如实验例3所示的油红O(Oil Red O)染色和免疫组化染色分别对两组小鼠的动脉组织进行染色，最终得到实验例2-3的治疗结果(为节约篇幅，本文不再一一罗列相关的染色结果图和统计图表)，给药组的每只小鼠的动脉粥样硬化的形成显著得到抑制、粥样硬化程度显著减小、粥样硬化处间质层体积(面积和厚度)显著减小、炎性细胞显著减少、炎症得到显著缓解，因此，本发明的药物在动物试验中的有效性为100％。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. (To save space, this article will not list the relevant staining results and statistical charts.) The formation of atherosclerosis was significantly inhibited in each mouse, the degree of atherosclerosis was significantly reduced, the volume (area and thickness) of stromal layer was significantly reduced, the number of inflammatory cells was significantly reduced, and inflammation was significantly relieved. Therefore, the effectiveness of the medicament of the present invention in an animal test is 100%.

Claims (10)

  1. 用于治疗单核细胞趋化蛋白-1参与的疾病的药物，其特征在于，以YB-1为药物靶点；且所述药物的活性成分包括能直接或间接调控YB-1磷酸化的物质。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 .
  2. 根据权利要求1所述的药物，其特征在于，所述单核细胞趋化蛋白1参与的疾病包括：炎症、动脉粥样硬化、2型糖尿病，肿瘤，自身免疫病，肥胖、及脑病(痴呆，癫痫等)。The medicament according to claim 1, wherein the diseases in which monocyte chemoattractant protein 1 is involved include inflammation, atherosclerosis, type 2 diabetes, tumor, autoimmune disease, obesity, and encephalopathy (dementia) , epilepsy, etc.).
  3. 根据权利要求1或2所述的药物，其特征在于，所述药物的活性成分包括直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质；The medicament according to claim 1 or 2, wherein the active ingredient of the medicament comprises a substance which is directly or indirectly down-regulated, and/or which directly or indirectly inhibits phosphorylation of YB-1;
    进一步地，所述直接或间接下调，和/或，直接或间接抑制YB-1磷酸化的物质包括YB-1磷酸化抑制剂。Further, the substance that directly or indirectly downregulates, and/or directly or indirectly inhibits YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor.
  4. 根据权利要求3所述的药物，其特征在于，所述YB-1磷酸化抑制剂选自由下述物质中的任一种或任几种组成的组：促使YB-1脱磷酸化的物质、阻断或延缓YB-1磷酸化的物质、下调或抑制YB－1的蛋白激酶活性的物质、上调或激发YB－1磷酸化酶活性的物质；The drug according to claim 3, wherein 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, a substance that down-regulates or inhibits protein kinase activity of YB-1, a substance that up-regulates or stimulates YB-1 phosphorylase activity;
    与所述YB-1磷酸化抑制剂相对的是YB-1磷酸化增强剂；Opposite to the YB-1 phosphorylation inhibitor is a YB-1 phosphorylation enhancer;
    所述YB-1磷酸化增强剂选自由下述物质中的任一种或任几种组成的组：阻断或延缓YB-1脱磷酸化的物质、上调或激发YB-1磷酸化的物质、上调或激发YB－1的蛋白激酶活性的物质、抑制或下调YB－1磷酸化酶活性的物质。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.
  5. 根据权利要求3或4所述的药物，其特征在于，所述YB-1磷酸化抑制剂包括：如式I所示的物质：The medicament according to claim 3 or 4, wherein the YB-1 phosphorylation inhibitor comprises: a substance as shown in formula I:
    Figure PCTCN2018080675-appb-100001
    Figure PCTCN2018080675-appb-100001
    ***；和/或，Dexamethasone; and/or,
    YB-1突变蛋白；YB-1 mutant protein;
    所述YB-1突变蛋白指，野生型YB-1蛋白的氨基酸序列的102位点丝氨酸突变后得到的突变蛋白。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.
  6. 根据权利要求3-5任一所述的药物，其特征在于，所述药物的活性成分还包括所述YB-1磷酸化抑制剂的聚合物，和/或，所述YB-1磷酸化抑制剂在药学上可接受的盐类化合物；和/或，酯类化合物；和/或；增效类化合物。The medicament according to any one of claims 3 to 5, wherein the active ingredient of the medicament further comprises a polymer of the YB-1 phosphorylation inhibitor, and/or the YB-1 phosphorylation inhibition The agent is a pharmaceutically acceptable salt compound; and/or an ester compound; and/or a synergistic compound.
  7. 根据权利要求1-6任一所述的药物，其特征在于，还包括药学上可接受的辅料和/或载体。The medicament according to any one of claims 1 to 6, which further comprises a pharmaceutically acceptable adjuvant and/or carrier.
  8. 用于治疗单核细胞趋化蛋白1参与的疾病的药物的筛选方法，其特征在于，采用可直接或间接调控 YB-1磷酸化的物质做为候选药物进行病理实验，和/或，临床试验，和/或，治疗，筛选出具有效果，和/或产生疗效的物质用作所述药物的活性成分。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.
  9. 根据权利要求8所述的筛选方法，其特征在于，所述可直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；The screening method according to claim 8, wherein the substance capable of directly or indirectly regulating YB-1 phosphorylation comprises a YB-1 phosphorylation inhibitor;
    所述YB-1磷酸化抑制剂优选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。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 protein kinase active substance, and/or a substance that up-regulates or stimulates YB-1 phosphorylase activity.
  10. 可直接或间接调控YB-1磷酸化的物质在制备用于治疗单核细胞趋化蛋白1参与的疾病的药物方面的用途，其特征在于，将所述可直接或间接调控YB-1磷酸化的物质放置在标有单核细胞趋化蛋白1参与的疾病治疗用途的商品包装盒内；Use of 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;
    在优选范围内，所述可直接或间接调控YB-1磷酸化的物质包括YB-1磷酸化抑制剂；Within the preferred range, the substance that directly or indirectly regulates YB-1 phosphorylation includes a YB-1 phosphorylation inhibitor;
    在更进一步优选范围内，所述YB-1磷酸化抑制剂选自：可促使YB-1脱磷酸化的物质，和/或，阻断或延缓YB-1磷酸化的物质，和/或，下调或抑制YB－1的蛋白激酶活性的物质，和/或，上调或激发YB－1磷酸化酶活性的物质。In a still further preferred range, 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.
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