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WO2009108005A2 - Composition et procédé pour cribler une matière active destinée à prévenir et traiter le diabète ou l'obésité au moyen de la senp2 et composition pour prévenir et traiter le diabète et l'obésité - Google Patents

Composition et procédé pour cribler une matière active destinée à prévenir et traiter le diabète ou l'obésité au moyen de la senp2 et composition pour prévenir et traiter le diabète et l'obésité Download PDF

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WO2009108005A2
WO2009108005A2 PCT/KR2009/000949 KR2009000949W WO2009108005A2 WO 2009108005 A2 WO2009108005 A2 WO 2009108005A2 KR 2009000949 W KR2009000949 W KR 2009000949W WO 2009108005 A2 WO2009108005 A2 WO 2009108005A2
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senp2
composition
obesity
expression
diabetes
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WO2009108005A3 (fr
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박경수
정성수
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Seoul National University Industry Foundation
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    • 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
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates to a composition for screening an active substance for preventing or treating diabetes or obesity, a screening method using the composition and a composition for preventing or treating diabetes or obesity, and more specifically, SUMO (small-ubiquitin-like modifier) specific Inhibits the expression of a gene for encoding a SENP2 encoding a composition for screening a composition for screening an active substance for preventing or treating diabetes mellitus or obesity, including a protease 2 (SENP2) or a gene encoding SENP2 as a target substance,
  • the present invention relates to a composition for preventing or treating diabetes or obesity, including an agent that inhibits the activity of SENP2.
  • “SUMOylation” refers to the formation of a covalent bond with a target protein by isopeptide linkage between the C-terminal glycine of the small ubiquitin-related modifier (SUMO) and the lysine residues of the target protein. It is one of the posttranslational modifications of proteins.
  • a hair protein is a regulatory factor of about 11 Kda covalently bound to lysine residues of various proteins.
  • the hair hair binding site is defined as ⁇ KEX ( ⁇ is a hydrophobic amino acid).
  • Hydrocyclization affects the function of the protein of interest, and a large number of transtciption factors are covalently modified by small-ubiquitin-like modifiers (SUMOs), and their transcriptional activity is regulated by this hydrocyclization It is known that this can be a target of hydrolysis.
  • SUMOs small-ubiquitin-like modifiers
  • SENP1 SUMO specific proteases
  • SENP3 SENP5 are found in the nucleolus.
  • SENP2 was originally known as a nuclear membrane-associated protease and has recently been reported to be capable of nuclear-cytoplasmic shuttling.
  • SENP1 can increase androgen receptor-dependent transcription and regulate c-jun-dependent transcription through dehydration of p300 by unbinding hair from histone deacetylase 1 (HDAC1). SENP1 may also modulate the rehydration of leptin involved in cancer metastasis.
  • HDAC1 histone deacetylase 1
  • SUSP4 a newly identified SENP in mice, positively regulates p53 through dehydration of Mdm2. Dehydrogenation activity of SENP2 is involved in down-regulation of beta-catenin.
  • Adipose tissue is an excess of energy storage and secretes a variety of factors called adipocaine.
  • Adipocaine affects physiological and pathological events, including energy metabolism, insulin sensitivity, atheroogenesis and immune responses.
  • Differentiation of adipocytes into adipocytes is caused by the sequential induction of several transcription factors, and this process is tightly regulated.
  • PPAR ⁇ and C / EBP family members play an important role in adipocyte differentiation.
  • hormonal stimulation initiates sequential expression of transcription factors. Expression of C / EBP ⁇ and C / EBP ⁇ is induced immediately after stimulation, followed by the expression of PPAR ⁇ and C / EBPa.
  • PPAR ⁇ induces adipocyte specific genes, including aP2 and adiponectin, and C / EBPa works with PPAR ⁇ .
  • PPAR ⁇ is known to be a major regulator of adipocyte differentiation.
  • CAMP-response element-binding protein CREB is involved in the induction of C / EBP ⁇ and PPAR ⁇ , and may act as a proactive factor for mediating the induction of adipocyte transcription factors exposed to differentiation signals.
  • C / EBPa and C / EB ⁇ can be hydrophilized, and their transcriptional activity is negatively regulated by hydrocyclization.
  • PPAR ⁇ is a substrate for hair-binding, and overexpressing the PPAR ⁇ variant, which replaced lysine 107, a major hydrophilic site with alanine, has been shown to more effectively stimulate the lipogenesis process.
  • Dehydrogenation activity of SENP2 has been reported to negatively regulate the Wnt / ⁇ -catenin signaling pathway.
  • Wnt signaling is known to inhibit adiogenesis.
  • PPAR ⁇ peroxisome proliferator activated receptor ⁇
  • PPAR ⁇ peroxisome proliferator activated receptor ⁇
  • PPAR ⁇ is an important factor in cell signaling pathways and is known to regulate transcription factors related to glucose metabolism and fat cell formation.
  • PPAR ⁇ is expressed in the form of PPAR ⁇ 1 and PPAR ⁇ 2 in adipocytes.
  • PPAR ⁇ is most expressed in adipocytes and low in skeletal muscle, heart, liver, intestine, kidney, vascular endothelial and smooth muscle cells and macrophages.
  • PPAR ⁇ is known to play a multifaceted role in vascular cell proliferation, migration and differentiation, macrophage activation and inflammatory action (Hsueh WA, et al., Diabetes Care 24: 392, 2001; Law RE, et al., Circulation 101: 1311, 2000; Ricote M, et al., Nature 391: 79, 1998; Blanquart C, et al., J Steroid Biochem Mol Biol 85: 267-273, 2003; Benson S, et al., Am J Hypertens 13: 74-82, 2000).
  • PPAR ⁇ has been known to be associated with obesity, diabetes, carcinogenesis and atherosclerosis, and efforts have been made to develop PPAR ⁇ ligands (or PPAR ⁇ agonists) as agents for treating atherosclerosis.
  • PPAR ⁇ ligands or PPAR ⁇ agonists
  • rosiglitazone Choi D, et al., 2004; 27: 2654
  • pioglitazone pioglitazone
  • PPAR ⁇ ligand therapeutics have been developed as PPAR ⁇ ligand therapeutics.
  • PPAR ⁇ activation of the PPAR ⁇ gene is required in the target tissue, and if PPAR ⁇ itself can be activated without the regulation of the PPAR ⁇ ligand, it will be able to stably relieve diabetes without the harmful effects of the PPAR ⁇ ligand.
  • Korean Patent Registration No. 10-532242 discloses mU1pB and its gene, a new hair-progease in the mouse brain, and this hair-protease may be a key for the development of a treatment for cancer including acute leukemia.
  • this hair-protease may be a key for the development of a treatment for cancer including acute leukemia.
  • Japanese Patent Laid-Open No. 2001-224373 relates to a novel SUMO-1 specific protease that is highly expressed in human reproductive organs, and discloses that they may play an important role in the reproductive process. These documents do not disclose any relationship between SENP2 and PPAR ⁇ and the relationship between SENP2 and diabetes or obesity-related diseases.
  • SENP2 promotes dehydration of several transcription factors such as C / EB ⁇ , C / EBPa and PPAR ⁇ and expression of SENP2 in adipocyte differentiation.
  • C / EB ⁇ C / EBPa
  • PPAR ⁇ PPAR ⁇
  • SUMO small-ubiquitin-like modifier
  • SENP2 specific protease 2
  • Another object of the present invention is to provide a method for screening an active substance for preventing or treating diabetes or obesity, characterized by using the composition as a target substance.
  • Still another object of the present invention is to provide a composition for inhibiting and treating diabetes or obesity, comprising an agent that inhibits the expression of a gene encoding SENP2 or inhibits the activity of SENP2.
  • Figure 1 is related to the dehydration of PPAR ⁇ by SENP2
  • A transformed COS-7 cells with HA-PPAR ⁇ , FLAG-SUMO and SENP expression vectors, and after 24 hours, the cells were collected to antagonize the cell lysate.
  • B shows that after transformation, PPAR ⁇ protein is immunoprecipitated with anti-HA antibody, and the immunoprecipitated protein is detected by Western blot analysis with anti-HA antibody or anti-FLAG antibody. Male-bound PPAR ⁇ is indicated by an arrow.
  • (C) is a result of Western blot analysis of cell lysates after transforming cells with HA-PPAR ⁇ , FLAG-SUMO, SENP2, and SENP2 variants (SENP2 C548S, SENP2 RK576LM).
  • (C) transforms cells with SENP1, 2 or 3 expression vectors, sets the luciferase activity of pM-PPAR ⁇ wild type to 1 when there is no overexpression of rosiglitazone and SENP2, and expresses other activities relative to it did.
  • 3 relates to SENP2 expression during adipogenesis.
  • 3T3-L1 adipocytes were differentiated by the method described in "Experimental procedures”.
  • Total RNA was isolated by date in (A) and time in (B) after induction of differentiation, and 10 ⁇ g of RNA was used for Northern blot analysis.
  • Probes specific to several genes were used, including acid binding protein 2 (aP2) and ribosomal protein s2 (RPS2).
  • FIG. 4 shows that raising cAMP levels induces SENP2 expression.
  • A is a result of processing one of insulin, dexamethasone, IBMX, which induces differentiation of adipose progenitor cells, and preparing RNA and performing Northern blot analysis 3, 6, 24 hours after adding these elements.
  • B shows that the progenitor cells were treated with DMI medium (a medium containing dexamethasone, IBMX and insulin, which is a medium for normal adipocyte differentiation) or DI medium (does not include IBMX), followed by RNA at the indicated time. The result is a Northern blot analysis using the prepared and specific probes.
  • DMI medium a medium containing dexamethasone, IBMX and insulin, which is a medium for normal adipocyte differentiation
  • DI medium does not include IBMX
  • (C) is the result of treating the progenitor cells with or without H89 in the IBMX, RNA prepared after 8 hours of treatment.
  • (D) is the result of measuring the SENP2 expression by Northern blot analysis after treatment with fatscholine (10 mM or 50 mM) to the fat precursor cells.
  • FIG. 5 to 7 show the effect of SENP2 siRNA on adipocyte differentiation.
  • Adipocytes were treated with SENP2 siRNA one day before addition of differentiation medium.
  • Figure 5 after the induction of adiogenesis process, the RNA of the indicated date is prepared, and the result of Northern blot analysis of 10 ⁇ g of RNA of each step.
  • Figure 6 shows the result of staining the cells of the indicated date with oil red O after induction of differentiation.
  • 7 is a result of Western blot analysis by preparing a protein of the cell.
  • C / EBP family member is a substrate of SENP2.
  • A transforms COS-7 cells with C / EBP ⁇ , SUMO and SENP (Myc-SENP1, FLAG-SENP2, FLAG-SENP3 or FLAG-SENP2 variant) expression vectors and expresses SENPs in myc or FLAG. The result was detected by Western blot using the antibody.
  • B is a result of transforming the cells with C / EBPa, SUMO and SENPs expression vectors and analyzing the C / EBPa constriction form by Western blot analysis.
  • FIG. 9 shows the effect of SENP2 siRNA on DNA binding activity.
  • NS siRNA control
  • SENP2 siRNA 5 ⁇ g of protein was labeled with a DNA fragment represented by the C / EBP binding site of the C / EBPa gene promoter.
  • EMSA was analyzed. Probes that did not bind to the protein are shown in lane 1, and the analysis results using the nuclear extract of day 0 cells are shown in lane 2.
  • the present invention comprises a small-ubiquitin-like modifier (SUMO) specific protease 2 (SENP2) or a gene encoding SENP2 as a target substance, preventing or treating diabetes or obesity A composition for screening materials.
  • SUMO small-ubiquitin-like modifier
  • SENP2 specific protease 2
  • the gene sequence of the SENP2 protein included in the composition of the present invention is publicly available from the NCBI database as NCBI accession No. AF151697 (human SENP2), and SENP2 encoded by the gene is known in the art. It can be easily manufactured by the known technology.
  • NCBI is a National Center for Biotechnology Information (mail: National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD 20894, USA; Internet address: www.ncbi.nhm.nih.gov).
  • the term "SENP2" of the present invention also encompasses sequences, variants, derivatives and fragments with any homology as long as they maintain the biological activity of SENP2 described herein.
  • the SENP2 protein or gene encoding it as a target substance in the composition of the invention is expressed in cells (SENP2 protein), in the form of a transcriptional activation system or translational activation system (gene encoding SENP2) It may be included in the composition of the present invention.
  • cells include cells that have the property of expressing SENP2 as well as cells that have been genetically modified to exhibit these properties.
  • a transcriptional activation system is any biochemical or cellular system that is capable of carrying out a transcriptional reaction of at least a transcriptional unit.
  • Such systems are well known in the art and include in vitro transcription systems that are commercially available as well as cells (eg, primary culture of eukaryotic or prokaryotic cells as well as common laboratory strains or cell lines).
  • a translation activation system is any biochemical or cellular system that is capable of at least performing a translation reaction of a transcript.
  • Such systems are well known in the art and include in vitro translation systems (which may be commercially available) as well as cells (eg, primary culture of eukaryotic or prokaryotic cells as well as common laboratory strains or cell lines). do.
  • composition of the present invention in addition to the SENP2 protein or a gene encoding the same may include a buffer or a reaction solution for maintaining a stable structure of the protein or nucleic acid.
  • the present inventors promote the dehydration of several transcription factors, such as PPAR ⁇ and C / EBP family members, in which SENP2 plays an important role in adipocyte differentiation, unlike SENP1 and SENP3, among SENPs, and SENP2 by siRNA. It was confirmed that the differentiation of adipocytes was inhibited when knock-down expression of. Therefore, the SENP2 protein plays an important role in increasing adipose tissue production and transcriptional activity of PPAR ⁇ , and thus is useful as a target material for diabetes and obesity related studies, and the composition of the present invention comprising such SENP2 protein or a gene encoding the same may be used for diabetes. Or are useful for screening substances with prophylactic or therapeutic activity for obesity related diseases.
  • the present invention relates to a method for screening an active substance for preventing or treating diabetes or obesity, characterized by using the composition of claim 1 as a target substance.
  • the confirmation of the reaction between the composition comprising the SENP2 and the test substance may use methods commonly used to confirm the reaction between the protein-protein or the protein-compound. For example, a method for measuring activity after reacting SENP2 with a test substance, yeast two-hybrid, searching for phage display peptide clones that bind to SENP2, and using HTS (natural and chemical libraries) Screening methods using high throughput screening, drug hit HTS or cell-based screening, etc. may be used, but the present invention is not limited thereto.
  • the confirmation of the reaction between the test substance and the composition containing the gene encoding SENP2 can be used the conventional methods used to confirm the reaction between the DNA-DNA, DNA-RNA, DNA-protein.
  • hybridization tests to check the binding between the gene and the test substance in vitro a method for measuring the expression rate of the gene through northern analysis after reacting the mammalian cell and the test substance, or a reporter gene to the gene. After introducing into the cell by connecting the reaction with the test material and measuring the expression rate of the reporter protein, etc. may be used, but is not limited to these methods.
  • the test substance is estimated to have the possibility of preventing or treating diabetes or obesity-related diseases according to a conventional selection method or randomly selected individual nucleic acids, proteins, other extracts or natural products, compounds And so on.
  • the material obtained by this screening method acts as a leading compound in the development of a preventive or therapeutic agent for diabetes or obesity in the future, and has a structure so that the leading material can exhibit an inhibitory effect on SENP2 or a gene encoding the same.
  • new diabetes or obesity prophylaxis or treatment can be developed, and these substances can have a partial or complete inhibitory effect on SENP2 or the gene encoding it, thereby preventing or treating diabetes or obesity related diseases.
  • the screening method of the present invention can be usefully used for the search and development of a preventive or therapeutic agent for diabetes or obesity-related diseases.
  • the present invention relates to a composition for inhibiting and treating diabetes or obesity comprising a substance that inhibits the expression of a gene encoding SENP2 or inhibits the activity of SENP2.
  • the inhibitory nucleic acid may be included as a substance that inhibits the expression of the gene encoding SENP2 included in the composition.
  • inhibitory nucleic acid refers to a nucleic acid compound that can result in gene-specific inhibition of gene expression.
  • Typical inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, triple helix DNA, RNA aptamers, ribozymes, and siRNAs.
  • knowledge of nucleotide sequences can be used to design siRNA or antisense molecules that strongly inhibit the expression of SENP2.
  • ribozymes can be synthesized to recognize and cleave specific nucleic acid sequences of genes. Techniques for designing the art for use in the inhibition of targeted gene expression are well known to those skilled in the art.
  • the inhibitory nucleic acid compound included in the composition of the present invention may be synthesized by conventional means on a commercially available automated DNA synthesizer, and may be a commercially available form.
  • the inhibitory nucleic acid comprised in the composition of the present invention is an antisense nucleic acid sequence complementary to the mRNA of siRNA or SENP2, and in one specific embodiment the inventors of the present invention are directed to the preparation of SENP2 from Dharmacon, Lafayette, CO, USA. siRNA was purchased and used.
  • siRNA refers to a short double-chain RNA capable of inducing RNA interference (RNAi) through specific cleavage of mRNA. It consists of a sense RNA strand having a sequence homologous to mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. siRNAs can be provided as an efficient gene knockdown method or as a method of gene therapy because they can inhibit the expression of a target gene.
  • RNAi RNA interference
  • siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included.
  • the total length is 10 to 100 bases, preferably 20 to 70 bases.
  • the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the target gene can be inhibited by the RNAi effect.
  • siRNA is a low-molecular RNA (for example, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene It may include.
  • the siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a step loop structure in which the terminal portion of the double chain RNA general is connected by linker RNA.
  • the siRNA used in the present invention is itself a complete form with polynucleotide pairing, that is, a form introduced into a cell through two transformation processes in which the siRNA is directly synthesized in vitro, or one form to have such a form after administration in vivo.
  • Single-chain oligonucleotide fragments and their reverse complements can be derived from single-chain polynucleotides separated by spacers, e.g. siRNA expression vectors or PCR-derived siRNAs prepared to express siRNA in cells
  • the expression cassette may be in a form introduced into a cell through a transformation or infection process. Determination of how to prepare an siRNA and introduce it into a cell or animal may depend on the purpose and cellular biological function of the target gene product.
  • antisense nucleic acid refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein.
  • Antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to SENP2 A mRNA and capable of binding SENP2 mRNA, and that translates into SENP2 mRNA, translocation into the cytoplasm, maturation or any other overall biological function. May inhibit the essential activity for.
  • the antisense nucleic acid is 6 to 100 bases in length, preferably 10 to 40 bases.
  • Antisense RNA can be synthesized in vitro and administered in vivo by conventional methods, or the antisense RNA can be synthesized in vivo.
  • One example of synthesizing antisense RNA in vitro is using RNA polymerase I.
  • One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin of the recognition site (MCS) is in the opposite direction.
  • MCS origin of the recognition site
  • the composition of the present invention may comprise an antibody specific for SENP2 as a substance that inhibits the activity of SENP2.
  • SENP2 an antibody specific for SENP2 as a substance that inhibits the activity of SENP2.
  • Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, or antigen-binding fragments thereof.
  • Antibodies specific for such SENP2 can be obtained from commercial sources or can use a variety of methods known in the art. Specifically, polyclonal antibodies can be prepared from the sera of animals immunized with SENP2 protein, in which case an appropriate adjuvant can be used together to promote immunization. In the case of monoclonal antibodies can be easily prepared by known monoclonal antibody production techniques.
  • a monoclonal antibody according to the present invention may be prepared by using an antibody library using phage display technology in which an antibody gene is directly obtained from B lymphocytes without expressing a hybridoma and expressing an antibody on a phage surface.
  • phage display technology in which an antibody gene is directly obtained from B lymphocytes without expressing a hybridoma and expressing an antibody on a phage surface.
  • Other chimeric antibodies, humanized antibodies and methods for preparing antigen-binding fragments with antigen binding functions such as Fab, F (ab '), F (ab') 2 and Fv are also well known in the art.
  • composition for preventing or treating diabetes mellitus or obesity of the present invention includes, in addition to the active substances, agents for promoting the influx of siRNA or antisense nucleic acid molecules, such as liposomes (US Pat. Nos. 4,897,355, 4,394,448, 4, 23,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270) or a lipophilic carrier of one of many sterols, including cholesterol, cholate and deoxycholic acid It can also be combined with.
  • Antisense nucleic acids can also be conjugated to peptides that are taken up by cells. Examples of useful peptides include peptide hormones, antigens or antibodies and peptide toxins.
  • composition for preventing or treating diabetes mellitus or obesity of the present invention may be used alone, or may be used in combination with existing agents or methods for preventing and / or treating diabetes or obesity.
  • composition of the present invention may be administered with a pharmaceutically acceptable carrier, and when used orally, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like may be used.
  • a binder a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like
  • buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used.
  • bases, excipients, lubricants, preservatives and the like can be used.
  • the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • oral administration in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.
  • the effective dosage range of a substance that inhibits the expression of the gene encoding SENP2 contained in the composition of the present invention or inhibits the activity of SENP2 is determined by various factors such as sex, severity, age, method of administration, target cell and expression level. And can be easily determined by those skilled in the art.
  • Mouse PPAR ⁇ cDNA was cloned into pcDNA-HA and pM vectors (Clontech, Mountain View, CA, USA).
  • the expression vectors pFLAG-SUMO, pMyc-SUMO, pMyc-SENP1, pFLAG-SENP2 and pFLAG-SENP3 were expressed by Dr. C.H. Received from Chung, and the vectors for expressing C / EBPa, ⁇ , ⁇ and CREB were: J.B. I got it from Kim.
  • the SENP2 variant forms C548S and RK576LM were made using site directed mutagenesis (site directed mutagenesis, Stratagene, La jolla, CA, USA).
  • SiRNA of SENP2 was purchased from Dharmacon, Lafayette, CO, USA.
  • Antibodies against PPAR ⁇ , C / EBPa, CEBP ⁇ , C / EBP ⁇ and c-Myc (9E10) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
  • HA antibodies were purchased from Roche (Mannheim, Germany) and antibodies against Flag from Sigma-Aldrich (Louis, MO, USA).
  • 3T3-L1 adipocytes were maintained in ⁇ DMEM (Dulbecco modified Eagle medium) containing 10% bovine serum (Invitrogen, Carlsbad, Calif., USA). Differentiation into adipocytes included 5 mg / ml insulin, 0.25 mmol / l dexamethasone and 0.5 nmol / l 3-isobutyl- ⁇ -methylxanthine (IBMX), with 10% bovine serum Induced by addition of DMEM. After 2 days, cells were maintained for up to 10 days with medium change every other day with DMEM containing 10% bovine serum and 1 mg / ml insulin. The degree of differentiation of adipose progenitor cells was measured by Oil Red O staining (Sigma-Aldrich, Louis, MO, USA). COS-7 cells were maintained in DMEM containing 10% bovine serum.
  • COS-7 cells were plated in 60 mm dishes for immunoprecipitation and transformed with lipofectamine plus (Invitrogen) with respective 1 mg pHA-PPAR ⁇ or pCMV-C / EBP, pFLAG-Sumo-1 and SENP expression vectors. .
  • COS-7 was plated in 12 well plates for reporter assays, transformed with 100ng pGal4-TK-Luc, 30ng pM, 30ng pM-PPAR ⁇ , 100ng pFLAG-SENP, pCMV- ⁇ -galactosidase, pcDNA was transformed by adding a total of 600ng per well. Luciferase reporter analysis was performed according to Invitrogen's preparation instructions. To transform siRNAs of mouse SENP2, 3T3-L1 adipocytes were transformed with siRNAs at 100 nM concentration or at concentrations described in Lipofectamine 2000 (Invitrogen).
  • RNA was prepared by Trizol (Invitrogen), electrophoresed 5-10 ⁇ g of RNA in a 1% agarose-formaldehyde gel, and then onto a Nytran membrane (Schleicher & Schuell, Stanford, ME, USA). Transfer. The membranes were hybridized with [ 32 P] -labeled probes in ExpressHyb solution (BD Stratagene) and then exposed to radiation.
  • PPAR ⁇ can be hydrophilized intracellularly, and that hair binding can negatively regulate the transcriptional activity of PPAR ⁇ .
  • Lysine residue 107 of PPAR ⁇ is the major hair binding site and we found lysine residue 68 as an additional site (not shown as data).
  • SENP1 SENP1
  • SENP3 did not promote dehydration of PPAR ⁇ .
  • SENP2 variants that lacked dehydrogenation activity also did not induce dehydrogenation of PPAR ⁇ (FIG. 1C).
  • SENP2 dehydration by SENP2 can positively regulate the transcriptional activity of PPAR ⁇ .
  • Wild-type PPAR ⁇ showed lower transcriptional activity than PPAR ⁇ K107R variant.
  • co-expression of SENP2 elevated the activity of wild type PPAR ⁇ to a level similar to that of PPAR ⁇ K107R variant (FIG. 2A).
  • Two other SENP2 variants did not increase PPAR ⁇ activity (FIG. 2B).
  • Overexpression of other SENPs, SENP1 and SENP3 showed little effect on the transcriptional activity of PPAR ⁇ (FIG. 2C).
  • SENP2 was examined in more detail the expression of SENP2 in the early stage of adipocyte differentiation (FIG. 3B).
  • SENP2 expression increased within a few hours after adipocyte derivative addition and reached a maximum level within 4 hours. Since 1 hour after induction of induced adipocyte differentiation, C / EBP ⁇ and C / EBP ⁇ were shown to be expressed maximally, an increase in C / EBP ⁇ and C / EBP ⁇ transcription was preceded before SENP2 expression was induced. Expression of PPAR ⁇ and C / EBPa occurred after an increase in SENP2 expression.
  • SENP2 plays an important role in adiogenesis
  • cells were treated with siNPs of SENP2 one day before induction of differentiation.
  • SENP2 transcript levels did not disappear completely by treating siNPs of SENP2, but were generally reduced by two days after induction of differentiation.
  • siRNA treatment of SENP2 inhibited the induction of PPAR ⁇ and aP2, but had little effect on the expression of C / EBP ⁇ and C / EBP ⁇ (FIG. 5).
  • SENP2 Since SENPs functioned before PPAR ⁇ expression and were shown to regulate protein levels of C / EB ⁇ , we tested whether SENP2 families were dehydrated by SENP2. SENP2 eliminated C / EBp ⁇ hairloids when they were exogenously expressed in COS-7 cells (FIG. 8A). SENP2 was also able to dehydrate C / EBP ⁇ (not shown as data), but SENP1, SENP3 or SENP2 variants did not dehydrate C / EBP ⁇ . The effect of SENP2 on C / EBPa hydrophilization was examined, and SENP2 completely eliminated the C / EBPa hydrophilic conjugate (FIG. 8B).
  • Nucleic proteins of cells were prepared on days 1 and 2 after induction of adiogenesis and the binding activity to the specific binding sequences of C / EB ⁇ was monitored. It showed lower activity against the C / EBP binding site in the nuclear extract of cells transformed with SENP2 siRNA (FIG. 9). This phenomenon is consistent with the results of Western blot analysis shown in FIG. 7. 7 shows that C / EB ⁇ protein levels decrease when SENP2 siRNA is introduced. Therefore, the reduction of C / EB ⁇ protein by SENP2 siRNA is a potential mechanism for inhibiting adipocyte differentiation.
  • SENP2 specifically dehydrogenated PPAR ⁇ and C / EBP family members, and that other SENPs, SENP1 or SENP3, did not dehydrogenate. These results support the hypothesis that each SENP has different substrate specificity. . It was also confirmed that overexpression of SENP2 increased PPAR ⁇ transcriptional activity. It was confirmed that SENP2 overexpression can improve the function of PPAR ⁇ as an insulin sensitizer, adipocaine expression regulator and inhibitor of atheromatous process.
  • SENP2 used in the present invention dehydrates transcription factors such as the PPAR ⁇ and C / EBP families and increases the transcriptional activity of these, in particular, PPAR ⁇ , and is involved in the differentiation of adipocytes and the production of adipose tissue. And screening methods are very useful for the identification of active substances for the prevention or treatment of diabetes or obesity-related diseases, and compositions of the invention comprising agents that inhibit the expression or activity of SENP2 are very useful for the prevention or treatment of diabetes or obesity.

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Abstract

L'invention concerne une composition pour cribler une matière active utilisée pour prévenir et traiter l'obésité, un procédé de criblage utilisant ladite composition et une composition pour prévenir ou traiter le diabète ou l'obésité. De manière plus spécifique, l'invention concerne une composition pour cribler une matière active utilisée pour prévenir et traiter le diabète ou l'obésité, cette composition contenant une protéase 2 (SENP2) spécifique du SUMO (modificateur de type petite ubiquitine) ou un gène codant pour la SENP2 en tant que matière cible; un procédé de criblage utilisant la composition comme matière cible; et une composition pour prévenir ou traiter le diabète ou l'obésité, qui contient un agent inhibant l'expression ou l'activation de la SENP2, et l'expression du gène qui code pour la SENP2. La SENP2 utilisée dans l'invention non seulement inhibe le SUMO des facteurs de transcription tels que PPARγ et la famille C/EBP et améliore leur activation de transcription, mais participe également à la différentiation d'un adipocyte et à la génération de tissu adipeux. De ce fait, la composition et le procédé de criblage sont utilisés pour identifier la matière active utilisée pour prévenir ou traiter le diabète ou les maladies associées à l'obésité, cette composition contenant en outre un agent inhibant l'expression et l'activation de la SENP2 utilisée pour prévenir ou traiter le diabète et l'obésité f.
PCT/KR2009/000949 2008-02-28 2009-02-27 Composition et procédé pour cribler une matière active destinée à prévenir et traiter le diabète ou l'obésité au moyen de la senp2 et composition pour prévenir et traiter le diabète et l'obésité Ceased WO2009108005A2 (fr)

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CN120114578A (zh) * 2025-05-12 2025-06-10 天津科技大学 Senp5在制备治疗血管损伤后内膜增生药物中的应用

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US7173119B2 (en) 2004-03-25 2007-02-06 Medical College Of Georgia Research Institute SUMO4 gene and methods of use for type 1 diabetes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120114578A (zh) * 2025-05-12 2025-06-10 天津科技大学 Senp5在制备治疗血管损伤后内膜增生药物中的应用

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