WO2006043701A1 - Procédé d'inhibition de la phosphorylation d'un facteur de transcription pour un gène associé à la gluconéogenèse et inhibiteur de phosphorylation - Google Patents

Procédé d'inhibition de la phosphorylation d'un facteur de transcription pour un gène associé à la gluconéogenèse et inhibiteur de phosphorylation Download PDF

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WO2006043701A1
WO2006043701A1 PCT/JP2005/019518 JP2005019518W WO2006043701A1 WO 2006043701 A1 WO2006043701 A1 WO 2006043701A1 JP 2005019518 W JP2005019518 W JP 2005019518W WO 2006043701 A1 WO2006043701 A1 WO 2006043701A1
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rskb
hnf
gene
phosphorylation
kinase
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PCT/JP2005/019518
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English (en)
Japanese (ja)
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Hirofumi Doi
Yuka Shozaki
Gen Kudo
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Daiichi Pharmaceutical Co., Ltd.
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Publication of WO2006043701A1 publication Critical patent/WO2006043701A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for phosphorylating hepatocyte nuclear factor 4a (hereinafter referred to as HNF-4a), a phosphorylating agent, a method for inhibiting phosphorylation, and phosphoric acid Relates to inhibitors. More specifically, the present invention relates to a method for phosphorylating HNF-4 ⁇ and a phosphorylating agent characterized by using ribosome 1 S6 kinase B (abbreviated as RSKB hereinafter). The present invention also relates to a method for inhibiting HNF-4 Q; Furthermore, the present invention relates to a method for promoting the production of a gene product of a gene in which HNF-4 ⁇ acts as a transcription factor.
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosome 1 S6 kinase B
  • the present invention also relates to a method for inhibiting the production of a gene product of a gene for which HNF-4a acts as a transcription factor and a production inhibitor. Further, the present invention relates to prevention and Z or treatment methods and prevention and Z or treatment agents for diseases caused by phosphate lees from HNF-4. Further, the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4 ⁇ , the binding of RSKB and HNF-4a, or phosphorylation of HNF-4a by RSKB, and the compound identified by the identification method. The Furthermore, the present invention relates to a polynucleotide encoding RSKB, HNF-4a, and a reagent kit comprising a vector containing the polynucleotide.
  • the liver is an important organ for maintaining glucose homeostasis, and maintains the balance of glucose in the living body by gluconeogenesis that produces glucose and glycogen that produces glycogen from glucose. Yes. In diabetic patients, excessive glucose production occurs in the liver, which is considered to be one of the causes of hyperglycemia (Non-patent Document 1).
  • Glucogenesis is a pathway in which pyruvate also synthesizes glucose, most of which is performed in the liver.
  • phosphoenol pyruvate carboxy kinase hereinafter abbreviated as PEPCK.
  • PEPCK phosphoenol pyruvate carboxy kinase
  • Non-patent Document 8 This transcription is regulated by hormones, darcocorticoids promote the transcription of the PEPCK gene (Non-patent Document 8), and insulin represses the transcription of the PEPCK gene (Non-patent Documents 4, 6, and 8).
  • the half-life of PEPCK gene mRNA is as short as 40 minutes, and the amount of transcription dramatically increases or decreases in units of several hours (Non-patent Document 3).
  • PE PCK enzyme activity, gluconeogenesis, and blood glucose level are regulated by the amount of transcription from the PEPCK gene (Non-patent Document 3).
  • PEPCK gene expression In the liver in the diabetic state, PEPCK gene expression is increased by darcocorticoids, and as a result, gluconeogenesis is enhanced (Non-patent Documents 8 and 9).
  • Type 1 diabetes insulin-dependent diabetes mellitus, IDDM
  • type 2 diabetes non-insulin-dependent diabetes mellitus, NIDDM
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • Type 2 diabetes is further divided into two types. One is diabetes mainly due to a decrease in insulin secretion, and the other is diabetes where insulin is secreted but the insulin sensitivity to glucose in target cells is mainly reduced. The latter is particularly said to be insulin resistant.
  • the target tissues of insulin are the liver, fat, and muscle, and blood glucose levels are lowered by suppressing gluconeogenesis and sugar release in the liver. Sustained hyperglycemia reduces the sensitivity of the liver to insulin, thereby making it impossible to expect an insulin-dependent inhibitory effect on gluconeogenesis.
  • the mechanism that lowers insulin sensitivity in the liver due to persistent hyperglycemia has not been clarified yet, such as the potential for insulin receptor down-regulation.
  • HNF-4a is one of the nuclear receptors and is expressed in ⁇ cells, kidney and small intestine of liver and spleen. HNF-4 ⁇ is known to act as a transcription factor, and is involved in the expression of various genes encoding proteins involved in metabolism and liver development and differentiation, such as cholesterol, fatty acids and glucose. In gluconeogenesis, HNF-4a binds to the AF1 site present in the promoter of the PEPCK gene and is involved in the expression of the PEPCK gene (Non-patent Document 16). Transcription factors have been shown to change their DNA-binding ability by phosphorylation (Non-patent Document 17).
  • HNF-4 ⁇ is phosphorylated by PKA (cAMP-dependent protein kinase), and as a result, the ability of L-type pyruvate kinase to bind to the promoter decreases. 1 is reported (Non-patent Document 10).
  • RSKB is a kinase activated by the p38 kinase family, which is a stress-responsive MAPK (mitogen activated kinase), and is distributed in the nucleus (Non-patent Document 11).
  • MAPK mitogen activated kinase
  • P38 kinase is activated in various pathological conditions (Non-patent Document 12).
  • P38 kinase is also activated by oxidative stress caused by active oxygen in cells that are continuously exposed to hyperglycemia under diabetic conditions (Non-patent Documents 13 and 14).
  • Non-patent Document 15 vascular smooth muscle cells cultured under hyperglycemia (16.5 mM)
  • Non-patent Document 29 obZob mouse livers of diabetes model animals
  • Patent Document 1 International Publication No. WO01Z67299 pamphlet.
  • Non-patent literature l “Nature” 2001, No. 413, No. 13, p. 131-138.
  • Non-Patent Document 2 Japanese Clinical” 2002, 60th, Special Issue 7, p. 121-128.
  • Non-Patent Document 3 “J. Biol. Chem.” 1982, No. 257, No. 13, p. 7629-7636 0
  • Non-patent Reference 4 1983, No. 305, No. 5934, p. 549— 551.
  • Non-Patent Document 5 “Biochem.” 1974, 138th, p. 387-394.
  • Non-Patent Document 6 “Mol. Endocrinol.” 1993, No. 7, No. 11, p. 1456–1462.
  • Non-Patent Document 7 “Proc. Natl. Acad. Sci. USA” 1994, 91st pp. 9151-915
  • Non-Patent Document 8 “J. Biol. Chem.” 1993, No. 268, No. 17, p.
  • Non-Patent Document 9 “Lab. Anim. Sci.” 1993, 42nd pp. 473-477.
  • Non-Patent Document 10 “Mol. Cell. Biol.” 1997, Vol. 17, No. 8, p. 4208-4219.
  • Non-patent document 11 “J. Biol. Chem.” 1998, 273, 45, p. 29661-2967
  • Non-patent document 12 “Crit. Care Med.” 2000, 28, N67— 77.
  • Non-Patent Document 13 “Japanese Clinical” 2002, 60th, Special Issue 7, p. 395-398.
  • Non-Patent Document 14 “Endocr. Rev.” 2002, No. 23, No. 5, p. 599-622.
  • Non-Patent Document 15 “J. Clin. Invest.” 1999, No. 103, p. 185-195.
  • Non-Patent Document 16 “Proc. Natl. Acad. Sci. USA” 1995, 92nd, p. 412-416
  • Non-patent document 17 -Cell, 1992, 70th, No. 3, p. 375 — 387.
  • Non-Patent Document 18 "Mol. Cell. Biol.” 1988, VIII, p. 3467-3475.
  • Non-Patent Document 19 J. Biol. Chem., 1995, 270, p. 8225-8232.
  • Non-Patent Document 20 “Proc. Natl. Acad. Sci. USA” 1994, 91st, p.
  • Non-Patent Document 21 -Science "1995, 269th, p. 1108-1112.
  • Non-Patent Document 22 J. Biol. Chem., 1997, 272, p. 26306-26312.
  • Non-Patent Document 23 J. Biol. Chem., 2000, Vol. 275, p. 5804-5809.
  • Non-Patent Document 24 “Mol. Endocrinol.” 1999, 13th, p. 604-618.
  • Non-Patent Document 25 J. Biol. Chem., 1999, No. 274, No. 9, p. 5880-5887.
  • Non-Patent Document 26 -Diabetes ", 1989, 38th page, p. 550-557.
  • Non-Patent Document 27 "Annu Rev Biochem.” 1997, 66th, p. 581-611.
  • Non-Patent Document 28 J. Biol. Chem., 2002, No. 277, No. 35, p.
  • Non-patent document 29 “Mol. Endocrinol.” 2003, Vol. 17, No. 6, p. 1131— 1143
  • Non-patent document 30 “J. Biol. Chem.” 2004, [Epub ahead of pront].
  • Non-Patent Document 31 Ulmer, K. M. “Science”, 1983, 219, p. 666-671.
  • Non-Patent Document 32 “Peptide Synthesis” (Japan), Maruzen Co., Ltd., 1975.
  • Non-Patent Document 33 “Peptide Synthesis” (USA), Interscience, 1996.
  • Non-Patent Document 34 “J. Biol. Chem.” 2000, No. 275, No. 31, p.
  • Non-Patent Document 35 “Mol. Endocrinol.” 1999, 13th, p. 604-618.
  • Non-Patent Document 36 “DNA”, 1989, VIII, p. 127-133.
  • Non-Patent Document 37 “Mol. Endocrinol.” 1990, No. 4, No. 9, pl302-1310.
  • Non-Patent Document 38 “Diabetes” 2001, 50th, p. 131-138.
  • Non-Patent Document 39 “Arch Biochem Biophys.” 1995, No. 323, No. 2, p477 483.
  • Non-Patent Document 40 “Diabetes” 2000, 49th, p. 1165–1168.
  • Non-Patent Document 41 “: Biol. Pharm. Bull.” 2005, Vol. 28, No. 4, p. 565-568. Disclosure of the invention
  • An object of the present invention is to phosphorylate HNF-4a involved in transcription of a gene encoding PEPCK, which is considered to be a gluconeogenic rate-determining enzyme, and to bind the PEPCK gene promoter to a region.
  • a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme
  • bind the PEPCK gene promoter to a region.
  • HNF-4a and RSKB are combined, HKB-4a is cleaved by RSKB, HNF-4a is phosphorylated by RSKB, and the PEPCK gene promoter It was demonstrated that the ability to bind to AF1 (accessory factor binding site 1, accessory factor binding sitel) within the region is promoted, and that the transcriptional activity of the PEPCK gene is enhanced by vigorous promotion. It was also clarified that RSKB activity is required for phosphorylation of HNF-4a by RSKB.
  • the PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4 ⁇ to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
  • RSKB is known to be activated by p38 kinase!
  • p38 kinase is It is activated under various pathological conditions.
  • p38 kinase such as oxidative stress caused by active oxygen, is activated in cells that are continuously exposed to hyperglycemia under diabetic conditions. Therefore, it can be considered that p38 kinase is activated and thereby RSKB is activated under diabetic conditions.
  • RSKB the active I spoon has been P 38 kinase is active I spoon, can be considered to be further HNF- 4 a mosquito ⁇ phosphorylated by RSKB. It can be considered that HNF-4Q; phosphorylated by RSKB enhances gluconeogenesis and worsens hyperglycemia by promoting the transcriptional activity of the PEPCK gene.
  • HNF-4a is known to act as a transcription factor.
  • HNF-4a is also involved in the metabolism of various genes, such as cholesterol, fatty acids and glucose, and the development and differentiation of the liver. It is involved in the expression of genes that code for the proteins involved. Therefore, the ability of HNF-4a to bind to the promoter region of a gene other than the PEPCK gene, where HNF-4 ⁇ acts as a transcription factor, is promoted by the phosphate of HNF-4 ⁇ by RSKB, As a result, it can be considered that expression of the gene may be promoted.
  • HNF-4a phosphorylates HNF-4a by interacting RSKB with HNF-4a, thereby promoting a gene for which HNF-4 ⁇ acts as a transcription factor.
  • the ability to bind HNF-4a to the region can be promoted, and further the production of the gene product of the gene can be promoted.
  • HNF-4 ⁇ can be phosphorylated by interacting with RSKB and HNF-4, thereby promoting a promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene.
  • the ability to bind HNF4a to the AF1 site of can be promoted, and further the production of the gene product of the gene can be promoted.
  • HNF-4a by inhibiting phosphorylation of HNF-4a by RSKB, the ability of HNF-4a to bind to the promoter region of a gene in which HNF-4a acts as a transcription factor can be reduced. As a result, production of the gene product of the gene can be inhibited. Specifically, by inhibiting the phosphorylation of HNF-4a by RSKB, the ability of HNF4 ⁇ to bind to AF1 in the promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene, is reduced. Production of the gene product of the gene And can be inhibited, and can inhibit gluconeogenesis.
  • HNF-4a can inhibit the production of a gene product of a gene that acts as a transcription factor
  • prevention and Z or treatment of a disease caused by an increase in the gene product can be performed.
  • diseases caused by an increase in gene products of gluconeogenesis-related genes, more specifically diseases caused by an increase in gene products of PEPCK genes such as diabetes can be prevented and Z or treated.
  • the present invention is characterized in that ribosome S6 kinase B (RSKB) and hematocytonuclear factor 4 a (HNF-4 a) are allowed to coexist under conditions that allow interaction. It relates to the phosphorylation method of HNF-4a by RSKB.
  • RSKB ribosome S6 kinase B
  • HNF-4 a hematocytonuclear factor 4 a
  • the present invention also relates to a method for inhibiting phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RKSB, characterized by inhibiting the activity of ribosomal S6 kinase B (RSKB) .
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosomal S6 kinase B
  • the present invention relates to HNF-4a by RSKB, characterized in that it inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a).
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 1a
  • the present invention relates to a method for inhibiting phosphorylation.
  • the present invention provides treatment of a cell expressing at least ribosomal S6 kinase B (RSKB) and henocytonuclear factor 4a (HNF-4a) with an inhibitor of RSKB activity. It relates to a method for inhibiting HNF-4a phosphorylation by RSKB.
  • RSKB ribosomal S6 kinase B
  • HNF-4a henocytonuclear factor 4a
  • the present invention also selects an antibody that recognizes RSKB, an inhibitory force of ribosomal S6 kinase B (RSKB) activity, and an antibody that recognizes hetocytonuclea factor 4 ⁇ (HNF-4) 1
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 4 ⁇
  • the present invention relates to a method for inhibiting phosphorylation of HNF-4a by RSKB, which is one or more antibodies.
  • the present invention relates to a phosphorylation inhibitor of hepatocyte nuclear factor 4a (HNF-4a) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity.
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosomal S6 kinase B
  • the present invention relates to HNF-4 by RSKB, characterized by inhibiting the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 4a (HNF-4a). It relates to a phosphate inhibitor of a.
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 4a
  • the present invention also includes an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity, and the phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RSKB. It relates to an anther inhibitor.
  • RSKB ribosomal S6 kinase B
  • HNF-4a hepatocyte nuclear factor 4a
  • the present invention is selected from an antibody that recognizes the inhibitory power RSKB of ribosomal S6 kinase B (RSKB) activity and an antibody that recognizes hetocytonuclea factor 4 ⁇ (HNF-4).
  • RSKB ribosomal S6 kinase B
  • HNF-4 hetocytonuclea factor 4 ⁇
  • the present invention relates to a phosphate inhibitor of HNF-4a by RSKB, which is one or more antibodies.
  • the present invention relates to a method for promoting gene product production of a gene involved in gluconeogenesis, which comprises phosphorylating hepatocyte nuclear factor 4a using ribosome S6 kinase B.
  • the present invention also relates to a method for promoting gene product production of the gluconeogenesis-related gene, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene.
  • the present invention relates to a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclease factor 4a by ribosomal S6 kinase B.
  • the present invention relates to a method for inhibiting the production of a gene product of a phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of henocytocytolase factor 14a by ribosome S6 kinase B. About.
  • the present invention also relates to a method for inhibiting the production of a glycoprotein-related gene product, characterized by using any one of the methods for inhibiting phosphate kneading.
  • the present invention relates to a method for inhibiting the production of a gene product of a gene that acts as a transcription factor, which is characterized in that any one of the above-described methods for inhibiting phosphate kneading is used. .
  • the present invention relates to a method for inhibiting the gene product production of a phosphoenolpyruvate carboxykinase gene, characterized in that any one of the above-described methods for inhibiting phosphate kine is used.
  • the present invention provides a ribozyme characterized by using any one of the methods for inhibiting phosphorylation.
  • the present invention relates to a method for preventing and / or treating a disease caused by phosphorylation of hetocytonuclea factor 4a by S6 kinase B.
  • the present invention relates to a method for the prevention and Z or treatment of a disease caused by an increase in the gene product of a gluconeogenesis-related gene, characterized by using any one of the above-described methods for inhibiting phosphorylation.
  • the present invention provides a method for preventing and / or treating a disease caused by an increase in the gene product of the phosphoenolpyruvate carboxykinase gene, characterized by using any one of the methods for inhibiting phosphate kneading. About.
  • the present invention also relates to prevention and / or treatment of diabetes, characterized by using the method for inhibiting hepatocyte nuclear factor 1a phosphorylation by any one of the above-mentioned ribosome S6 kinase B. Regarding the method.
  • the present invention uses a phosphate inhibitor of hepatocyte nuclear factor 4a and an inhibitor of Z or RSKB activity by any one of the above-mentioned ribosomal S6 kinase B. Prevention and Z or treatment methods.
  • the present invention provides a phosphoprotein comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphorylation inhibitors of henocytonuclear factor 4a and an inhibitor of Z or RSKB activity.
  • the present invention relates to an inhibitor of production of the gene product of the enolpyruvate carboxykinase gene.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 1a by any one of the ribosome S6 kinase B and an inhibitor of Z or RSKB activity. About.
  • the present invention provides a phosphoenolpyruvate comprising an effective amount of any one of the aforementioned ribosomal S6 kinase B hepatocyte nuclear factor 4a phosphate inhibitors and inhibitors of Z or RSKB activity.
  • the present invention relates to a preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a carboxykinase gene.
  • the present invention comprises a diabetic comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphoric acid phosphatase inhibitors of cytoplasmic factor 1a 4a and inhibitors of Z or RSKB activity. Prevention and Z or treatment.
  • the present invention also relates to a method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hetocytonuclea factor 4a (HNF-4a), wherein RSKB and HNF A system that uses a signal and Z or marker to detect the binding of RSKB and HNF-4a by contacting RSKB and Z or HNF-4a with the test compound under conditions that allow -4a interaction Use to determine whether the test compound inhibits the interaction between RSKB and HNF-4a by detecting the presence or absence of this signal and the Z or marker and the Z or change.
  • the present invention relates to an identification method.
  • the present invention provides a method for identifying a compound that inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a), comprising RSKB and HNF4 Using a system using RSKB and Z or HNF-4a in contact with a test compound under conditions that allow binding of a, and using a signal that detects the binding of RSKB and HNF-4a and Z or marker
  • the present invention relates to an identification method comprising determining whether or not a compound has a test compound strength 3 ⁇ 4 SKB and a binding to HNF-4a by detecting the presence or absence of this signal and Z or a marker and Z or a change.
  • the present invention provides a method for identifying a compound that inhibits phosphorylation of hepatocyte nuclear factor 1a (HNF-4 ⁇ ) by ribosomal S6 kinase B (RSKB), comprising: — RSKB and Z or HNF-4a in contact with the test compound under conditions that allow phosphorylation of 4a, and HNF by RSKB — Uses signal and Z or marker to detect phosphorylation of 4a To determine whether the test compound inhibits phosphorylation of HNF-4a by RSKB by detecting the presence or absence and Z or change of this signal and Z or marker. It relates to identification methods including
  • the present invention also relates to at least one of ribosomal S6 kinase B (RSKB), a polynucleotide encoding RSKB, and a vector containing a polynucleotide encoding RSKB, and hepatocyte nuclear factor 4
  • RSKB ribosomal S6 kinase B
  • the present invention relates to a kit comprising at least one of a (HNF-4a), a polynucleotide encoding HNF-4a, and a vector containing the polynucleotide encoding HNF-4a.
  • the invention's effect it is possible to provide a method for phosphorylating HNF-4a by RSKB, a method for inhibiting phosphorylation, and a phosphoric acid inhibitor. Further, according to the present invention, it is possible to provide a productivity method, a production inhibition method, and a productivity inhibitor of a gene product of a gene on which HNF-4a acts.
  • a method for inhibiting the production of a gene product of a gene on which HNF-4a acts can be carried out by using a phosphoric acid inhibitor of HNF-4a and a Z or phosphoric acid inhibitor method by RSKB.
  • a preventive and Z or therapeutic method and preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a gene on which HNF-4a acts.
  • diseases caused by an increase in gene products of gluconeogenesis-related genes more specifically diseases caused by an increase in the gene product of PEPCK gene diabetes, such as diabetes prevention and Z or treatment methods and prevention and Z
  • a therapeutic agent can be provided.
  • the present invention is thus very useful for the prevention and Z or treatment of diseases caused by excessive phosphorylation of HNF-4a involved in the expression of gluconeogenesis-related genes such as PEPCK gene.
  • diseases caused by excessive phosphorylation of HNF-4a involved in the expression of gluconeogenesis-related genes such as PEPCK gene.
  • Preferred examples of such diseases include diseases caused by increased gluconeogenesis due to expression of the PEPCK gene, specifically diabetes.
  • PEPCK gene expression is repressively controlled by insulin, and gene expression occurs in units of several hours as necessary.
  • the PE PCK gene is overexpressed continuously for some reason, gluconeogenesis in the liver is increased and hyperglycemia occurs, causing insulin-resistant diabetes.
  • ⁇ -cells in the spleen have collapsed and the amount of insulin in the blood has decreased, so that it is considered that the inhibitory effect of insulin on the PEPCK gene is decreased in end-stage patients with diabetes.
  • the expression of the PEPCK gene can be inhibited in an insulin-independent manner by the phosphate inhibitor of HNF-4a and Z or phosphate inhibitory method by RSKB according to the present invention, insulin resistant diabetes
  • gluconeogenesis involving the PEPCK gene can be suppressed. That is, in the liver of an insulin resistant patient with type 2 diabetes who can not be expected to have an insulin effect by the phosphate inhibitor of HNF-4 ⁇ and the method of inhibiting phosphate or phosphate by RSKB according to the present invention
  • gluconeogenesis can be suppressed, and as a result, hyperglycemia can be suppressed.
  • thiazolidinedione derivatives are used in clinical settings.
  • the thiazolidinedione derivative has an action of changing large adipocytes having low insulin sensitivity in adipose tissue to small adipocytes having high insulin sensitivity.
  • the phosphate inhibitor of HNF-4a by RSKB according to the present invention has an action of inhibiting the expression of PEPCK gene in the liver, and as a result, can suppress gluconeogenesis.
  • the phosphorylation inhibitor of HNF-4a by RSK B according to the present invention and the thiazolidinedione derivative differ in the target organ and the mechanism of action.
  • FIG. 1 is a diagram showing the results of in silico prediction of the interaction between RSKB and HNF-4a.
  • FIG. 1 and 2 show the elution fraction 1 and elution fraction 2 by FLAG, respectively. Elution fraction 1 was used for the experiment. Arrowheads indicate purified FLAG-RSKB.
  • the numerical values shown in the left column of the figure are the molecular weights of the molecular weight markers (indicated as M in the figure).
  • FIG. 3 shows that HNF-4a was phosphorylated by RSKB as a result of in vitro immunoprecipitation phosphorylation test of HNF-4a.
  • Panel A shows the results of the phosphate control test with the positive control PKA
  • Panel B shows the results of the phosphate test with RSKB.
  • CREB1 cyclic AMP responsive element binding protein 1, cAMP responsive element binding protein 1
  • phosphate by RSKB indicates that the purified RSKB has activity.
  • Figure Inside + and one indicate the presence or absence of each protein.
  • the arrowhead indicates HNF-4 ⁇ , and the star (*) indicates CREB1 phosphate.
  • the numbers listed in the left column of the figure are the molecular weights of the molecular weight markers. (Example 2)
  • FIG. 4 shows the nucleotide sequence of the promoter region of the human PEPCK gene (NCBI accession number U31519).
  • the AF1 sequence used in the gel shift assembly is shown in bold.
  • PEPCK AF1 promoter ZpGL3 is the 882 to 140 6th base sequence (bold display) separated by arrows 1 and 3
  • PEPCK ⁇ AF1 promoter ZpGL3 is the 934 to 1406 bases separated by arrows 2 and 3 Each sequence was used.
  • the numbers in the left column indicate the base numbers in U31519. (Example 3)
  • FIG. 5 FLAG-HNF-4a (DC BB stained image, transiently expressed in HEK293T cells using an N-terminal FLAG-tagged animal cell expression plasmid and purified using FLAG M2 affinity gel.
  • 1 and 2 indicate FLAG elution fraction 1 and elution fraction 2, respectively, and elution fraction 1 was used in the experiment, and arrowheads indicate purified FLAG-HNF-4a, as shown in the right column of the figure.
  • the numerical value is the molecular weight of the molecular weight marker (shown as M in the figure) (Example 3)
  • FIG. 6 is a diagram showing the results of examining the binding ability of HNF-4a to the AF1 sequence in the PEPCK gene promoter region using EMSA (electrophoretic mobility shift assay).
  • Panel A shows that HNF-4a bound to the AF1 sequence. By adding anti-HNF-4 ⁇ antibody, a supershift was observed in which the mobility of the HNF-4a 'DNA complex was reduced.
  • Panel B shows the effect of phosphorylation by RSKB on the ability of HNF-4 ⁇ to bind DNA to the A F 1 sequence. Phosphate treatment with RSKB enhanced the ability of HNF-4a to bind to the AF1 sequence. Phosphate treatment with PKA showed no such enhancement.
  • the amount of HNF-4 ⁇ used in Panel B is about one-tenth that of Panel A. In the figure, + and — indicate the presence or absence of anti-HNF-4 ⁇ antibody supplements. (Example 3)
  • FIG. 7 shows that the binding of HNF-4a to the AF1 sequence is enhanced in a dose-dependent manner by the phosphorylation of HNF-4a by RSKB.
  • RSKB used for HNF-4a phosphate treatment
  • the binding activity of HNF-4 ⁇ to AF1 decreased.
  • ⁇ In the A treatment indicated as PKA in the figure
  • IX, 1/2, and 1Z4 show the EMS results of the phosphorylated samples with RSKB diluted 2 and 4 times, respectively.
  • the star mark (*) indicates the addition of anti-HNF-4a antibody.
  • FIG. 8 shows the results of reporter assembly using PEPCK AF1 promoter. Transcriptional activity increased depending on the amount of HNF-4a introduced. This result indicates that HNF-4a force SPEPCK gene is positively controlled. Even when RSKB was expressed, the transcriptional activity of HNF-4a was not affected.
  • the vertical axis is a multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0.05 ⁇ g of F-4a expression plasmid).
  • FIG. 9 shows the results of reporter assembly using PEPCK AF1 promoter.
  • Transcriptional activity by HNF-4a Significantly enhanced by RSKB activated by MAPK11. Since only the expression of MAPK11 except RSKB had no effect on the transcriptional activity of HNF-4a, this action can be attributed to RSKB.
  • the vertical axis is the multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0. g of HNF-4 ⁇ expression plasmid).
  • FIG. 10 is a diagram showing the results of reporter assembly using PEPCK AF1 promoter.
  • the Transcriptional activity by HNF-4 ⁇ was significantly enhanced by RSKB activated by MAPK11.
  • dominant negative RSKB S343A
  • the vertical axis is a multiple of the transcriptional activity of the control group (only PEPCK AF1 promoter ZpGL3 introduced) is assumed to be 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 introduced 0.05 g of HNF-4 ⁇ expression plasmid). *: Significantly different from control ( ⁇ ⁇ 0. 05), #: HNF—4 Significantly different from 4 ⁇ 0.05 ( ⁇ ⁇ 0.05), $: HNF-4 a 0. 05 + Significantly different from RSKB 0.25 + MAPK11 0. 05 (p ⁇ 0. 05). (Example 4)
  • FIG. 11 shows the results of reporter assembly using PEPCK A AF1 promoter.
  • HNF-4a promotes transcriptional activity by co-expressing HNF-4a, RS KB and MAPK11 even in cells into which HNF-4a has been introduced. It was not recognized even in the cells!
  • HNF-4 ⁇ is dependent on AF1 within the promoter region of the SPEPCK gene promoter.
  • the vertical axis is a multiple when the transcriptional activity of the control group (in which only PEPCK A AF1 promoter ZpGL3 is introduced) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4 ⁇ 0.05 was introduced with 0.05 g of HNF-4 ⁇ expression plasmid). *: Significantly different from the control ( ⁇ 0. 05), #: Significantly different from HNF— 4 a 0. 05 (p ⁇ 0. 05), $: HNF — 4 ⁇ 0.2 There is a significant difference ( ⁇ ⁇ 0.05). (Example 4)
  • FIG. 12 shows the results of reporter assembly using the PEPCK AF1 promoter in HepG2 cells. This result was obtained under the condition that the darcocorticoid receptor was expressed by transfecting pMMGR into HepG2 cells. Transcriptional activity by HNF-4a was promoted by RSKB activated with MAPK11. When the MAPK11 inhibitor SB203580 was added, co-expression of MAPK11 and RSKB did not promote the transcriptional activity of HNF-4a. This result shows that the activity of RSKB is HNF-4 ⁇ It is important for transcriptional activity.
  • the vertical axis is a multiple of the transfer activity of the HNF-4 and 0.5 treatment groups as 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.5 introduced 0.5 g of HNF-4a expression plasmid). *: Significantly different from HNF— 4 ⁇ 0.5 ( ⁇ 0. 05), #: Significantly different from HNF— 4 ⁇ 0.5 + R SKB 0.25 (p 0. 05) ), $: HNF— 4 ⁇ 0.5 + RSKB 0.5 (p ⁇ 0.05), +: RSKB 0.5 (p ⁇ 0.05) ⁇ : HNF-4 a 0.
  • FIG. 13 is a drawing showing that expression of PEPCK gene is enhanced by RSKB depending on its kinase activity.
  • RSKB wild-type RSKB
  • MAPK11 MAPK11
  • the expression level of endogenous PE PCK gene increased compared to cells that did not express them.
  • an inactive mutant RSKB RSKB (S 196A / S343A / T568A)
  • RSKB S 196A / S343A / T568A
  • MAPK11 co-expressing cells showed an increase in the expression level of endogenous PEPCK gene. I was helped.
  • the expression level of the endogenous control GAPDH gene was almost the same in all cells.
  • each gene was detected by RT-PCR using the total RNA prepared by the cells.
  • the PCR product derived from the PEPCK gene is 575 bp
  • the PCR product derived from the GAPDH gene is 209 bp.
  • the 50 bp DNA ladder (1 adder) shown in the left lane in the figure is a DNA size marker during electrophoresis. (Example 5)
  • FIG. 14 HepG2 cells co-expressed with wild-type RSKB (shown as RSKB (WT)) and MAPK11, inactive mutant RSKB (RSKB (S196AZS343AZT568A)) and HepG2 co-expressed with M APK11
  • FIG. 3 is a view showing the expression level of each protein in cells and cells in which these were not expressed.
  • the upper panel shows the results of detecting the expression of FLAG-RSK B and MAPKll-FLAG in each cell by immunoblotting (ib) using an anti-FLAG antibody.
  • N-terminal FLAG tag wild type RSKB and N-terminal FLAG tag inactivation detected by immunoblotting (ib) using anti-FLAG antibody The band indicating the sexual variant RSKB! Is indicated by the FLAG-RSKB display on the right of the panel.
  • the middle panel shows the results of detecting phosphorylated RSKB with anti-phosphorylated RSKB antibody. This anti-phosphorylated RSKB antibody recognizes the 360th serine phosphate that is important for the kinase activity of RSKB.
  • FIG. 15 is a diagram showing that HNF-4a and RSKB bind in cells.
  • Cell force in which FLAG—HNF-4a and FLAG—RSKB were transiently co-expressed Prepared cell lysate was prepared by immunoprecipitation using anti-HNF-4a antibody and detection using anti-FLAG M2 antibody. A band indicating FLAG-HNF-4a and a band indicating FLAG—RSKB were both detected (Panel A). This result indicates that RSKB co-precipitated in a HNF-4 ⁇ -dependent manner.
  • Panel A shows the results of immunoblotting (i. B.) Of an immunoprecipitation sample (i. P.) With anti-HNF-4 ⁇ antibody with anti-FLA G M2 antibody.
  • Panel B shows the results of immunoblotting (i.b.) the cell lysate with anti-FLAG M2 antibody.
  • + and 1 indicate the presence or absence of each expression plasmid, respectively.
  • amino acids may be represented by one letter or three letters.
  • Peptide also means any peptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Peptide Short-chain peptides such as isolated or synthetic full-length oligopeptides, referred to as oligomers, and long chains such as isolated or synthetic full-length polypeptides and isolated or synthetic full-length proteins Also means peptide.
  • HNF-4a and RSKB bind, HNF-4a cation is converted by RSKB, HNF-4a is phosphorylated by RSKB, and PEPCK gene promoter It was demonstrated that the ability to bind to AF1 within the region was promoted, and that the transcriptional activity of the PEPCK gene was enhanced by vigorous promotion. It was also clarified that the activity of RSKB is required for phosphorylation of HNF-4a by RSKB.
  • the phosphorylation power of HNF-4a by RSKB can be used to positively regulate the transcriptional activity of the PEPCK gene through promoting the binding of HNF-4a to AF1 in the PEPCK gene promoter region. Turned out to be involved.
  • the PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4 ⁇ to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
  • Non-patent Documents 8 and 27 Transcription of the PEPCK gene is regulated by hormones, promoted by darcocorticoids, and repressed by insulin. Insulin regulates gluconeogenesis in the liver by suppressing transcription of PEPCK gene through LIP (liver-enriched transcriptional inhibitory protein), a transcriptional regulator in PEPC K gene promoter. (Non-patent document 28). There are also reports that insulin stimulation acts directly on the PEPCK gene promoter to suppress transcriptional activity (Non-patent Documents 6 and 37).
  • PEPCK gene is repressively controlled by insulin. For some reason, if overexpression of PEPCK gene persists, It is thought that diabetes develops when gluconeogenesis is increased and hyperglycemia occurs and insulin resistance is further developed. In fact, gluconeogenesis by PEPCK in non-insulin-dependent diabetic patients is about 3 times higher than that in normal individuals, and this gluconeogenesis is correlated with the fasting plasma glucose concentration in patients. (Non-patent Document 26).
  • Overexpression of the PEPCK gene includes phosphorylation by RSKB, which promotes the ability of HNF-4a to bind to AF1 in the PEPC K gene promoter region and thereby increases the transcriptional activity of the PEP CK gene. It is thought to be involved.
  • the PEPCK gene promoter has a region called GRU (Glucocorticoid Response Unit), and AF1, AF2 (accessory factor binding site 2, accessory factor binding site 2) in the region , GR1, 2 (glucocorticoreticoid receptor binding ⁇
  • GRU Glucocorticoid Response Unit
  • AF1, AF2 accessory factor binding site 2, accessory factor binding site 2
  • GR1, 2 glucocorticoreticoid receptor binding ⁇
  • CRE cyclic AMP response element, cAMP responive element
  • CREB cyclic AMP response element binding protein, cAMP responsive element binding protein
  • CREB is known as a transcription factor that is phosphorylated by RSKB and binds to the promoter.
  • CREB is a transcription factor that binds to CRE of PEPCK gene promoter (Non-patent Document 19).
  • CRE is a region that exists in the PEPCK gene promoter and positively regulates PEPCK gene expression (Non-patent Document 18).
  • CZEBP CCAATZenhancer binding protein
  • CZEBP CCAATZenhancer binding protein
  • RSKB phosphorylates CREB, and phosphorylated CREB enhances the transcriptional activity of a promoter having CRE (Non-patent Document 11). Therefore, when RSKB is activated, CREB is phosphorylated without passing through HNF-4a, and CREB binds to CRE of PEPCK gene promoter, which may promote PEPCK gene expression.
  • AF1 to which HNF-4a binds has a greater effect on the expression of the PEPCK gene than CRE. Specifically, substitution or deletion of AF1 reduces the transcriptional activity of the PEPCK gene promoter to about one-fourth that of the wild-type PEPCK gene promoter (Non-patent Document 24). On the other hand, depending on CRE substitution and deletion, the transcriptional activity of the PEPCK gene promoter can be reduced to about half that of the wild-type PEPCK gene promoter! (Non-patent Document 25).
  • AF1 to which HNF-4a binds can be considered to be a more important factor in the regulation of PEPCK gene expression compared to CRE.
  • RSKB does not promote the transcriptional activity of the PEPCK gene promoter.
  • a reporter gene having RSKB, MAPK11 that activates RSKB, and PEPCK A AF1 promoter lacking AF1 in the PEPCK gene promoter region was expressed in cells, the reporter gene Transcriptional activity was hardly promoted (see Example 4 and FIG. 11).
  • AFNF of the PEPCK gene promoter region involved in transcription binds to HNF-4a by being converted to HNF-4a by RSKB, resulting in transcription of the PE PCK gene promoter. Activity is promoted.
  • HNF-4a is phosphorylated by RSKB, HNF-4a cannot bind to the PEPCK gene promoter. Therefore, it can be considered that the transcription activity of the PEPCK gene promoter by RSKB was not promoted.
  • RSKB is activated by p38, an upstream kinase, and exhibits kinase activity.
  • Activity RSKB is known to phosphorylate CREB. Therefore, the activity of RSKB can be determined using the activity of P38 and the phosphate of CREB as indicators.
  • the p38 phosphoric acid type active p38
  • the p38 phosphoric acid type is about 2.5 to 3 times higher than that of normal individuals, and the phosphorylated form of CREB is also about 2 times higher. Have been reported (Non-Patent Documents 29 and 30).
  • PEPCK activity is regulated at the mRNA level, and PEPCK mRNA level, enzyme activity, gluconeogenic potential, and blood glucose level increase are well correlated. Specifically, in experiments using hepatocytes, it was reported that when the amount of PEPCK gene mRNA was increased 2-fold, enzyme activity increased 2.8-fold and gluconeogenesis increased 2.1-fold. (Non-patent document 6). In mice, when the gluconeogenic potential in the liver is increased by about 2.5 times, the blood glucose level rises by a factor of 2 (Non-patent Document 7), and in humans, the glucose gluconeogenic potential by PEPCK is 2.5 times higher in humans. Is 3 times higher than normal (Non-patent Document 26).
  • the present inventors considered the degree of increase in blood glucose level due to RSKB activity as follows. Assuming that the RSNF HNF-4a phosphate is similar to the CREB phosphate, the liver KBN HNF-4a phosphate is normal in the pathogenesis of diabetes It can be estimated that it is about 2 times higher. This is because, as described above, the phosphorylated form of CREB is increased about 2-fold in the liver of diabetic model animals, and RSKB activated by p38 is involved in phosphorylation of CREB. (Non-Patent Documents 29 and 30).
  • phosphorylation of HNF-4 ⁇ by RSKB increased the PEPCK gene promoter activity involving HNF-4 ⁇ force S by about 2-3 times (Example 4). And Figures 9, 10 and 12). If the promoter activity of the PEPCK gene is replaced with the amount of mRNA, it can be estimated that the amount of mRNA of the PEPCK gene increases by about 2 to 3 times due to the activity of RSKB in diabetic conditions. The increase in the amount of PEPCK gene mRNA is as described above. It is directly reflected in the enzyme activity, leading to increased gluconeogenic potential and increased blood glucose level.
  • One embodiment of the present invention achieved by these findings relates to a method for phosphorylating HNF-4a, characterized by using RSKB.
  • the HNF-4a phosphate method according to the present invention can be carried out by allowing RS KB and HNF-4a to coexist under conditions that allow their interaction.
  • an HNF-a phosphate inhibitor containing an effective amount of RSKB can be provided.
  • RSKB and HNF-4a interaction means that RSKB and HNF-4a are directly related.
  • the direct association between RSKB and HNF-4a includes a reaction in which RSKB and HNF-4a bind, and a reaction in which HNF-4 ⁇ is phosphorylated by RSKB as a result of the binding.
  • the reaction of RSKB first binds to HNF4a, and as a result, the reaction of HNF-4a cation is caused by the action of RSKB.
  • reaction of RSKB and HNF-4a binding can be called the interaction of RSKB and HNF-4 ⁇ , and as a result of the binding reaction, the reaction of RSKB causes HNF-4a to be oxidized. This reaction can be called the interaction between RSKB and HNF-4a.
  • the bond between RSKB and HNF-4a is closer to non-covalent bonds such as hydrogen bonds, hydrophobic bonds, or electrostatic interactions to form RSKB and HNF-4a force complexes. Means that. In this case, the bond is sufficient if RSKB and HNF-4a are bonded in part. For example, it means a bond between RSKB or part thereof and HNF-4 ⁇ or part thereof. In addition, the complex formed by RSKB and HNF-4 ⁇ contains a different type of protein! /, Or even! /.
  • phosphorylation of HNF-4a by RSKB refers to the transfer of the ⁇ -phosphate group of ATP to the hydroxyl group of the serine, threonine or tyrosine residue of HNF-4a by the kinase activity of RSKB. As a result, it means a reaction in which an HNF-4 ⁇ protein having a phosphate group bound thereto is produced.
  • RSKB kinase activity refers to an RSKB force that binds to another protein (hereinafter sometimes referred to as a substrate protein) and binds to the hydroxyl group of an amino acid residue in the substrate protein with adenosine triphosphate ( It means a function of phosphorylating the substrate protein by catalyzing a reaction of transferring the ⁇ -phosphate group of (ATP).
  • substrate means a compound or molecule that is catalyzed by an enzyme.
  • the conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions. That is, the HNF-4 ⁇ phosphate method according to the present invention is used in vitro and in vivo. However, it can also be implemented.
  • the HNF-4a phosphate method according to the present invention can be preferably performed in an in vitro sample or a non-human mammal.
  • “In vitro sample” refers to cells and tissues prepared from animals such as mammals, cultured cells derived from animals, and solutions containing proteins and genes prepared from the cells, tissues and cultured cells. Means sample. Preferred examples of cells include liver cells and liver cell lines.
  • “Non-human mammal” means mammals other than human, for example, mammals such as mouse, rat, rabbit, nu, goat and the like. Preferably a mouse is used.
  • the method for phosphorylating HNF-4a according to the present invention specifically includes expressing RSKB in a cell expressing HNF-4a, or coexisting RSKB and HNF-4a in the cell. It can be implemented by expressing. Alternatively, the phosphorylation method of HNF-4a can be performed by coexisting RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
  • HNF-4a phosphate system characterized by coexistence of RSKB and HNF-4a.
  • the phosphorylation system of HNF-4a according to the present invention may be either in vitro or in vivo.
  • the HNF-4 phosphate system by RSKB can preferably be a system utilizing in vitro samples or non-human mammals.
  • the phosphorylation system of HNF-4a according to the present invention specifically allows RSKB to be expressed in cells expressing HNF-4a, or allows RSKB and HNF-4a to be co-expressed in cells. It can be constructed by expressing it.
  • the phosphorylation system of HNF-4a can be constructed by phosphorylating RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
  • RSKB expression and HNF-4a expression can be performed by a conventional genetic engineering technique using an appropriate vector containing a gene encoding RSKB and an appropriate vector containing a gene encoding HNF-4a, respectively. These can be achieved by transfecting the cells. Phosphorylation of HNF-4a by RSKB is achieved by the interaction of RSKB and HNF-4a in the cell.
  • a cell used for expression a cell generally used for protein expression can be used.
  • eukaryotic cells are used.
  • Eukaryotic cells can be any of cells prepared from eukaryotes, primary cultured cells, and cultured cell lines.
  • a mammalian cell line more preferably a human cell line is used.
  • a cultured cell line derived from a mammalian liver more preferably a cultured cell line derived from a human liver is used.
  • human-derived cultured cell lines include HeLa cells (cervical cancer-derived cell lines) and HepG2 cells (human liver cancer-derived cell lines).
  • Detection of HNF-4 ⁇ phosphate by RSKB was carried out by contacting RSKB with HNF-4a using a well-known protein phosphate assay method. After that, it can be carried out by measuring phosphoric acid type HNF-4.
  • the detection of phosphate-type HNF-4 can be performed, for example, by Western blotting using an antibody against phosphate-type HNF-4.
  • detection of phosphoric acid-type HNF-4 was performed using ATP labeled with a radioisotope for phosphorylation, such as [y 32 P] ATP.
  • amino acid residues of HNF-4 a It can be carried out by measuring the radioactivity of [ ⁇ - 32 P] transferred to. Specifically, with reference to Example 2 described later, detection of HNF-4a phosphate by RSKB can be performed.
  • RSKB and HNF-4 ⁇ are preferably human-derived proteins, but mammal-derived proteins having the same functions as the human-derived proteins and having sequence homology, eg, For example, it can be a protein derived from mouse, horse, hidge, ushi, nu, monkey, cat, rat or rabbit.
  • the genes encoding RSKB and HNF-4a are preferably human-derived genes, but mammal-derived proteins having the same function and sequence homology as the human-derived proteins.
  • Examples of RSKB functions include kinase activity.
  • functions of HNF-4 ⁇ include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
  • RSKB-encoding gene and RSKB include the human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 1 and the human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 2, respectively.
  • the gene encoding RSKB is not limited to the above-exemplified gene, and any gene may be used as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoding the gene. Denko is also included.
  • RSKB is not limited to the above-exemplified proteins, and any protein is included as long as it has a sequence homology with the protein and has the same function as the protein.
  • sequence homology is usually 50% or more, preferably at least 70% of the entire amino acid sequence or base sequence. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more.
  • RSKB functions include kinase activity.
  • HNF-4a and HNF-4a a human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 3 and the human represented by the amino acid sequence set forth in SEQ ID NO: 4, respectively Preferred examples are derived proteins.
  • the gene encoding HNF-4a is not limited to the gene exemplified above, as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoded by the gene. In this regard, any gene is included.
  • HNF-4Q is not limited to the above-exemplified proteins, and any protein is included as long as it is a protein having sequence homology with the protein and having the same function as the protein.
  • the Sequence homology is usually amino acids It is appropriate that the total sequence or base sequence is 50% or more, preferably at least 70%. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more.
  • Examples of the function of HNF-4a include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
  • RSKB and HNF-4a may each be a cell sample prepared by genetic engineering techniques, or may be a cell-free synthetic product or a chemical synthesis product. It may be further purified. In addition, cells in which at least one of RSKB and HNF-4a is expressed by a genetic engineering technique can also be used.
  • RSKB and HNF-4a can be directly or phosphorylated with other types of labeling substances such as proteins and polypeptides on the N-terminal side and C-terminal side, as long as their properties and functions are not affected. It can be added indirectly via a carpeptide or the like using genetic engineering techniques. By measuring the labeling substance itself or its function, it is possible to easily detect the binding of RSKB and HNF-4 and the detection of phosphate of HNF-4a by RSKB.
  • labeling substances enzymes (Dartathione S-transferase, horseradish peroxidase, anolekali phosphatase or 13 galactosidase), tag peptides (His-tag, Myc-tag, HA-tag, FLAG) — Tag or Xpress— tag), fluorescent proteins (green fluorescent protein, fluoresceinis othiocyanate or phycoerythrin, etc.), maltose binding protein, immunoglobulin Fc fragment, Alternatively, piotin can be exemplified, but not limited thereto. Alternatively, it can be labeled with a radioisotope. When labeling is performed, one type of labeling substance may be added, or a plurality of labeling substances may be added in combination.
  • the genes encoding RSKB and HNF-4a are, for example, from an appropriate origin (for example, liver tissue and cells derived from liver tissue) from which each gene is expressed, or from a human cDNA library. It can be easily obtained using a known clawing method or the like. Proteins encoded by these genes can be obtained by, for example, a known genetic engineering technique using each gene. Specifically, each gene An appropriate expression vector DNA, for example, a vector derived from a bacterial plasmid is introduced by a known genetic engineering technique to obtain a vector containing the gene, and the vector is introduced into an appropriate host cell. A cell expressing the gene can be obtained, and a protein encoded by the gene can be obtained from the cell.
  • an appropriate expression vector DNA for example, a vector derived from a bacterial plasmid is introduced by a known genetic engineering technique to obtain a vector containing the gene, and the vector is introduced into an appropriate host cell. A cell expressing the gene can be obtained, and a protein encoded by the gene can be obtained
  • RSKB can be prepared as an immunoprecipitate obtained from a cell lysate of a cell expressing a gene encoding RSKB using an anti-RSKB antibody or the like.
  • HNF-4a can be similarly prepared using a gene encoding HNF-4a and an anti-HNF-4 ⁇ antibody.
  • the phosphorylating agent of HNF-4a, the phosphorylating method, and the phosphoric acid salt system according to the present invention include
  • a method for identifying a compound that inhibits phosphorylation of HNF-4a by RSKB can be constructed using the phosphorylation method and the phosphoric acid-containing system.
  • Another embodiment of the present invention relates to a phosphorylation inhibitor and a method for inhibiting HNF-4a by RSKB.
  • the phosphorylation inhibitor and inhibition method of HNF-4a according to the present invention inhibits RSKB activity or inhibits the interaction between RSKB and HNF-4 ⁇ , that is, RSKB and HNF-4a. By inhibiting the binding of or by inhibiting the phosphate of HNF-4a by RSKB.
  • Examples of the target to which the inhibitor and the inhibition method according to the present invention are applied include a target including at least RSKB and HNF-4a, for example, an in vitro sample including at least these.
  • Examples thereof include cells expressing at least RSKB and HNF-4 ⁇ , such as liver cells, and non-human mammals carrying such cells.
  • RSKB activity means a function of RSKB, a function to bind to other proteins, for example, a function to bind to HNF-4a, and a function to phosphorylate other proteins as kinases. For example, a function of phosphorylating HNF-4a is included.
  • “Inhibiting RSKB activity” means inhibiting the function of RSKB, for example, inhibiting the function of binding to HNF-4a and phosphorylating HNF-4a Inhibiting function, ie kinase activity against HNF4a.
  • the phosphate inhibitor of HNF-4a by RSKB according to the present invention contains at least one compound (RSKB activity inhibitor) having an effect of inhibiting RSKB activity in one aspect thereof.
  • RSKB activity inhibitor means a compound having a function of inhibiting RSKB activity. That is, “RSKB activity inhibitors” include RSKB kinase activity inhibitors and RSKBH NF-4a binding inhibitors. The “RSKB activity inhibitor” may be a composition comprising one or more compounds having a function of inhibiting RSKB activity.
  • Examples of RSKB activity inhibitors include antibodies having a competitive inhibitory effect and low molecular weight compounds.
  • Examples of the antibody include an antibody that recognizes and binds to RSKB or HNF-4a and inhibits phosphorylation of HNF-4 ⁇ by RSKB.
  • the antibody can be obtained by an antibody production method known per se using RSKB or HNF-4a itself, a partial peptide derived therefrom, or a peptide having an amino acid sequence at the site where they interact as an antigen.
  • Low molecular weight compounds include peptides, peptide-like substances, polypeptides, polynucleotides, organic compounds, and inorganic compounds, and the molecular weight is preferably 10,000 or less, more preferably 5000 or less, and even more preferably 1000. Hereinafter, even more preferably 500 or less compounds are meant.
  • Low molecular weight compounds include compounds that inhibit the kinase activity of RSKB, preferably compounds that specifically inhibit the kinase activity.
  • the compound to be obtained can be identified, for example, by determining whether it can inhibit the phosphorylation of HNF-4a by RSKB using the phosphorylation method or phosphorylation system according to the present invention.
  • To specifically inhibit the kinase activity of RSKB means to strongly inhibit the kinase activity of RSKB, but not or weakly inhibit the activity of other enzymes.
  • a peptide having an amino acid sequence at the site where RSKB and HNF-4a interact can be exemplified.
  • peptides include peptides containing the amino acid sequences of the sites to which these proteins bind in the amino acid sequences of RSKB and HNF-4a.
  • An example of such a peptide is a peptide containing the amino acid sequence of the site phosphorylated by RSKB in the amino acid sequence of HNF-4a.
  • Such peptides compete for phosphorylation of HNF-4 ⁇ by RSKB It is thought to inhibit.
  • peptides were designed from the amino acid sequence of RSKB or HNF-4Q; and synthesized by a peptide synthesis method known per se, phosphorylation of HNF-4a by RSKB and Z or RSKB and HNF-4a It can be obtained by selecting those that inhibit the binding of.
  • the peptide having a mutation may be a naturally occurring peptide or a mutation introduced.
  • Means for introducing mutation such as deletion, substitution, addition or insertion are known per se, and for example, Ulmer's technology (Non-patent Document 31) can be used.
  • Ulmer's technology Ulmer's technology
  • homologous amino acids polar amino acids, nonpolar amino acids, hydrophobic amino acids, Mutual substitution between hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids, etc.
  • a peptide that inhibits the interaction between RSKB and HNF-4a can be altered to such an extent that it does not undergo a significant change in function, such as modification of its constituent amino group or carboxyl group, for example, by amido.
  • modifications generally used to stabilize the interaction between peptides and other proteins and make it difficult to dissociate peptides such as C-terminal aldehydes or N-terminal acetylation Is useful to increase the effectiveness of peptides that inhibit the interaction between RSKB and HNF-4a.
  • the peptide can be produced by a general method known in peptide science.
  • methods Non-Patent Documents 32 and 33
  • known literatures can be mentioned, but the known methods can be widely used without being limited thereto.
  • HNF-4a is known to be involved in gluconeogenesis-related gene expression as a transcription factor in the liver in one of its functions.
  • the “gluconeogenesis-related gene” is a gene that encodes a substance that regulates gluconeogenesis in a living body, and preferably includes, for example, the PEPCK gene. Activation of HNF-4a during diabetic conditions is thought to further increase gluconeogenesis and cause exacerbation of hyperglycemia.
  • Another aspect of the present invention relates to a method for regulating the production of a gene product of a gluconeogenesis-related gene.
  • the method for regulating the production of gene products of gluconeogenesis-related genes regulates the phosphorylation by RSKB of HNF-4a related to the expression of genes encoding them, thereby regulating the production of the gene products of the genes. It is characterized by adjusting.
  • One aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for promoting the production of a gene product of a gluconeogenesis-related gene, and phosphorylating HNF-4a related to the gene expression using RSKB It is characterized by.
  • Another aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, and the phosphorylation by RSK B of HNF-4a related to the gene expression. It is characterized by inhibiting oxidation.
  • the production inhibition method can be achieved by using a phosphorylation inhibitor of HNF-4a by RS KB or a phosphorylation inhibition method of HNF-4a by RSKB.
  • Examples of a gene product of a gene on which HNF-4a acts on gluconeogenesis-related gene expression include a gene product of a gene having a binding site of HNF-4 ⁇ in a promoter or enhancer.
  • a gene on which HNF-4 ⁇ acts includes a gene having AF1 which is a binding site of HNF-4a phosphorylated by RSKB in a promoter or enhancer, for example, PEPCK gene.
  • Yet another embodiment of the present invention relates to a production inhibitor of a gene product of a gluconeogenesis-related gene.
  • the gene product production inhibitor comprises the phosphorylation inhibitor as an active ingredient in an effective amount thereof.
  • the production inhibitor of this gene product is preferably a PEPCK gene product production inhibitor comprising the above phosphorylation inhibitor as an active ingredient and an effective amount thereof.
  • Another embodiment of the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4a.
  • This identification method can be constructed using a pharmaceutical screening system known per se. Moreover, this identification method can be implemented using the phosphorylation system or phosphorylation method according to the present invention.
  • a condition that enables interaction between RSKB and HNF-4a is selected, and a compound (test compound) with RSKB and Z or HNF-4a is brought into contact under these conditions.
  • R Using a system that uses a signal and Z or marker to detect the interaction between SKB and HNF-4a, by detecting the presence or absence or change of this signal and Z or marker, RSKB and HNF-4a Identify compounds that inhibit the interaction.
  • whether a test compound inhibits the interaction between RSKB and HNF-4a is determined by the binding of RSKB and HNF-4a in the presence of the test compound and Z or RSKB.
  • the signal generated by phosphorylation of HNF-4a or the binding and Z or phosphate marker and the signal generated by the binding and Z or phosphate in the absence of the test compound are This can be done by comparing the binding and Z or phosphorylation markers. In the absence of a test compound, there is a signal generated by the binding and Z or the phosphoric acid, and the binding and Z in the presence of the test compound are compared with the binding and Z or the phosphorylation marker. Alternatively, when the signal generated by the phosphorylation or the binding and Z or phosphate markers are reduced or eliminated, the test compound can be determined to inhibit the interaction between RSKB and HNF-4a. .
  • the conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • the contact between RSKB and NF or HNF-4a and the test compound may be performed before the interaction between RSKB and HNF-4a, or may be performed by coexisting with the interaction.
  • a signal is a signal that itself can be directly detected by its physical or chemical properties, and a marker is a signal that can be detected indirectly using its physical or biological properties as an indicator. Point to.
  • signals include enzymes such as luciferase and green fluorescent protein, and radioactive isotopes.
  • the marker include a reporter gene such as chloramphenicol acetyl transferase gene, or a detection epitope tag such as 6 X His-tag. Signals and markers are not limited to these exemplified substances, and any labeling substance generally used in compound identification methods can be used. Can be. These signals or markers may be used alone or in combination of two or more. Methods for detecting these signals or markers are well known to those skilled in the art.
  • Still another embodiment of the present invention relates to a method for identifying a compound that inhibits phosphate phosphate of HNF-4a by RSKB.
  • This identification method can be constructed using a pharmaceutical screening system known per se.
  • this identification method can be carried out using the phosphorylation system or phosphorylation method according to the present invention.
  • RSKB and Z or HNF-4a are brought into contact with the test compound under the condition, and RSKB is used.
  • HNF-4 by RSKB by detecting the presence or absence or change of this signal and Z or marker using a system that uses the signal and Z or marker to detect HNF-4a phosphate
  • a compound that inhibits the phosphate of a can be identified.
  • whether or not the test compound inhibits HNF-4a phosphate by RSKB is determined by RSKB in the presence of the test compound. It can be carried out by comparing the signal generated by ⁇ or the phosphate marker and the signal or phosphate marker generated by the phosphate in the absence of the test compound. The signal generated by the phosphorylation in the absence of the test compound or the marker of phosphorylation is reduced or the signal generated by the phosphorylation in the presence of the test compound or the phosphate marker is reduced or When it disappears, it can be determined that the test compound inhibits phosphorylation of HNF-4a by R SKB.
  • RSKB and HNF-4a may be in vitro or in vivo.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • RSKB and ⁇ ⁇ ⁇ or HNF-4a can be contacted with the test compound before the phosphorylation reaction of RSKB and HNF-4 ⁇ ! Also good.
  • Phosphorylation of HNF-4a by RSKB can be conveniently detected by measuring the presence or absence and the Z or change of the amount of phosphorylated HNF-4a.
  • Phosphorylated HNF— 4 a Quantification of the amount can be carried out using a well-known method for measuring phosphate protein. For example, the amount of phosphate-type HNF-4 ⁇ can be quantified by Western blotting using an antibody against phosphate-type HNF-4a.
  • the detection of phosphorylated HNF- 4 ⁇ is, Arufatauro labeled radioisotope Rinsani ⁇ reaction, for example using [ ⁇ - 32 ⁇ ] ⁇ , amino acid residues RESULTS HNF- 4 a of the reaction This can be done by measuring the radioactivity of [ ⁇ - 32 ⁇ ⁇ ] transferred to the group. Specifically, referring to Example 2 to be described later, detection of phosphate salt of HNF-4 Q; by RSKB can be performed.
  • the detection of phosphorylation of HNF-4 ⁇ by RSKB can also be performed by measuring the presence or absence of HNF-4 ⁇ activity and wrinkles or changes.
  • the phosphorylation of HNF-4a by RSKB can be detected by detecting the transcriptional activity of the PEPCK gene promoter.
  • Detection of the transcriptional activity of the PEPCK gene promoter can be carried out by reporter assembly using a reporter gene having a PEPCK gene promoter.
  • cells expressing HNF-4 ⁇ and RSKB are transfected with a plasmid containing a reporter gene incorporating the PEPCK gene promoter region upstream, and the expression level of the reporter gene is measured. By doing so, detection of HNF-4 ⁇ phosphate by RSKB can be performed.
  • the expression level of the reporter gene when the test compound is brought into contact with the cell is compared with the expression level of the reporter gene when the test compound is not brought into contact with the test compound.
  • the expression level when the test compound is brought into contact with the cells is reduced or eliminated, it can be determined that phosphorylation of HNF-4a by RSKB is inhibited by the test compound.
  • Another embodiment of the present invention relates to a method for identifying a compound that inhibits the binding between RSKB and HNF-4a.
  • This identification method can be constructed using a pharmaceutical screening system known per se. Further, this identification method can be carried out using the phosphoric acid system or the phosphoric acid method according to the present invention.
  • whether or not the test compound inhibits the binding between RSKB and HNF-4a is determined by the signal generated by the binding of RSKB and HNF-4a in the presence of the test compound or the This can be carried out by comparing the marker of binding with the signal generated by the binding in the absence of the test compound or the marker of binding.
  • the test compound is It can be determined that the binding between RSKB and HNF-4a is inhibited.
  • the conditions enabling the binding of RSKB and HNF-4a may be any of in vitro and in vivo conditions.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • the contact between RSKB and ⁇ or HNF-4a and the test compound may be performed before the binding reaction of RSKB and HNF-4a, or may be carried out by coexisting in the binding reaction.
  • the binding between RSKB and HNF-4 ⁇ can be performed using a protein detection method known per se.
  • the binding between RSKB and HNF-4 ⁇ can be detected by detecting a complex containing RSKB and HNF-4a by a known protein detection method.
  • the protein detection method include Westamplot method, immunoprecipitation method, pull-down method, two-hybrid method, and fluorescence resonance energy transfer method. These methods can be used alone, or a combination of these methods can be used to detect the desired protein.
  • a complex containing RSKB and HNF-4a can be easily detected.
  • Preferred examples of the labeling substance include tag peptides such as HA-tag and FLAG-tag.
  • test compound examples include compounds derived from chemical libraries and natural products, or compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a.
  • test compound examples include compounds derived from chemical libraries and natural products, or compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a.
  • compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a Alternatively, based on the peptide structure of the binding site of HNF-4 ⁇ and RSKB and the phosphorylation site of HNF-4a by Z or RSKB, The compound obtained by in-situ is also suitable as the test compound.
  • the compound obtained by the identification method according to the present invention can be used as a phosphate inhibitor of HNF-4a by RSKB or a gene product production inhibitor of a gene on which HNF-4a acts.
  • the inhibitor of phosphorylation of HNF-4a by RSKB or the gene product production inhibitor of the gene that HNF-4a acts on is selected by considering the balance between biological usefulness and toxicity. It can be prepared as a composition. In the preparation of the pharmaceutical composition, these inhibitors can be used alone or in combination.
  • Yet another embodiment of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the phosphate inhibitor according to the present invention as an active ingredient.
  • the pharmaceutical composition according to the present invention can be used as a preventive and Z or therapeutic agent for diseases caused by phosphate knot of HNF-4a by RSKB. It can also be used for prevention and Z or treatment of the disease.
  • another embodiment of the present invention relates to a preventive and Z or therapeutic agent for a disease caused by phosphorylation of HNF-4a by RSKB, and a preventive and Z or therapeutic method for the disease.
  • the preventive and Z or therapeutic agent for the disease contains the phosphorylation inhibitor.
  • the prevention and Z or treatment method of the disease can be achieved by using the phosphorylation inhibitor or the phosphorylation inhibition method.
  • Examples of diseases caused by phosphorylation of HNF-4a by RSKB include diseases caused by an increase in the gene product of a gene on which HNF-4a acts.
  • HNF-4a phosphorylated by RSKB promotes binding to the transcription site upstream of the PEPCK gene and contributes to the gene product production of the gene.
  • the disease caused by the phosphorylation of HNF-4a by RSKB is caused by an increase in the gene product of a gene involved in gluconeogenesis, for example, an increase in the gene product of the PEPCK gene.
  • diseases caused by abnormal gluconeogenesis such as diabetes.
  • the pharmaceutical composition according to the present invention is usually preferably prepared as a pharmaceutical composition containing one or more pharmaceutical carriers in addition to the active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical preparation according to the present invention is appropriately selected from a wide range. . Usually, it is appropriate that the amount is in the range of about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight.
  • Examples of pharmaceutical carriers include fillers, fillers, binders, moisturizers, disintegrants, lubricants, diluents and excipients that are generally used depending on the form of use of the preparation. it can. These are appropriately selected and used depending on the dosage form of the preparation to be obtained.
  • water pharmaceutically acceptable organic solvent
  • collagen polyvinyl alcohol, polybutylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin Xanthan gum, gum arabic gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, ratatose and the like.
  • These may be used alone or in combination of two or more according to the dosage form of the pharmaceutical composition.
  • the stabilizer examples include human serum albumin, ordinary L amino acids, sugars, and cellulose derivatives. These can be used alone or in combination with a surfactant or the like. In particular, according to this combination, the stability of the active ingredient may be further improved.
  • the L-amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like.
  • Sugars are not particularly limited, for example, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, dextran, hydroxypropyl starch, chondroitin Any deviation from polysaccharides such as sulfuric acid and hyaluronic acid, and their derivatives.
  • Cellulose derivatives are not particularly limited, such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropenoresenorelose, hydroxypropinoremethenoresenorelose, sodium canoleboximethylcellulose, etc. ,.
  • surfactant there are no particular limitations on the surfactant, and both an ionic surfactant and a nonionic surfactant can be used.
  • the surfactant include polyoxyethylene glycol solvent. Vitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, fatty acid glyceride and the like are included.
  • Buffering agents include boric acid, phosphoric acid, acetic acid, citrate, ⁇ -aminocaproic acid, glutamic acid and sputum or their corresponding salts (for example, sodium salts, potassium salts, calcium salts, magnesium salts thereof) Examples thereof include alkali metal salts and alkaline earth metal salts.
  • Examples of the isotonic agent include sodium chloride sodium, potassium salt potassium, sugars, and glycerin.
  • Examples of the chelating agent include sodium edetate and citrate.
  • the pharmaceutical and pharmaceutical composition according to the present invention can be used as a solution preparation, and after freezing, drying and storing it, it contains water, physiological saline and the like at the time of use. It can be used after being dissolved in a buffer solution or the like to prepare an appropriate concentration.
  • the dose range of the medicine and the pharmaceutical composition is not particularly limited, and the effectiveness of the contained ingredients, the administration form, the administration route, the type of the disease, the nature of the subject (weight, age, medical condition and use of other medicines Or the like) and the judgment of the doctor in charge.
  • a suitable dose is, for example, in the range of about 0.01 ⁇ g to 100 mg, preferably about 0.1: g to lmg, per kg of body weight of the subject.
  • these dosage changes can be made using general routine experimentation for optimization well known in the art.
  • the above dose can be administered once to several times a day, and may be administered intermittently at a rate of once every several days or weeks.
  • the pharmaceutical composition When administering the pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicines necessary for the prevention and Z or treatment of the target disease. May be. For example, you may mix
  • the administration route can be selected from systemic administration or local administration! In this case, an appropriate administration route is selected according to the disease, symptoms and the like.
  • parenteral routes include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Or it can be administered by the oral route.
  • transmucosal administration or transdermal administration can be performed.
  • Various administration forms can be selected according to the purpose. Typical examples are solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules, and aqueous solutions. Solutions, ethanol solution preparations, suspensions, fat emulsions, ribosome preparations, inclusions such as cyclodextrins, and liquid dosage forms such as syrups and elixirs. Depending on the route of administration, these preparations can also be administered orally, parenterally (infusions, injections), nasal preparations, inhalants, vaginal preparations, suppositories, sublingual, eye drops, ear drops, ointments, creams. And can be prepared, molded and prepared according to ordinary methods.
  • Powders, pills, capsules, and tablets are excipients such as ratatoses, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc.
  • it can be produced using a binder such as polybutyl alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin.
  • solid pharmaceutical carriers are used for the production of tablets and capsules.
  • Suspending agents can be produced using water, sugars such as sucrose, sorbitol and fructose, dallicols such as polyethylene glycol, and oils.
  • Solutions for injection can be prepared using a carrier that also has a salt solution, a glucose solution, or a mixture of saline and glucose solution.
  • Ribosomeization is performed by, for example, adding a solution in which the above active ingredient is dissolved in a solvent (such as ethanol) to a solution in which phospholipid is dissolved in an organic solvent (such as chloroform), and then distilling off the solvent. This can be carried out by adding a phosphate buffer, shaking, sonicating and centrifuging, and then collecting the supernatant by filtration.
  • a solvent such as ethanol
  • an organic solvent such as chloroform
  • Fat emulsification is carried out, for example, by mixing and heating the above active ingredients, oil components (vegetable oils such as soybean oil, sesame oil and olive oil, MCT, etc.), and emulsifiers (phospholipids etc.) It can be carried out by adding the required amount of water and emulsifying and homogenizing using an emulsifier (homogenizer, such as a high-pressure jet type or ultrasonic type). It can also be lyophilized.
  • emulsification aids that may be added with emulsification aids when making fat emulsions include dariserine and saccharides (eg, glucose, sorbitol, fructose, etc.).
  • cyclodextrin clathrate is a precipitate formed by adding a solution in which cyclodextrin is heated and dissolved in water to a solution in which the above active ingredient is dissolved in a solvent (ethanol or the like), and then cooling to precipitate. Can be carried out by filtering and sterilizing and drying.
  • cyclodex used For the string cyclodextrins ( ⁇ , 13, ⁇ type) having different pore diameters may be appropriately selected according to the size of the active ingredient.
  • reagent kit comprising a polynucleotide encoding a and at least one of a vector containing the polynucleotide.
  • the reagent kit can be used, for example, in the identification method according to the present invention.
  • RSKB or HNF-4a-derived partial peptide for example, a peptide having an amino acid sequence ability at a site where RSKB and HNF-4a interact, or a reagent having a combined force obtained by the above identification method , And reagent kits containing these are also included within the scope of the present invention.
  • This reagent kit measures signals and Z or markers for detecting phosphorylation of HNF-4a by RSKB, their detection agents, reaction diluents, buffers, detergents, and reaction stop solutions. It may contain substances required for the implementation of In addition, the reagent kit can include stabilizers and substances such as Z or preservatives. For formulation, it is sufficient to introduce formulation means for each substance to be used. These reagents and reagent kits are useful, for example, for molecular studies on the involvement of HNF-4a and RSKB in diseases caused by HKB-4 ⁇ phosphate caused by RSKB, such as diabetes.
  • Non-patent Document 34 ((p- Bromocmnamyl) ammo no etnyl] — 5— isoquino lines ulfonamide) (Non-patent Document 34) Judge the degree. The blood glucose concentration is reduced to about half as described above.
  • RSKB activity can be measured by preparing an anti-activated RSKB antibody and performing immunoprecipitation or immunoprecipitation with an anti-RSKB antibody and measuring in vitro kinase activity.
  • Prediction of the protein that interacts with RSKB was carried out as follows according to the in silico prediction method described in Patent Document 1: (i) The amino acid sequence of RSKB was decomposed into oligopeptides of a certain length, and (ii ) Search the database for the amino acid sequence of each oligopeptide or a protein having an amino acid sequence homologous to the amino acid sequence, (iii) perform local alignment between the obtained protein and RSKB, and (iv ) We predicted that proteins with high local alignment scores interacted with R SKB.
  • the local alignment score was set to 25.0 or higher, as in the method described in International Publication No. W001Z67299.
  • HNF is predicted to have a function of interacting with RSKB.
  • Human HNF-4a was phosphorylated by RSKB was examined in an in vitro phosphorylation test.
  • Human HNF-4 ⁇ and RSKB were each transiently expressed in cultured mammalian cells and then prepared from the cells. The activity of RSKB was confirmed using CREB1, and PKA known to phosphorylate HNF-4 was used as a positive control for phosphorylase. [0169] ⁇ Material>
  • RSKB expression plasmid (RSKB / pCMV-Tag2) was constructed as shown below.
  • Human RSKB cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, manufactured by Clontech), and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an animal cell expression plasmid pCMV-Tag2 (Stratagene), to which a FLAG tag was added at the N-terminus, to construct an RSKB expression plasmid (R SKB / pCMV-Tag2).
  • the amino acid sequence encoded by the cloned RSKB cDNA was identical to that disclosed in the NCBI protein database as accession number NP-003933 (protein name is RPS6KA4).
  • HNF-4a ZpcDNA3.1ZHis A human HNF-4 ⁇ expression plasmid (HNF-4a ZpcDNA3.1ZHis) was constructed as shown below.
  • Human HNF-4a cDNA was obtained from human brain polyA + RNA by RT-PCR.
  • an expression plasmid for animal cells to which a (6 X His) —Xpress tag is added at the N-terminus, incorporated into pcDNA3.1 / His (Invitrogen) HNF-4 ⁇ expression plasmid (HNF-4 a ZpcDNA 3.lZHis) was constructed.
  • the amino acid sequence encoded by the cloned HNF-4a cDNA was identical to that disclosed in the Swiss-Prot database as accession number P41235 (protein name HNF4A).
  • a human CREB1 expression plasmid (CREBlZpcDNA3.lZHis) was constructed as shown below.
  • Human CREB1 cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, Clontech), and the sequence was confirmed by sequencing. Then, add (6 X His) -Xpress tag to the N-terminal to the expression plasmid for animal cells pcDNA3. L / His (Invitrogen) to construct a CREB1 expression plasmid (CR EBl / pcDNA3. L / His) did.
  • the amino acid sequence encoded by the cloned CREB1 cDNA was identical to that disclosed in the NCBI protein database as accession number NP-004370 (protein name CREB1).
  • RSKBZpCMV— Tag2 A 10 gZ Petri dish RSKB expression plasmid (RSKBZpCMV— Tag2) The cells were tranfected using 6 (Roche Diagnostics). After culturing for 2 days, sodium arsenite was added to the medium to a final concentration of 50 mM and treated at 37 ° C. for 30 minutes to recover the cells.
  • buffer A 50 mM Tris-HCl (pH 7.5) / lmM esteramine tetraacetic acid (EDTA) / lmM Ethylene glycol bis (2-aminoethyl ether) —N, N, ⁇ ', ,, tetraacetic acid (EGTA) ⁇ 10% glycerol ⁇ 500 ⁇ dithiothreitol (DTT) / 5 mM NaPPi / lmM Na VO / 50 mM NaF / 1% Triton—XI 00) 4ml
  • FLAG M2 affinity gel (manufactured by Sigma) was added in an amount of 100 1 and mixed by inversion at 4 ° C for 1 hour. After mixing, the gel is washed twice with 10 times the volume of buffer B, and 10 times the volume of buffer C (50 mM Tris—HCl (pH 7.5) / 0. ImM EGTA / 10% Glycerol ⁇ . 5 M DTT / 150 mM NaCl ) Washed once. The gel was then washed with 400 / zl of buffer D (50 mM Tris—HCl (pH 7.5) / 0.
  • cAMP-dependent protein kinase As the cAMP-dependent protein kinase (PKA), Ushi PKA (cAMP-dependent protein kinase, catalytic subunit, manufactured by Promega) was used.
  • the immunoprecipitation phosphate test was performed as shown below.
  • HEK293T cells with 7 x 10 5 cells were sputum cultured at 37 ° C in the presence of 5% CO (60 mm diameter petri dish), 5 g HNF-4a expression plasmid (HNF-4a / pcDNA3.l / His) or CREB1 expression plasmid (CREBlZpcDNA3.l / His) was transfected into cells using FuGENE6 (Roche's Diagnostatus) . After culturing for 2 days, the cells were washed with chilled PBS (—) and RIPA buffer (50 mM Tris—HCl (pH 8.0) / 150 mM).
  • adenosine triphosphate 0.5 1 ⁇ 32 ⁇ — ⁇ (5 Ci)
  • 8.5 1 distilled water totaled 29 1 and reacted at 30 ° C for 30 minutes I let you.
  • 10 HNF-4a / protein G-sepharose prepared as above, was added to 1.5 1 ⁇ , 3 1 10X kinase buffer, 2 1 ⁇ ⁇ ATP, 0. ⁇ 32 ⁇ —ATP (5 / zCi), 3.5 / zl IX kinase buffer, 8.5 1 distilled water totaled 29 ⁇ 1 and incubated at 30 ° C for 30 minutes .
  • EMSA The effect of phosphate on the ability of HNF-4a to bind to the AF1 sequence in the PEPCK gene promoter region was examined by EMSA. Phosphorylation of HNF-4a was performed using RSKB. In addition, in order to investigate the specificity of RSKB, EMSA was similarly performed using PKA, which is known to phosphorylate HNF-4a.
  • the AF1 probe used was the following two probes designed and synthesized by the AF1 equivalent sequence force, which is the HNF-4a binding site in the promoter region of the human PEPCK gene (Sigma Genosis Japan). ): AF1 / S / HNF4 (5′-GTG ACCTTTG ACTA-3 ′) (SEQ ID NO: 7) and AF1ZASZHNF4 (5′—AT AGTCAAAGGTCA-3 ′) (SEQ ID NO: 8) (FIG. 4).
  • the promoter region (SEQ ID NO: 15) of the human PEPCK gene (NCBI accession number U31519) is shown in FIG.
  • the AF1 equivalent sequence is shown in FIG. 4 by surrounding it with a bold frame.
  • HNF-4 ⁇ expression plasmid (HNF-4a ZpCMV-Tag2) was constructed from the HNF-4 ⁇ expression plasmid (HNF-4a ZpcDNA 3. lZHis) constructed in Example 1 at the Eco RI site.
  • the cDNA sequence of HNF-4a was recombined into an expression plasmid for animal cells (pCMV-Tag2, manufactured by Stratagene) with an N-terminal FLAG tag.
  • HNF-4a expression of HNF-4a in cells and preparation and purification of HNF-4a from the cells were performed as follows. Cell count 1.2 ⁇ 10 6 HEK293T cells at 37 ° C, 5% CO
  • the F-4a expression plasmid (HNF-4a ZpCMV-Tag2) was transfected into cells using FuGENE6 (Roche Diagnostics).
  • the cells were washed with chilled PBS (-), and 4 ml of buffer A (50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycerol Z500 M DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 1% Triton—XI 00) was added and pipetted well, and the mixture was stirred at 4 ° C. for 30 minutes. Thereafter, NaCl was added to a final concentration of 150 mM, and the mixture was centrifuged at 10,000 g for 10 minutes at 4 ° C.
  • buffer A 50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycerol Z500 M DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 1% Triton—X
  • Radiolabeling and purification of the AF1 probe was performed as follows.
  • the synthesized A F1 probes were dissolved in distilled water so that each of them was lOOpmolZ ⁇ 1, heated at 100 ° C for 2 minutes and at 38 ° C for 1 hour, and then naturally cooled to anneal the DNA. Dilute this 10-fold to obtain 0.5 ⁇ l, 1 ⁇ l of ⁇ 4 polynucleotide kinase (manufactured by Koeisha), 6 1 ⁇ ” 2 P—ATP (60 Ci), 2 1 10 X Protruding End Kinase Buffer (500mM Tris—HCl (pH8.0) / 100mM MgCl / lOOmM 2—Mercaptoethanol
  • Protruding End means ⁇ protruding end '' and Protruding End Kinase Buffer is a T4 polynucleotide. This buffer is used when the photokinase adds a phosphate group at the ⁇ position of ⁇ -ATP to the ⁇ -end of the protruding end of double-stranded DNA.
  • HNF-4 ⁇ ′ DNA complex a band indicating a complex of HNF-4a and AF1 probe (hereinafter referred to as HNF-4 ⁇ ′ DNA complex) was detected.
  • the addition of the anti-HNF-4 ⁇ antibody showed a supershift in which the mobility of the HNF-4 ⁇ 'DNA complex was reduced. From this result, the band detected in the non-anti-HNF-4 ⁇ antibody-supplemented band was HNF-4 It was confirmed that the a-DNA complex was shown.
  • the amount of HNF-4a ′ DNA complex was increased by the phosphorylation treatment with RSKB. On the other hand, phosphorylation treatment with sputum did not detect an increase in the amount of HNF-4 ⁇ .DNA complex.
  • the amount of HNF-4a 'DNA complex decreased as the amount of RSKB used for the phosphorylation of HNF-4a decreased. From this result, it can be considered that the binding ability of HNF-4 ⁇ to the AF1 sequence decreased as the amount of RSKB used for the phosphorylation treatment of HNF-4a decreased.
  • phosphoric acid treatment with PKA increases the amount of HNF-4a 'DN A complex! That is, no enhancement of the binding ability of HNF-4 ⁇ to the AF1 sequence was observed.
  • HNF-4a phosphorylated with RSKB was found to have enhanced DNA binding ability to the AF1 sequence compared to the untreated sample. It was also found that the enhancement of the DNA binding ability of HNF-4a phosphorylated with RSKB was dependent on the dose of RSKB used for phosphorylation. On the other hand, HNF-4 Q; that had been phosphated with PKA showed only the same DNA binding ability as the untreated sample. This indicates that the DNA-binding ability of HNF-4a by RSKB treatment is specific.
  • the effect of RSKB on the transcriptional activity of HNF-4a in the PEPCK gene promoter was determined using HeLa cells (human cervical cancer-derived cell line) and HepG2 cells (human liver cancer-derived cell line) using luciferase reporter assembly. investigated.
  • a reporter gene a gene (PE PCK AF1 promoter-dependent luciferase reporter) in which a firefly luciferase gene is fused downstream of a region containing AF 1 which is a cis element of HNF-4a in the human PEPCK gene promoter region was used. .
  • RSKB expression plasmid used was RSKB / pCMV-Tag2 prepared in Example 2 [0189] Dominant negative RSKB (S343A) expression plasmid (RSKB (S343A) / pCMV- Tag2) is RSKB / pCMV- Tag2 as a saddle, using QuikChange Site-Directed Mutagenesis kit (Stratagene) RSKB The amino acid substitution at position 343 was performed. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine.
  • the protein RSKB (S343 A) expressed by this expression plasmid is an inactive mutant RSKB in which the kinase activity disappears by substitution of the 343rd serine of RSKB with alanine.
  • HNF-4a expression plasmid used was HNF-4a / pCMV-Tag2 prepared in Example 3.
  • a MAPK11 expression plasmid (MAPK11 / pCMV-Tag4) was constructed as shown below.
  • Human MAPK11 (p38-j82) cDNA was obtained from human brain whole cDNA (Human brain whole Quick-clone cDNA, Clontech) by PCR, and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an expression plasmid for animal cells pCMV-Tag4 (Stratagene) to which a FLAG tag was added at the C terminus, and a MAPK11 expression plasmid (p38-beta2 / pCMV-Tag4) was constructed.
  • the amino acid sequence encoded by the cloned MAPK11 cDNA is the same as the Swiss-Prot database accession number Q 15759 (protein name is MAPK11ZSAPK2B).
  • PEPCK AF1 promoter-dependent luciferase reporter plasmid PEPCK
  • AF1 promoter / pGL3 The construction of AF1 promoter / pGL3) was performed as shown below.
  • PEPCK A AF1 promoter-dependent luciferase reporter plasmid (PEPC
  • the KA AFl promoter ZpGL3) was constructed as shown below. From the cloned human PEPCK gene promoter region, a portion not containing AF1 (base sequence 934-1406 of U31519) was obtained by PCR. After confirming the sequence by sequence
  • PGL3 Base (manufactured by Promega) and inserted.
  • pMMGR that expresses a rat glucocorticoid receptor was used (Non-patent Document 36).
  • phRL null plasmid or pRL—expressing Renilla luciferase as a reporter gene
  • SV40 plasmid (Promega) was used.
  • SB203580 was purchased from Promega.
  • Reporter assembly in HeLa cells was performed as follows. 6 X 10 4 Zwell HeLa cells at 37 ° C in the presence of 5% CO
  • the cells were washed with chilled PBS (—), and the firefly luciferase activity and the renilla luciferase activity in the cell lysate were measured with Dual-Lusif erase Reporter Assay kit (Promega).
  • the firefly luciferase activity value is divided by the Renilla luciferase activity value. After, viewed in multiples relative to controls group (PEPCK AF1 promoter ZpGL3 only).
  • Reporter assembly in HepG2 cells was performed as follows. 1 x 10 4 Zwell HepG2 cells at 37 ° C in the presence of 5% CO After sputum culture (24 well plate (2.0 cm 2 Z well), 0.5 g PEPCK AF1 promoter ZpGL3, 0.5 ⁇ g (HNF-4 a / pCMV ⁇ Tag2, 0.25 g and 0 5 ⁇ g RSKBZpCMV— Tag2, 0.5 ⁇ g MAPKl lZpCMV— Tag4, 0.5 ⁇ g pMMGR and 0.05 ⁇ g internal control pRL—SV40 with a preset combination of FuGENE6 (Roche The cells were transferred using the “Diagnostics Co., Ltd.” using the empty vector pC MV-Tag2 (Stratagene) so that the total amount of DNA was constant in each experimental group.
  • SB203580 was added to the medium so that the final concentration was 10 ⁇ immediately before transfection with FuGENE 6.
  • Dexamethasone was added to the final concentration at 1 hour 20 hours after the transfection.
  • PBS was added to the culture medium and cultured for 24 hours. After washing, the firefly luciferase activity and renilla luciferase activity in the cell lysate were measured with the Dual-Lusiferase Reporter Assay kit (Promega) .
  • the transcriptional activity was the value of the luciferase activity value. After dividing by RS, it was expressed as a multiple of the RSKB non-expression group (HNF-4a 0.5).
  • HNF-4a phosphorylated by RSKB promotes the transcriptional activity of the promoter through binding of the PEPCK gene promoter to AF1.
  • HNF-4 ⁇ from the group that co-expressed MAPK11, RSKB, and HNF-4a (HNF-4 ⁇ 0.5 + RSKB 0.5 + MAPK11 0.5)
  • Transcriptional activity against PEPCK AF1 promoter increased by 3 times or more compared to the RSKB non-expression group (HNF-4 ⁇ 0.5) (FIG. 12).
  • HNF-4 ⁇ phosphorylated by RSKB also promotes the transcriptional activity of the PEPCK gene promoter including AF1 in HepG2 cells.
  • HepG2 cells (human liver cancer-derived cell line) are transiently expressed with wild type RSKB or inactive mutant RS KB and MAPK11, an upstream kinase of RSKB, and the PEPCK gene The expression of was measured.
  • RSKB / pCMV-Tag2 prepared in Example 2 was used as the wild-type RSKB expression plasmid.
  • SKBZpCMV—Tag2 causes N-terminal FLAG tag RSKB (sometimes referred to as FLAG—RSKB) force S to be expressed.
  • the inactive mutant RSKB expression plasmid (RSKB—S196AZS343AZT568AZP CMV-Tag2) is the wild type RSKB expression plasmid RSKBZpCMV— Tag2 is used as a saddle and the QuikChange Site-Directed Mutagenesis kit (Stratagene) is used. The three amino acid residues 196, 343 and 568 were replaced with alanine. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine. (Non-patent document 34).
  • RSKB expressed by RSKB—S196A / S343A / T568A / pCMV—Tag has its kinase activity lost by substituting its 196th and 343th serine and 568th threonine with alanine.
  • This inactive mutant RSKB has a FLAG tag attached to its N-terminus.
  • MAPK11 expression plasmid As the MAPK11 expression plasmid, MAPKlZpCMV-Tag4 prepared in Example 4 was used.
  • the anti-phosphorylated RSKB antibody was prepared according to a previous report (Non-patent Document 34) by SCRAM, which produced anti-phosphorylated RSKB antibody PS360 that recognizes phosphorylation of the 360th serine of RS KB.
  • Hep G2 cells were seeded on a 6-well plate at a cell number of 5 ⁇ 10 5 Zwell and cultured in a MEM medium containing 10% FBS at 37 ° C. under conditions of 5% CO 2/95% air. -After sputum culture, wild type
  • RSKB expression plasmid (RSKBZpCMV—Tag2) or inactive mutant RSKB expression plasmid (RSKB—S196A / S343A / T568A / pCMV—Tag2) 2 ⁇ g 11 Expression plasmids (MAPKlZpCMV—Tag4, C-terminal FLAG tag) were transfected into cells using 2 ⁇ g of Lipofectamine 2000 (invitrogen). The total DNA amount was corrected by empty vector (P CMV-Tag2) such that 4 mu g in each experimental group. After transfection, the cells were cultured for 2 days.
  • PEPCK gene PCR was detected by RT-PCR. Specifically, the cells were washed with PBS, collected using trypsin ZEDTA, and total RNA was extracted from the cells using RNeasy Mini Kit (manufactured by Quiagen). CDNA was synthesized from the collected total RNA using Omniscript RT kit (manufactured by Qiagen) using random primer 9mer and oligo go dt primer. PEPCK gene PCR was performed using the obtained cDNA as a template. In addition, GAPDH gene PCR was performed as an internal control. Primer sequences and PCR cycle conditions were as previously reported (Non-patent Documents 40 and 41). Under these conditions, the PEPCK gene produces a 575 bp PCR product and the GAPDH gene produces a 209 bp PCR product.
  • each gene transfected into the cells was detected by Western blotting. Specifically, after washing the cells with PBS, the cells were collected using trypsin ZEDTA, 1501 of a cell lysis buffer (manufactured by Cell Signaling) was added, and the cells were allowed to stand for 15 minutes on ice to lyse the cells. . Thereafter, centrifugation was performed at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was recovered and used as a cell lysate. The cell lysate was supplemented with an equal volume of 2 X SDS sample buffer and heated at 100 ° C for 5 minutes, and used as an SDS-PAGE sample.
  • a cell lysis buffer manufactured by Cell Signaling
  • Proteins were separated by SDS-PAGE using a 5 to 20% gradient gels, after transfer to Immobiron- P SQ membrane (Millipore (Millipore) Co. Ltd.) was detected for each protein.
  • FLAG—RSKB and MAPK11—FLAG were detected with an anti-FLAG M2 antibody (manufactured by Sigma).
  • Phosphorylated RSKB was detected with an anti-PS360 antibody, and / 3-tubulin was detected with an anti-tubulin antibody (-235 (manufactured by Santa Cruz). Detection was carried out using a fluorescently labeled secondary antibody using an Odyssey imaging system (Aloka).
  • This anti-phosphorylated RSKB antibody is an antibody recognizing the phosphate of the 360th serine of RSKB.
  • the RSKB kinase activity is important for the 360th serine phosphate, and the presence or absence of phosphate at this amino acid site is consistent with the kinase activity. (Non-patent Document 34). From this result, it was clarified that the inactive mutant RSKB used in this example lost its kinase activity.
  • HepG2 cells co-expressing wild-type RSKB and MAPK11, and inactive mutant RSKB (RSKB (S 196AZS343AZT5 68A)) and wild-type RSKB and inactive mutant RSKB in HepG2 cells co-expressing MAPK11
  • RSKB S 196AZS343AZT5 68A
  • MAPK11 wild-type RSKB and inactive mutant RSKB in HepG2 cells co-expressing MAPK11
  • RSKB and HNF-4a were transiently co-expressed in HeLa cells and immunorecipitated with RSKB. The binding of HNF-4a was examined.
  • RSKB / pCMV-Tag 2 prepared in Example 2 was used as the RSKB expression plasmid.
  • HNF-4a ZpCMV-Tag2 prepared in Example 3 was used.
  • RSKBZpCMV—Tag2 expresses the N-terminal FLAG tag HNF-4a (sometimes referred to as FLAG-HNF-4a).
  • B expression plasmid (RSKBZpCMV—Tag2) 0.5 ⁇ g and HNF-4 ⁇ expression plasmid ( ⁇ NF-4a / pCMV-Tag2) 0.5g or empty vector (pCMV-Tag2, Stratagene) ) 0.5 ⁇ g was transferred into cells using FuGENE6 (Roche Diagnostics) (total DNA amount was 1 ⁇ g). After transfection, the cells were cultured for 2 days.
  • the binding between RSKB and HNF-4a in the cells was detected by immunoprecipitation. Specifically, the cells were washed with cold PBS, and cell lysis buffer (20 mM HEPES (pH 7.5) / 150 mM NaCl / lmM EDTA / 1% Triton X—100Z Protea Zein Hibita Kuttel (manufactured by Sigma) )) 500 ⁇ l was added and left on ice for 15 minutes to lyse the cells. Thereafter, the lysed cells were centrifuged at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was collected and used as a cell lysate.
  • cell lysis buffer (20 mM HEPES (pH 7.5) / 150 mM NaCl / lmM EDTA / 1% Triton X—100Z Protea Zein Hibita Kuttel (manufactured by Sigma)
  • HNF-4a was precipitated with an anti-HNF-4a antibody, and as a result, co-precipitation of RSKB was detected depending on HNF-4a (Panel A in Fig. 15). From these results, it was found that HNF-4 binds to RSKB in cells.
  • the liver was lysed under ice cooling (cell lysis buffer (manufactured by Cell Signaling) (20 mM Tris—HCl (pH 7.5), 150 mM NaCl / lmM Na EDTA / lmM EGTA / 1% Triton / 2. 5 mM pyrroline)
  • Anti-HNF-4a antibody (manufactured by Santa Cruz Co., Ltd.) was added to the prepared cell lysate and mixed by inverting for 1 hour at 4 ° C. Add protein G sepharose 4 Fast Flow (Amersham 'Falmacia' manufactured by Biotech) blocked with Saline (TBS) (50 mM Tris-HCl / 150 mM N aCl)) (pH 8.0), and add 1 at 4 ° C. Mix by inversion for an hour.
  • wash protein G sepharose twice with cell lysis buffer add 2X SDS sample buffer, heat at 100 ° C for 5 minutes, and use as SDS-PAGE sample.
  • Liver cell lysates are prepared from diabetic animals (ZDF faZfa) and normal animals (ZDF-lean) in the same manner as in Experimental Example 1 above.
  • Anti-RSKB antibody and protein G seph arose are added thereto, and RSKB is recovered by the same immunoprecipitation method as in Experimental Example 1 above.
  • the kinase activity of the recovered RSKB was measured using the synthetic substrate talebitide (CREBtide (manufactured by Santa Cruz). ;)) Detected by in vitro kinase assay using (SEQ ID NO: 9) (Non-patent Document 34).
  • RSKB-binding resin was added to CREBtide (33 ⁇ M) and kinase buffer (Kinase buffer (50 mM Tris-HCl (pH 7.5) / 0. ImM EGTA / 140 mM KCl / 5 mM NaPPi / 10 mM MgCl)). Mix with ⁇ - 32 P- ATP and bring to 22 ° C
  • H-8 9 blood glucose concentration and insulin concentration (Levis Insulin Kit, manufactured by Shibayagi) are measured.
  • the HNF-4 ⁇ phosphate and RSKB activities are measured in the same manner as in Experimental Examples 1 and 2 above, and the expression level of PEPCK is detected by Western blotting.
  • Administration of H-89 decreases both RSKB activity, phosphorylation of HNF-4 ⁇ , and expression of PE PCK, and gluconeogenesis is suppressed, resulting in a decrease in blood glucose concentration.
  • Inhibition of production of a gene product of a gene on which HNF-4a acts by the phosphorylation inhibitor of HNF-4a and / or the method of inhibiting phosphorylation by RSKB provided in the present invention, such as the PEPCK gene Inhibition of the production of the gene product can be carried out. It also makes it possible to prevent and / or treat diseases caused by increased gene products of genes that act on HNF-4 ⁇ . More specifically, for example, diseases caused by an increase in PEPCK gene products, more specifically, diabetes and the like can be prevented and Z or treated. Thus, the present invention is very useful for the prevention and Z or treatment of diseases caused by excessive HNF-4a phosphate involved in gluconeogenesis-related gene expression.
  • SEQ ID NO: 1 RSKB cDNA.
  • Sequence number 2 RSKB.
  • SEQ ID NO: 3 HNF-4 ⁇ cDNA.
  • SEQ ID NO: 4 HNF—4 ⁇ .
  • SEQ ID NO: 5 CREB 1 cDNA 0
  • SEQ ID NO: 6 CREB1.
  • SEQ ID NO: 7 Probe oligonucleotide designed based on the sequence of AF1.
  • SEQ ID NO: 8 Probe oligonucleotide designed based on the sequence of AF1.
  • SEQ ID NO: 9 Synthetic substrate CREBtide.
  • SEQ ID NO: 10 Partial RSKB oligopeptide showing high score in local alignment between RSKB and HNF-4 ⁇ .
  • SEQ ID NO: 11 A partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4 ⁇ for local alignment!
  • SEQ ID NO: 12 Partial oligopeptide of HNF-4a showing a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 13 RSKB partial oligopeptide showing a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 14 Partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 15 Promoter region of the PEPCK gene, which corresponds to the sequence represented by nucleotides 841 to 1440 of the gene published as accession number U31519 in the NCBI nucleotide database .

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Abstract

L'invention concerne un procédé d'inhibition de la phosphorylation d'un facteur de transcription pour un gène associé à la gluconéogenèse et un inhibiteur de phosphorylation. En utilisant la découverte qu'un facteur de transcription HNF-4α est phosphorylé par le RSKB et que la liaison de celui-ci à la région promoteur d'un gène associé à la gluconéogenèse est favorisée par celui-ci, l'invention a pour objet : un procédé de phosphorylation du HNF-4α par le RSKB, un procédé d'inhibition de la phosphorylation et un inhibiteur de phosphorylation ; un procédé de production d'un produit génétique d'un gène sur lequel le HNF-4α agit comme un facteur de transcription, un inhibiteur de production et un procédé d'inhibition de la production ; un médicament servant à prévenir et à traiter une maladie provoquée par la phosphorylation du HNF-4α par le RSKB et un procédé servant à prévenir et à traiter celle-ci ; un procédé d'identification d'un composé inhibant l'interaction entre le RSKB et le HNF-4α, la phosphorylation du HNF-4α par le RSKB ou la liaison du RSKB au HNF-4α ; un composé obtenu par le procédé d'identification ; un polynucléotide codant pour le RSKB, HNF-4α ou le RSKB ou HNF-4α ; et un kit de réactifs contenant un vecteur ayant ce polynucléotide.
PCT/JP2005/019518 2004-10-22 2005-10-24 Procédé d'inhibition de la phosphorylation d'un facteur de transcription pour un gène associé à la gluconéogenèse et inhibiteur de phosphorylation WO2006043701A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HALL R.K. ET AL: "The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids", PROC. NATL. ACAD. SCI. USA, vol. 92, no. 2, 1995, pages 412 - 416, XP002994007 *
HUNTER T. ET AL: "The regulation of transcription by phosphorylation", CELL, vol. 70, no. 3, 1992, pages 375 - 387, XP002994008 *
PIERRAT B. ET AL: "RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK)", J. BIOL. CHEM., vol. 273, no. 45, 1998, pages 29661 - 29671, XP002120478 *
TOMAS-ZUBER M. ET AL: "Control sites of ribosomal S6 kinase B and persistent activation through tumor necrosis factor", J. BIOL. CHEM., vol. 275, no. 31, 2000, pages 23549 - 23558, XP002994005 *
VIOLLET B. ET AL: "Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4", MOLL. CELL. BIOL., vol. 17, no. 8, 1997, pages 4208 - 4219, XP002994006 *

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