WO2003062427A1 - Methode de criblage de medicaments ameliorant la resistance a l'insuline - Google Patents
Methode de criblage de medicaments ameliorant la resistance a l'insuline Download PDFInfo
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- WO2003062427A1 WO2003062427A1 PCT/JP2003/000546 JP0300546W WO03062427A1 WO 2003062427 A1 WO2003062427 A1 WO 2003062427A1 JP 0300546 W JP0300546 W JP 0300546W WO 03062427 A1 WO03062427 A1 WO 03062427A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
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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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
Definitions
- the present invention relates to a method for screening a protein that interacts with PPAR in a ligand-dependent manner, and a method for screening an insulin sensitizer using the protein.
- thiazolidine derivatives which have been shown to be effective as insulin sensitizers, act as agonists of the peroxisomal proliferator-activated receptor gamma (PPAR r).
- PPAR r peroxisomal proliferator-activated receptor gamma
- Non-Patent Document 1 Since the affinity of the thiazolidine derivative for PPAR ⁇ correlates with the hypoglycemic action in vivo, the action to improve insulin resistance of this group of compounds is considered to be an action via PPAR ⁇ (Non-Patent Document 2). reference). For this reason, the method of detecting agonists of PPAR r has been considered to be an effective method for screening drugs for treating insulin-resistant diabetes.
- Type 1 diabetes is caused by the destruction of beta cells in the brain and requires insulin for treatment.
- type 2 diabetes non-insulin dependent diabetes mellitus
- type 1 diabetes is very small and type 2 accounts for the majority.
- type 2 diabetic patients insulin metabolism is hardly promoted by insulin, and insulin resistance is caused. For this reason, research has been conducted not only on simple hypoglycemic drugs for diabetes but also on the treatment of type 2 diabetes, which promotes glycemic metabolism by improving insulin resistance.
- PPAR belongs to the nuclear receptor superfamily, and binds to a response element upstream of the target gene as a transcription promoter activated by ligand binding, and regulates its transcription. Induction is known (see Non-Patent Document 3).
- PPAR polypeptide containing at least one subtype of PPAR.
- PPAR polypeptide containing at least one PPAR.
- PPARr a thiazolidine derivative, which is a therapeutic agent for diabetes
- fibrate-based drugs which have long been used as lipid-lowering drugs, are known to have a PPAR ligand effect.
- a strong decrease in serum triacylglycerol levels has been observed (non-patented). References 10-11).
- Non-Patent Document 12 It has been reported that PPAR agonists stop cell proliferation and promote cell differentiation (see Non-Patent Document 12). PPAR r is particularly expressed in adipose tissue (see Non-Patent Documents 13 to 14), and homozygous mice do not induce adipocyte differentiation. Also
- Non-Patent Document 15 the mechanism by which thiazolidine derivatives improve insulin resistance is a substance that induces insulin resistance as a result of PPAR ragonist rapidly promoting adipocyte differentiation.
- Non-Patent Document 16 It is thought that suppression of TNF of production, promotion of glucose transporter expression in peripheral tissues, and suppression of free fatty acid production occur. As a result, it is thought that glucose uptake into cells is increased and hyperglycemia is improved. (See Non-Patent Document 16).
- PPAR transcription factor activity like other nuclear receptors, interacts with transcriptional coactivators Attempts have been made to identify factors that are necessary and interact with PPAR.
- biochemical methods have been used to study the binding of existing nuclear receptor interacting factors to PPAR r, and SRG-1 (see Non-Patent Document 19), GBP / p300 ( 20), ⁇ 205, TRAP220 (see non-patent document 21), SMRT (see non-patent document 22), Gadd45 (see non-patent document 23), and RIP140 (see non-patent document 24). It has been reported to interact.
- RXR retinoid X receptor
- the above-mentioned Gadd45 and PGG-1 show that, despite the fact that ligand-dependent interactions with nuclear receptors including subtype PPAR ⁇ have been detected in the yeast hybrid system, Regarding PPAR r, ligand dependence is observed only by a biochemical method (see Non-Patent Document 24).
- Sensitivity, probe-pair for biochemical and yeast-based methods It has been described that the effect of PPAR r ligand cannot be detected efficiently by the yeast two-hybrid system due to the difference in the ratio of interacting factors (see Non-Patent Document 24).
- biochemical methods are suitable for detecting the interaction between a pair of proteins, but it is difficult to comprehensively search for proteins that interact with a specific protein.
- the yeast two-hybrid system it is possible to search the library for proteins that interact with a specific protein.
- EGHLP / Echl has a structure that is expected to have two types of enzyme active regions in the molecule, enol CoA hydrolase and dienoy GoA isomerase that act on fatty acid metabolism.
- Patent Document 31 See Non-Patent Document 31
- A0P2 has a peroxidase-like sequence in the molecule, it is called anti-oxidant protein 2 (anti-oxidant protein 2 in Genbank accession number XM-001415), and there are various reports on the sequence. (See Patent Documents 8 to 12).
- Non-Patent Document 32 It has been reported that the actual physiological activity of the protein functions as calcium-independent phospholipase A2 (see Non-Patent Document 32). In mice, the locus of the Aop2 protein has been reported as a causative gene for polycystic nephropathy. (See Non-Patent Document 33). Thus, it is clear that A0P2 has a different function from the molecular function expected from its amino acid sequence structure, and its original physiological function has not been determined.
- FLJ13111 is a protein whose function is unknown, and its nuclear target sequence ⁇ glycosylation suggesting the presence in the cell nucleus in the molecule from the amino acid sequence There was no information suggesting a molecular function from the amino acid sequence structure except for the existence of a site that can be subjected to nucleosides.
- Patent Document 1 JP-A-11-56369
- Patent Document 2 WO 00/55350 pamphlet
- Patent Document 3 WO 02/29103 pamphlet
- Patent Document 4 International Publication No. 02/00677 Pamphlet
- Patent Document 5 WO 01/49716 pamphlet
- Patent Document 6 WO 00/37643 pamphlet
- Patent Document 7 WO 01/75067 pamphlet
- Patent Document 8 WO 98/43666 pamphlet
- Patent Document 9 International Publication No. 02/12328 Pamphlet
- Patent Document 10 WO 02/29086 Pamphlet
- Patent Document 11 WO 02/06317 Breadlet
- Patent Document 12 International Patent Publication No. WO 01/55301
- Patent Document 13 European Patent Application Publication No. 1074617
- Patent Document 14 WO 00/58473
- Non-Patent Document 1 J. Biol. Ghem., 1995, Vol. 270, p.12953-12956
- Non-Patent Document 2 J. Med. Ghem., 1996, Vol. 39, p. 665-668
- Non-Patent Document 3 Gel U 995, Vol. 83, p. 835-839
- Non-Patent Document 4 Proc. Natl. Acad. Sci. USA, 1994, Vol. 91, p. 7355-7359
- Non-Patent Document 6 Diabetes, 1997, Vol. 46, p. 433-439
- Non-Patent Document 7 Diabetes Care, 1996, Volume 19, Issue 2, p. 151-156
- Non-Patent Document 8 Diabetes Care, 1992, Vol. 15, No. 2, p. 193-203
- Non-Patent Document 9 Diabetologia, 1996, Vol. 39, p. 701-709
- Non-Patent Document 10 Proc. Natl. Acad. Sci. USA, 1997, Vol. 94, ⁇ ⁇ 4312-4317
- Non-Patent Document 11 Drugs, 1990 'Vol. 40, No. 2, p. 260-290
- Non-Patent Document 12 Jpn. J. Cancer Res., 1999, Vol. 90, p. 75
- Non-patent Document 13 Genes and Dev., 1994, Vol. 8, p. 1224-1234
- Non-Patent Document 14 Gel 1, 1994, Vol. 79, p. 1147-1156
- Non-Patent Document 15 ol. Cell, 1999, Volume 4, p.597-609
- Non-Patent Document 16 J. Biol. Chem., 1995, Vol. 270, p.12953-12956
- Non-Patent Document 17 Diabetes Frontier, 1999, Vol. 10, p. 811-818
- Non-Patent Document 18 Diabetes Frontier, 1999, Vol. 10, p. 819-824
- Non-Patent Document 19 Gene Expr., 1996, Volume 6, p.185-195
- Non-Patent Document 20 vJ. Biol. Chem., 1999, Vol. 274, p.7681-7688
- Non-Patent Document 21 Mol. Gel I. Biol., 2000, Volume 20, p. 8008-8017
- Non-Patent Document 22 Pro. Nat I Acad. Sci. USA, 1998, Vol. 95, p. 2920-2925
- Non-Patent Document 23 Biochem. Biophys. Res. Commun., 2000, Vol. 272, No. 1, ⁇ ⁇ 193-198
- Non-Patent Document 24 Mol Endocrinol., 1998, Vol. 12, No. 6, p. 864-881
- Non-Patent Document 25 Ann. Rev. Cell Dev. Biol., 1996, Volume 12, p.335-363
- Non-Patent Document 26 Proc. Natl. Acad. Sci. USA, 1991, Vol. 88, p. 9578-9582
- Non-Patent Document 27 J. Biol. Chem., 1999, Vol. 274, p.7681-7688
- Non-Patent Document 28 Cell, 1998, Vol. 92, p. 829-839
- Non-Patent Document 29 EMB0 J., 1999, Vol. 18, No. 13, p. 3676-3687
- Non-Patent Document 30 Biochim. Biophys. Acta., 1997, Volume 1, No. 1350, p.27-32
- Non-Patent Document 31 J. Biol. Ghem., 1998, 273: 1: p.349-355
- Non-Patent Document 32 J. Biol. Ghem., 1997, 272, 16 p.10981
- Non-Patent Document 33 Genomics, 1997, Vol. 42, No. 3, p. 474-478 Disclosure of the Invention
- the present inventors have developed a unique method of intercalating a highly active PPARragonist at a high concentration in the yeast two-hybrid system, and have developed a PPART based on the presence of an agonist with a high effect of improving glucose metabolism (main effect).
- a group of proteins that bind to this and a group of proteins that bind to PPAR ⁇ depending on the presence of an agonist with a high edema (side effect) -inducing effect were identified.
- EGHLP ECH-1 (enoyl-CoA hydratase) -like protein (enoy-GoA hydratase like protein: EGHLP) is converted to PPARr as a molecule that binds to PPARy depending on the main action agonist.
- human antioxidant protein 2 antioxidant protein 2 or selenium glutathione peroxidase, acidic calcium-independent phospholipase A2; Genbank Accession No. XI001415, hereinafter abbreviated as A0P2 was found. It was found that overexpression of EGHLP in cells markedly suppressed ligand-dependent PPAR r transcription-inducing activity.
- the present invention has been completed by providing a method for identifying and screening a novel insulin sensitizer that detects a substance that specifically contributes to the main effect via PPAR and does not cause side effects, and has completed the present invention. .
- a poly (bar) encoding a region containing at least the region from position 204 to position 505 of the PPAR r protein represented by SEQ ID NO: 2
- a region containing at least the region from position 204 to position 505 of the PPAR r protein represented by SEQ ID NO: 2 as a bait is coded.
- a polypeptide comprising a lost, substituted, or missing or inserted amino acid sequence, and which interacts with PPAR in a ligand-dependent manner andb) PPAR represented by SEQ ID NO: 2 or SEQ ID NO: 6 Cells expressing the white matter
- Polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8, or amino acid having ⁇ to 10 amino acids deleted, substituted, and Z or inserted in the amino acid sequence represented by SEQ ID NO: 8 A polynucleotide that contains an acid sequence and encodes a polypeptide that interacts with PPAR in a ligand-dependent manner, and ii) a response element to which the PPAR protein represented by SEQ ID NO: 2 or SEQ ID NO: 6 can bind
- A) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 or 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 8 Comprises a deletion, substitution, and Z or inserted amino acid sequence, and further interacts with PPAR in a ligand-dependent manner; andb) PPAR represented by SEQ ID NO: 2 or SEQ ID NO: 6 Cells expressing the white matter,
- a method for detecting whether a test substance promotes PPAR-mediated amelioration of glucose metabolism comprising a step of analyzing a change in PPAR-dependent transcription activity-inducing activity;
- a method for screening for an insulin sensitizer comprising a step of analyzing a change in the transcriptional activity-inducing activity of PPAR.
- (10) i) a step of bringing a test substance into contact with the cell according to (4), andU) a change in interaction with the test substance or a change in the interaction with the test substance using the expression of a reporter gene as an index. Analyzing the change in PPAR transcriptional activity-inducing activity by the method, wherein the method comprises detecting whether or not the test substance promotes PPAR-mediated edema-inducing activity,
- (11) i) a step of bringing a test substance into contact with the cell according to (4); ii) a change in interaction with the test substance or a change in PPAR transcriptional activity-inducing activity by the test substance using reporter gene expression as an index. And iii) selecting a test substance that does not increase reporter activity, a method for screening an insulin sensitizer having no edema-inducing activity,
- polypeptide comprising the amino acid sequence represented by SEQ ID NO: 17, or an amino acid having 1 to 10 amino acids deleted, substituted, and / or inserted in the amino acid sequence represented by SEQ ID NO: 17
- a method for screening for an insulin sensitizer comprising a step of analyzing a change in activity
- W001 / 55301 shows the same sequence as A0P2 identified by the present inventors, and treatments for a number of diseases are listed as uses of substances that modulate the function of the sequence, including treatment for diabetes mellitus. However, there is no working example or description that the sequence is involved in diabetes.
- the amino acid sequence identical to FLJ13111 consisting of SEQ ID NO: 17 and the nucleotide sequence encoding this sequence are disclosed in ⁇ 10746 ⁇ , but there is no description of the name of a specific disease involving FLJ13111 in this report.
- a sequence having homology to the nucleotide sequence of FLJ13111 is disclosed in W000 / 58473, and the use of a substance that modulates the function of the sequence is listed for the treatment of many diseases, including treatment for diabetes. There is no working example or description that the sequence is involved in diabetes. Therefore, the fact that EGHLP, A0P2, and FLJ13111 bind to PPAR is a finding discovered by the present inventors for the first time, and furthermore, by using these, PGH specifically contributes to the main effect via PPAR, thereby causing side effects.
- a novel insulin sensitizer, screening which detects a substance that does not cause lipase, is an invention made by the present inventors for the first time.
- FIG. 1 is a diagram showing agonist selectivity in binding of a ligand-dependent PPARr interacting factor to PPARr.
- FIG. 2 is a diagram showing a comparison between the expression levels of Ech1 in diabetic model mice KKAVTa (KKA y ) and G57BL / KsJ-db / db (db / db) and normal mice.
- FIG. 3 is a view showing the expression distribution of EGhl by tissue.
- FIG. 4 is a graph showing the inhibitory effect of EGHLP on the ligand-dependent transcription induction ability of PPARr.
- FIG. 5 is a diagram showing the promoting action of A0P2 on the ligand-dependent transcription induction ability of PPARr.
- FIG. 6 is a diagram showing screening of a main action-specific PPARr ligand using the action of EGHLP and A0P2 on the ligand-dependent transcription inducibility of PPARr.
- Figure 7 shows the effect of FLJ13111 on PPARr's ability to induce ligand-dependent transcription.
- FIG. 8 shows the diabetes model mice KKA y / Ta (KKA y ), G57BL / Ks J-db / db (db / db) and normal mice (G57BL / 6J (C57BL), C57BL / KsJ- + m / + m ( FIG. 2 is a diagram showing a comparison of the expression level of FLJ131 11 in m + / m +))).
- FIG. 9 is a diagram showing the transcription inducing activity of the FLJ131 11 promoter and the effect of pioglitazone or FU131 11 overexpression on the activity.
- FIG. 10 is a graph showing the effect of EGHLP overexpression on pioglitazone-induced increase in triglyceride content in mouse 3T3-1J cells.
- FIG. 11 is a graph showing the ability of PPAR r to induce transcription of PPARr depending on pioglitazone or compound XF in the presence or absence of FU131 11.
- FIG. 12 is a graph showing the effect of piodaritazone or compound XF on the expression level of sodium- rhodium ATPase in renal epithelial cells.
- main effect means “improving glucose metabolism”
- side effect j means “action causing edema”.
- the action of improving glucose metabolism refers to an action of promoting the function of taking sugar (glucose) in blood into cells and consuming it as energy, and accumulating it as an energy storage substance such as glycogen.
- “Edema-inducing action” refers to the effect that extracellular fluid accumulates and accumulates in the interstitium to induce edema (swelling).
- Mainnly acting ligand has a high effect of improving glucose metabolism (main action).
- ligand means "a ligand having a high edema (side effect) inducing effect”.
- Ligands having a high glucose metabolism improving effect-inducing effect are more preferably prepared by the method of Miwa I et al. (Glin Chim Acta 37, 538, 1972) under the conditions of Example 1.
- the concentration of the compound required to reduce blood glucose by 25 ⁇ 1 ⁇ 2 compared to the control group is lower than that of a conventional PPAR r ligand (for example, pioglitazone) by a factor of 5 or less, more preferably It is preferable that the concentration is as low as 1/10 or less.
- GW-7282 and G262262570 described later can be exemplified.
- the blood glucose level is measured by an enzymatic method combined with lucose oxidase.
- a ligand having a high edema-inducing effect the circulating plasma volume measurement method of Brizzee BL et al. (J. Appl. Physiol. 69 ( 6): In 2091-2096, 1990), more preferably, under the conditions of Example 1, when 100 mgZkg of the compound was administered, the circulating plasma volume of 25% or more compared to the control group in 2 weeks was increased. Preference is given to those that result in an increase or an increase in circulating plasma volume of at least 15% compared to conventional PPAR r ligands (eg pioglitazone). For example, GW-7282 and GL-100085 described later can be exemplified.
- Test cell is a cell that can measure ligand-dependent interaction between rppAR and PPAR EGHLP using the expression of reporter gene as an index ", and reports the ligand-dependent interaction between rppAR and PPAR A0P2 as a reporter gene Cells that can be measured using the expression of rppAR as an index, or cells that can measure the ligand-dependent interaction between rppAR and PPAR interaction FLJ13111 using the expression of a reporter gene as an index.
- the yeast one-hybrid system utilizes the fact that yeast transcriptional activators have a DNA-binding domain and a transcriptional activation domain, and the initiation of transcriptional activation requires interaction between the two.
- This system detects the interaction between (1) a target protein bound to the DNA binding region and (2) a protein bound to the transcription activation region.
- a bait indicates a target protein bound to a DNA binding region
- a prey indicates a protein bound to a transcription activation region.
- the “ G DNA library” extracts and separates tens of thousands of mRNAs (instructing the amino acid sequence of proteins by copying genetic information) synthesized in cells, and complements them with reverse transcriptase. It synthesizes DNA and incorporates the terminal processing into the vector.
- the rpPAR ligand binding region j is a region to which a ligand of PPAR binds, and is a region including the region from No.
- the “DNA binding region” is a region that functions to bind to DNA, and has a DNA binding ability to a response sequence, but does not independently have a transcription activation ability.
- the DMA-binding region of the GAL4 transcription factor is located on the N-terminal side (the region containing amino acids 1 to 147).
- the PPAR-interacting polypeptides encoded by the polynucleotides contained in the gene for the PPAR-interacting protein for preparing test cells herein include:
- polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17;
- SEQ ID NO: 4 In the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17.Includes an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, and / or inserted, and is ligand-dependent.
- a polypeptide that binds to PPAR hereinafter referred to as a functionally equivalent variant
- a protein consisting of an amino acid sequence having a homology of 90 ⁇ 1 ⁇ 2 or more of the amino acid sequence ⁇ : represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17, and which binds to PPAR in a ligand-dependent manner.
- a polypeptide hereinafter, referred to as a homologous polypeptide
- Functional equivalent variants include “polypeptides that are proteins that contain the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17 and that bind to PPAR in a ligand-dependent manner”; In the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 17, 1 to 10, preferably 1 to 7, and more preferably 1 to 5 amino acids are deleted, substituted, and / or inserted. And a polypeptide that binds to PPAR in a primary ligand-dependent manner ”or“ 1 to 10, preferably 1 to 7, more preferably 1 to 10, in the amino acid sequence represented by SEQ ID NO: 8.
- Homologous polypeptide is preferably a polypeptide j that contains a deleted, substituted, and / or inserted amino acid sequence of up to 5 amino acids and that binds to PPAR in a side effect ligand-dependent manner.
- Tide is a protein consisting of an amino acid sequence having 90% or more homology to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17, and which binds to PPAR in a ligand-dependent manner. Is not particularly limited as long as the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 17 is preferably 90% or more, more preferably 95 ⁇ 0 or more, and still more preferably 980/0 or more.
- a protein that binds to PPAR in a main-acting ligand-dependent manner with respect to the amino acid sequence represented by SEQ ID NO: 8, preferably 90% or more, more preferably An amino acid having a homology of preferably 95% or more, more preferably 980/0 or more.
- the “homology j” is a default by a Glustal program (Hig ins and Sharp, Gene 73, 237-244, 1998; Thompson et al. Nucleic Acid Res. 22, 4673-4680, 1994). Means the values obtained using the parameters provided in the above.The above parameters are as follows.
- the PPAR-interacting polypeptide contained in the test cell of the present specification has been described.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, its functional equivalent variant, and its homologous polypeptide are collectively referred to as PAR interaction ECHLPJ below.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8, its functionally equivalent variant, and its homologous polypeptide are hereinafter collectively referred to as “PAR interaction A0P2”.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, its functional equivalent variant, and its homologous polypeptide are hereinafter collectively referred to as rpPAR-interacting FLJ13111J.
- polynucleotide having the nucleotide sequence encoding PPAR-interacting EGHLP, PPAR-interacting A0P2, or PPAR-interacting FLJ13111 is a polynucleotide having the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17. Any nucleotide, any functionally equivalent variant thereof, or any polynucleotide comprising a base sequence encoding the homologous polypeptide thereof may be used.
- it is a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 17, and more preferably, SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 16.
- Screening for a protein that interacts with PPAR in a ligand-dependent manner which is a useful tool for screening for an insulin sensitizer deviating from side effects according to the present invention based on the described nucleotide sequence.
- a method for screening and a method for screening for an insulin sensitizer different from the side effect utilizing the protein are described below.
- a factor that interacts with PPAR r in a ligand-dependent manner can be comprehensively identified from a cDNA library using the expression of a reporter gene using the yeast two-hybrid system as an index.
- the present invention detects the ligand-dependent interaction between PPAR and its transcription-coupling factor, and does not require detection of the transcription-inducing ability of PPAR itself. No need for existence. Therefore, it is not necessary to use mammalian cells as test cells, and eukaryotic cells such as yeast cells, insect cells, and mammalian cells may be used. Of these, yeast cells can be easily cultured and cultivated quickly, and it is easy to apply genetic recombination techniques such as introduction of foreign genes. In addition, the dependence of ligand on the binding between PPAR and the interacting factor can be efficiently replicated and detected using the same yeast two-hybrid system.
- the yeast two-hybrid system is a method for detecting protein-protein interaction using the expression of a reporter gene as a marker.
- a transcription factor has two regions with different functions, a DNA binding region and a transcription activation region.
- the transcription factor DNA binding A fusion protein consisting of a region and X and a fusion protein consisting of a transcription activation region of transcription factor and Y are simultaneously expressed in yeast cells.
- proteins X and Y interact, the two types of fusion proteins form a transcription complex, which binds to the transcription factor response element (specifically binding DMA site) in the nucleus of the cell. Activates the transcription of a reporter gene located downstream. In this way, the interaction between the two proteins can be detected by replacing the expression of the reporter gene.
- the yeast two-hybrid system is usually used for identifying genes of unknown proteins that interact with a specific protein as a probe.
- binding between the nuclear receptor and some of its transcription coupling factors depends on the presence of the receptor ligand, use a two-hybrid system with externally added ligand.
- the yeast With the mother-to-hybrid system it was difficult to detect the ligand-dependency of PPARr and interacting factors, and exhaustive screening of ligand-dependent PPARr-interacting factors has not been successful.
- the present inventors have foreseen that the reason for this is that, due to the nature of yeast, the permeability of the PPAR ⁇ agonist into cells is low and the sensitivity of detection of ligand dependence is low, and it has been reported that the PPAR ⁇ agonist is a PPAR ragonist.
- the group of the most active compounds act on yeast at a high concentration, we have completed a unique method for the yeast two-hybrid system that can be applied to screening of ligand dependence of PPAR ⁇ and interacting factors. More specifically, this screening can be performed by the method described in the second embodiment.
- Another embodiment of the method comprising detecting a ligand-dependent interacting factor of PPAR r and measuring the effect of a test substance on the interaction includes, for example, a ligand-dependent interaction between PPARy and the interacting factor.
- a method of biochemically detecting the binding for example, glutathione-S-transferase (GST), protein A, -galactosidase, malto-s-binding protein (MBP), etc. are extracted from an extract of cultured cells labeled with RI or the like.
- It can be carried out by directly detecting a protein that binds to a fusion protein comprising a tag binding protein and a ligand binding region of PPARr in the presence of a test substance, purifying the binding protein, and identifying the protein by amino acid sequencing.
- One embodiment of the present invention includes: (i) a fusion gene of at least the ligand binding region of PPARa or r and the DNA binding region of a transcription factor, or a gene encoding the full length region of PPAR or r molecule; Interaction a gene encoding EGHLP, and (iii) a reporter gene linked to a response element capable of binding to the DNA binding region of the transcription factor, or a response element capable of binding PPAR o? Or r.
- the test substance is used in the presence of PPAR ligand.
- PPAR interaction in test cells Selects the main PPAR-mediated effect, which consists of detecting and measuring changes in the inhibitory effect of EGHLP on the transcriptional activation of PPAR caused by the test substance using reporter gene expression as an index. There is a method of detecting whether or not to promote the promotion. In addition, a method of screening a compound that selectively promotes a PPAR-mediated main action by selecting a compound that increases the reporter activity detected by the same detection method may be used.
- a fusion gene of at least a ligand binding region of PPAR ⁇ or r and a DNA binding region of a transcription factor, or a code gene of the full length region of PPAR or r molecule (ii) PPAR interaction A0P2 code gene, and (iii) a reporter gene linked to a response element capable of binding to the DNA binding region of the transcription factor, or a response element capable of binding PPAR a or r.
- a method for detecting a compound that has a PPAR-mediated side effect by detecting and measuring the expression of a reporter gene as an index, and selecting a main effect that is separated from the side effect by the reporter system. Selecting a compound that promotes a method of screening mentioned et be.
- One embodiment of the present invention includes: (i) a fusion gene of at least a ligand binding region of PPAR r and a DNA binding region of a transcription factor, or a gene encoding the full length region of a PPAR r molecule, (ii) PPAR interaction A gene encoding FLJ13111, and (iii) a reporter gene linked to a response element capable of binding to the DNA binding region of the transcription factor, or a reporter gene linked to a response element capable of binding PPAR ⁇ or r.
- PPAR which consists of detecting and measuring A method of detecting whether or not to selectively promote the main action of the subject.
- a method of screening a compound that selectively promotes a PPAR-mediated main effect by selecting a compound that increases the reporter activity detected by the same detection method can also be mentioned.
- the transcription factor used for detecting the transcription inducing ability of PPAR is not limited as long as it is a eukaryotic transcription factor having a region that binds to a specific DNA sequence in the cell nucleus.
- the DNA binding region of the transcription factor may have a DNA binding ability to a response sequence, but may have no ability to activate transcription by itself.
- transcription factors include, for example, the yeast GAL4 protein (Keegan et al.,
- the DNA binding region and the transcription activation region of the GAL4 transcription factor are located on the N-terminal side (the region containing amino acids from the first to 147th positions).
- a DNA sequence to which the DNA binding region of the transcription factor can bind is used.
- the region may be cut out from the upstream region of the gene and used, or the sequence may be synthesized by chemical synthesis and used.
- the reporter gene located downstream of the response element is not particularly limited as long as it is a commonly used one, but is preferably an enzyme gene or the like which is easily quantitatively measured. Examples include the chloramphenicol acetyltransferase gene (GAT) derived from a bacterial transposon, the luciferase gene (LUG) derived from a firefly, and the green fluorescent protein gene (GFP) derived from a jellyfish.
- GAT chloramphenicol acetyltransferase gene
- LEG luciferase gene
- GFP green fluorescent protein gene
- the polynucleotide encoding PPAR o! Or r, the DNA-binding domain of the transcription factor, PPAR interaction EGHLP, PPAR interaction A0P2, or PPAR interaction FLJ13111 contains known amino acid sequence and nucleotide sequence information. It can be isolated from a GDNA library by PCR (Polymerase Cha- in Reaction) or hybridization screening using primers and probes designed and synthesized based on them.
- PPAR interaction EGHLP is identified as the same molecular species, and can be derived from any species that interacts with PPAR in a ligand-dependent manner and affects the transcription-inducing ability of the receptor.
- human L0G115289; GenBank accession number XM—008904, HPXEL GenBank accession number U16660, FitzPatrick DR et al., Genomics 1995, Vol. 27
- human L0G115289; GenBank accession number XM—008904, HPXEL GenBank
- PPAR interaction A0P2 is identified as the same molecular species, and can be derived from any species that interacts with PPAR in a ligand-dependent manner and affects the receptor's ability to induce transcription.
- PPAR interaction FLJ13111 is identified as the same molecular species, and can be derived from any species that interacts with PPAR in a ligand-dependent manner and affects the transcription-inducing ability of the receptor.
- human FLJ13111; GenBank accession number AK023173, related 025082
- mouse human FLJ13111-like protein; GenBank
- accession number XM J 34598 accession number XM J 34598.
- PPARr is identified as the same molecular species, and may be derived from any species as long as it functions in vivo as a nuclear receptor, such as mammals such as humans, mice, and rats. In addition to those derived from animals, they include those derived from African Megafrog. PPAR ⁇ (Dreyer et al., Cel. Vol. 68, 879-887, 1992, Zhu et al., Journal of Biological Chemistry, Vol. 268, pp. 26817-26820, 1993, Kliewer et al., Proc. Natl. Acad Sci. USA, Vol. 91, pp. 7355-7359, 1994, Mukherjee et al., Journal of Biological Chemistry, Vol. 272, pp. 8071-8076, 1997, Elbrecht et al., Biochem. Biophys. Res. Commun. 224, 431-437, 1996, Ghem et al., Biochem. Biophys. Res. Commun. 224
- PPAR rl There are two isoforms of PPAR rl and PPAR r2. Compared to PPAR r2, PPAR rl lacks 30 amino acids at the N-terminal side, but the other amino acid sequences are exactly the same. It is known that both are expressed in adipose tissue.
- a polynucleotide encoding PPAR or r, the DMA binding domain of a transcription factor, PPAR-interacting EGHLP, PPAR-interacting A0P2, or PPAR-interacting FLJ13111 can be obtained, for example, as follows. Without limitation, it can also be obtained by a well-known operation "Molecular Arginngj [Sambrook, J et al., Cold Spring Harbor Laboratory Press, 1989].
- mRNA including those encoding the protein is extracted by a known method.
- the extraction method include a guanidine-thiocyanate 'hot' phenol method, a guanidine-thiocyanate-guanidine-hydrochloric acid method and the like, and preferably a guanidine-thiocyanate cesium chloride method.
- Cells or tissues capable of producing PPAR or PPAR-interacting ECHLP, PPAR-interacting A0P2, or PPAR-interacting FLJ13111 can be produced by using a gene having a nucleotide sequence encoding the protein or a part thereof. It can be specified by a staining method, a stamp lotting method using an antibody specific to the protein, or the like.
- the mRNA may be purified according to a conventional method.
- the mRNA can be adsorbed on an oligo (dT) cellulose column and then purified to be purified.
- mRNA can be further fractionated by sucrose density gradient centrifugation or the like.
- a commercially available extracted mRNA may be used without extracting the mRNA.
- the purified mRNA is subjected to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer to synthesize first-strand cDNA.
- This synthesis can be performed by a conventional method.
- two types of primers sandwiching a partial region of the target gene for example, SEQ ID NO: 9 and SEQ ID NO: 10 for PPAR r, and SEQ ID NO: 2 for PPAR interacting EGHLP SEQ ID NO: 13, PPAR interaction SEQ ID NO: 14 and SEQ ID NO: 15 for A0P2, and PPAR interaction FLJ13111 are subjected to PGR using SEQ ID NO: 18 and SEQ ID NO: 19 to amplify the target gene sequence.
- the target gene sequence can also be amplified by subjecting it to PGR using two types of primers sandwiching a partial region of the target gene.
- the obtained DNA is fractionated by agarose gel electrophoresis or the like.
- the target DMA fragment can also be obtained by cutting the DNA with a restriction enzyme or the like and connecting it. Specifically, it can be obtained by the methods described in Examples 2, 4, 5, 7, 8, 10, and 11.
- DNA encoding each of these regions may be singly or ligated, and ligated downstream of an appropriate promoter.
- an expression system of PPAR or r and PPAR interaction EGHLP in a test cell and an expression system of PPAR or r and PPAR interaction A0P2 in a test cell can be constructed.
- an expression system of PPARr and PPAR interaction FLJ13111 in test cells can be constructed.
- the polynucleotide obtained as described above may be incorporated into an appropriate vector plasmid and introduced into a host cell in the form of a plasmid. These may be configured so that both are contained on one plasmid, or may be configured so that each is contained on a separate plasmid. Alternatively, cells having such a configuration integrated into chromosomal DNA may be obtained and used.
- the reporter gene linked to the response element was also constructed using a general gene recombination technique, this construct was incorporated into a vector plasmid, and the resulting recombinant plasmid was introduced into host cells. Is used. Alternatively, cells having such a configuration incorporated into chromosomal DNA may be obtained and used.
- PPAR may be introduced from the outside, but if adipose-derived cells or kidney-derived cells rich in endogenous PPAR ⁇ are used as host cells, PPARr should be omitted from the above configuration and the response element Only the configuration consisting of a reporter linked to PPAR interaction and EGHLP, omitting PPARr, and only the configuration consisting of reporter and PPAR interaction A0P2 linked to the response element, or only the composition consisting of A0P2 linked to the response element, omitting PPARr Repo It is also possible to introduce only the configuration consisting of one part and the PPAR interaction FLJ13111.
- fragments containing the isolated polynucleotides can be transformed into eukaryotic and prokaryotic host cells by reintegration into an appropriate vector plasmid. Furthermore, by introducing an appropriate promoter and a sequence related to expression into these vectors, the gene can be expressed in each host cell.
- eukaryotic host cells include cells such as vertebrates, insects, and yeast, and vertebrate cells include, for example, monkey COS cells (Gluzman, Y. (1981) Cell, 23, 175-182) and Chinese hamster ovary cell (GH0) dihydrofolate reductase deficient strains (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77, 4216-4220), human embryo For kidney-derived HEK293 cells and cells
- 293-EBNA cells into which the EBNA-1 gene of Epstein Barr Virus has been introduced are often used, but are not limited to these.
- Any substance can be used as long as it can detect the ability of EGHLP to inhibit the induction of PPAR or r transcription induction, the activity of PPAR interaction A0P2 to induce PPAR a or r transcription, or the activity of PPAR interaction FLJ13111 to induce PPAR ⁇ transcription induction.
- vertebrate cell expression vectors those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which are usually located upstream of the gene to be expressed, can be used. May be provided.
- expression vectors include the initial promoter of SV40.
- the expression vector has an SV40 origin of replication, is capable of autonomous replication in COS cells, and has a transcription promoter, a transcription termination signal, and an RNA splice site.
- pME18S (Maruyama, and and Takebe, Y. (1990) Med.
- the expression vector is prepared by the DEAE-dextran method (Luthtnan, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and van der Ed, AJ (1973) Virology, 52, 456-457), a method using FuGENE6 (Boeringer Mannheim), and an electric pulse perforation method (Neumann, E. et a (1982) EMBO J., 1, 841-845) and the like, so that COS cells can be taken up, and thus desired transformed cells can be obtained.
- DEAE-dextran method Lithtnan, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308
- calcium phosphate-DNA coprecipitation method Graham, FL and van der Ed, AJ (1973) Virology, 52, 456-457
- FuGENE6 Boeringer Mannheim
- an electric pulse perforation method
- a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector, for example,
- a desired transformed cell can be obtained using an expression vector such as pGEP4 (Invitrogen).
- the desired transformant obtained above can be cultured according to a conventional method, and the culture produces a target protein group in the cells.
- the medium used for the culture various types commonly used depending on the host cell used can be appropriately selected.
- the c can be used after the addition of serum components such as fetal bovine serum (FBS) if necessary to a medium such as if COS cells RPNH-1640 medium and Dar Beck co corrected Iguru minimal essential medium (DIEM), the For 293-EBNA cells, a medium obtained by adding G418 to a medium such as Dulbecco's Modified Eagle Minimum Essential Medium (DMEM) supplemented with serum components such as fetal bovine serum (FBS) can be used.
- FBS fetal bovine serum
- DMEM Dulbecco's Modified Eagle Minimum Essential Medium
- test cells are cultured in the presence of the test substance, and the PPAR interaction with the PPAR- or r-transcription-inducing ability is detected by expression of the reporter gene. And can be measured.
- Test substance interacts with PPAR
- EGHLP Acts on EGHLP or PPAR, depending on its action PPAR interaction EGHLP PPAR transcription When the inhibitory effect on the inducing activity is diminished, an increase in the expressed reporter activity is observed.
- a test substance is identified as a PPAR main action promoter.
- the test substance binds to PPAR to promote transcription induction ability, while inhibiting the inhibitory effect of PPAR-interacting EGHLP, an increase in the expressed reporter activity is observed.
- Such a test substance is identified as a main action-specific agonist of PPAR.
- the test substance when (3) the test substance binds to PPAR-interacting EGHLP and inhibits the effect of inhibiting the transcription-inducing ability of PPAR, or when the test substance promotes re-degradation that inhibited the expression of PPAR-interacting EGHLP, The increase in reporter activity is observed.
- Such substances have been identified as PPAR-interacting EGHLP inhibitors that promote the main effects of PPAR.
- Each of these (2), (3) and (4) is expected to act as an insulin sensitizer, which deviates from the side effects caused by PPARagonist. More specifically, an insulin sensitizer can be identified and screened by the methods described in Examples 5 and 9. For example, under the conditions described in Example 9, a substance having an IC50 of 10 M or less, preferably a substance having an IC50 of 1 ⁇ M or less can be selected as an insulin resistance improving agent.
- test cells are cultured in the presence of the test substance, and the PPAR interaction with the transcription-inducing ability of PPAR a or r is inhibited by the test substance. Can be detected and measured.
- test substance acts on PPAR-interaction A0P2 or PPARr, and the effect of the PPAR-interaction A0P2 on the PPARy transcription induction activity is attenuated depending on the effect, a decrease in the expressed reporter activity is observed.
- test substances are identified as substances that suppress the side effects of PPAR ⁇ .
- test substance when the test substance binds to PPAR r and promotes the ability to induce transcription and inhibits the promoting effect of PPAR-interacting A0P2, the reporter activity expressed is the same as when PPAR-interacting A0P2 is not co-expressed. It is observed to decrease to a level.
- test substances are identified as PPAR r-selective agonists that are separate from the side effects.
- the test substance when (3) the test substance binds to PPAR-interacting A0P2 and inhibits the effect of promoting the induction of PPAR r transcription, or when the test substance promotes re-degradation that inhibits the expression of PPAR-interacting A0P2. A decrease in re-expressed reporter activity is observed.
- Such substances are identified as PPAR-interacting A0P2 inhibitors that suppress the side effects of PPAR ⁇ . All of these are the side effects of PPAR ragonist. It is expected to act as an insulin sensitizer different from its action.
- the test substance acts on PPAR-interacting A0P2 or PPARr and, depending on the action, enhances the promoting effect of PPAR-interacting A0P2 on PPARr transcription-inducing activity, the increased reporter activity expressed Is observed. Since such a test substance is identified as a substance that strongly induces the side effect of PPARr, selecting a test substance that does not increase reporter activity makes it possible to screen for an insulin sensitizer that does not have edema-inducing activity. it can.
- the test cells are cultured in the presence of the test substance, and it can be detected and measured by the expression of the reporter gene that the promotion of PPAR interaction 11 on the transcription-inducing ability of PPAR ⁇ is enhanced by the test substance. it can.
- the test substance acts on PPAR-interaction FLJ13111 or PPAR ⁇ , and depending on the action, enhances the promoting effect of PPAR-interaction FLJ13111 on PPAR ⁇ transcription-inducing activity, an increase in the expressed reporter activity is observed. Is done.
- Such a test substance is identified as a main effect enhancer of PPAR r.
- test substance when the test substance binds to the PPAR to promote the transcription inducing ability by binding to PPAR and enhances the promoting effect of the PPAR interaction FLJ13111, an increase in the expressed reporter activity is observed.
- a test substance is identified as a principal agonist specific agonist of PPAR.
- test substance when (3) the test substance binds to PPAR-interacting FLJ13111 to enhance the effect of promoting PPAR transcription inducibility, or when the test substance suppresses the re-decomposition that promoted the expression of PPAR-interacting FLJ13111. An increase in reporter activity that is re-expressed is observed.
- Such a substance is identified as a PPAR-interacting FLJ13111 activator that promotes the main action of PPAR.
- Each of these 1, 2 and 3 is expected to act as an insulin sensitizer which is different from the side effect caused by PPARagonist.
- an insulin sensitizer can be identified and screened by the methods described in Examples 11 and 12. For example, under the conditions described in Example 12, a substance having an ED50 of 10 juM or less, preferably a substance having an ED50 of 1 M or less can be selected as an insulin resistance improving agent.
- a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO:
- SEQ ID NO: 26 or SEQ ID NO:
- 1 to 10 nucleotides are deleted, substituted, and transformed by a reporter gene fused to a polynucleotide having a Z or an inserted polynucleotide sequence and having a transcription promoter activity.
- Reporter gene Atsusei is a method for detecting gene regulation using the reporter gene expression as a marker.
- the regulation of gene expression is controlled by a part called the promoter region existing in the 5 'upstream region, and the gene expression level at the transcription stage can be estimated by measuring the activity of this promoter.
- the test substance activates the promoter, it activates the transcription of a reporter gene located downstream of the promoter region.
- the promoter activating effect that is, the expression enhancing effect can be detected by replacing the expression of the reporter gene.
- the reporter gene assay using the promoter region of PPAR-interacting FLJ13111 the effect of the test substance on the regulation of the expression of PPAR-interacting FLJ13111 can be detected by replacing the expression of the reporter gene.
- the ⁇ reporter gene '' fused to the promoter region of FLJ13111 consisting of the nucleotide sequence represented by SEQ ID NO: 26 is not particularly limited as long as it is a commonly used one, but is preferably an enzyme gene or the like which is easily quantitatively measured. .
- Examples thereof include a chloramphenicol acetyltransferase gene (GAT) derived from a bacterial transposon, a luciferase gene (Luc) derived from a firefly, and a green fluorescent protein gene (GFP) derived from a jellyfish.
- the reporter gene may be functionally fused to the promoter region of FLJ13111 consisting of the nucleotide sequence represented by SEQ ID NO: 26.
- PPAR interaction By comparing the expression levels of the reporter gene with and without contacting the test substance with cells transformed with the reporter gene fused to the promoter region of FLJ13111.
- (Ii) Analyzes of test substance-dependent changes in transcription induction activity can be analyzed.
- test substance used in the screening method of the present invention is not particularly limited. Examples thereof include commercially available compounds (including peptides) and various known compounds (including peptides) registered in a chemical file. A group of compounds obtained by a combinatorial technique (NK Terrett, M. Gardner, DW Gordon, RJ oby lecki, J.
- the present invention includes a step of screening using the screening method of the present invention, and a step of preparing a pharmaceutical composition using the substance obtained by the screening, and producing a pharmaceutical composition for improving insulin resistance.
- a method is included.
- Administration may be, for example, oral administration of tablets, pills, capsules, granules, fine granules, powders, or oral solutions, or injections such as intravenous, intramuscular, or joint injections, suppositories.
- parenteral administration such as intravenous injection is preferred for peptides digested in the stomach.
- compositions for oral administration one or more active substances and at least one inert diluent, such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch , Polyvinylpyrrolidone, or magnesium aluminate metasilicate.
- the composition may contain additives other than an inert diluent, for example, a lubricant, a disintegrant, a stabilizer, or a solubilizing or solubilizing agent, according to a conventional method.
- Tablets or The pills can be coated with a sugar coating or a film of a gastric or enteric substance, if necessary.
- Liquid compositions for oral use can include, for example, emulsions, solutions, suspensions, syrups, or elixirs; commonly used inert diluents, such as purified water Or it may include ethanol.
- the composition can contain additives other than inert diluents, for example, wetting agents, suspending agents, sweetening agents, fragrances, or preservatives.
- Parenteral injections may include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
- the aqueous solution or suspension may contain, for example, distilled water for injection or physiological saline as a diluent.
- examples of the diluent of the water-insoluble solution or suspension include propylene glycol, polyethylene glycol, vegetable oil (eg, olive oil), alcohols (eg, ethanol), and polysorbate 80. be able to.
- the composition may further contain a humectant, an emulsifier, a dispersant, a stabilizer, a solubilizing or solubilizing agent, a preservative, and the like.
- the composition can be sterilized by, for example, filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation.
- a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use.
- the dose is determined in consideration of the active ingredient, that is, the substance that inhibits the activation of the LTRPG2 protein, or the strength, symptom, age, sex, etc. of the activity of the substance obtained by the screening method of the present invention. Can be determined as appropriate.
- the dose in the case of oral administration, is usually about 0.1 to 100 mg, preferably 0.1 to 50 mg per day for an adult (assuming a body weight of 60 kg).
- the amount in the case of parenteral administration, in the form of an injection, is 0.01 to 50 mg, preferably 0.0 "! To 10 mg per day.
- GW7282 ((S) -3- [4- [2- (5-methyl-2-phenyloxazol-4-yl), a group of five thiazolidin derivatives that have been reported to act as agonists of PPARr ) Ethoxy] phenyl] -2- (1-pyrrolyl) propionic acid; GlaxoSmithKine, Drug Data Rep 2001, 23 (9): 889), GI-262570 ((S) -2-[(2-benzoylphenyl) ) Amino] -3- [4- [2- (5-methyl-2-phenyl-2-oxazole-4-yl) ethoxy] phenyl] propionic acid; GI axoSmithKIine, WOOO / 38811), GL-100085 (2- (3- (2- (5-Methyl-2-phenyloxazole-4-yl) ethoxy) phenylmethylthio) acetic acid; Ono Pharmaceutical, W099 / 46232), Rosi
- hypoglycemic effect as an indicator of the main effect and increase in circulating plasma volume as an indicator of the effect of causing edema (Arakawa, Masaaki, graduate School of Internal Medicine, Volume 3 Major symptoms-from symptoms to diagnosis-260-266, 1966 Kanazawa et al., Diabetes Frontier, Volume 10, 811-818, 1999; Iwamoto, Diabetes Frontier, Volume 10, 819-824, 1999).
- Test compounds were orally administered to rats (Sprague-Dawley rats; male, 3 weeks old) once daily for 2 weeks at a dose of 100 mg / kg (suspended in 0.5% ethylcel lulose). .
- the measurement of plasma volume was basically performed according to the method described in Appl. Physiol. 69 (6): 2091-2096, 1990. Under anesthesia with ether, 0.25 ml (0.625 mg) of 0.25% Evans Blue solution (physiological saline) was injected into rats via the lower leg vein, and 5 minutes later, blood was collected from the inferior vena cava.
- the plasma was diluted with water, and the plasma volume was determined by dividing the Evans blue concentration (mg / ml) obtained from the absorbance (620 nm) by the injection amount (0.625 mg). Further, the amount (%) of the control group (vehicle-administered group) was calculated based on the plasma volume corrected for body weight (Table 1).
- GW7282 strongly induced both main effects and side effects.
- GI-262570 elicits a relatively high value of the main effects but has weak side effects.
- GL-100085 caused only a minor effect, but strongly caused side effects.
- Example 2 Identification of a protein that interacts with PPARr in a ligand-dependent manner
- G-terminal 302 amino acid containing PPARr DNA binding region and ligand binding region The GDNA to be loaded is converted into a human adipose tissue-derived cDNA library (Clontech;
- Marathon Ready TM cDNA was obtained by polymerase 'chain reaction method (PGR method).
- PGR method polymerase 'chain reaction method
- Gene database Based on the human PPARr2 gene sequence described in GenBank accession number U79012, the yeast two-hybrid expression vector pDBtrp (Invitrogen, having the 77PV gene as a selectable marker) was used.
- homologous regions with 40 nucleotides before and after the multicloning site of the vector were added, and recognition sites for the restriction enzymes KpnI and SmaI were formed on both sides of the inserted PPARr gene fragment, respectively.
- the primers shown in SEQ ID NOS: 9 and 10 were designed as follows.
- the PGR uses a DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo Co., Ltd.) and cycles through 98 ° C (1 minute), 98 ° C (5 seconds), 55 ° C (30 seconds), and 72 ° C (3 minutes). Repeated 35 times.
- the resulting 1004 bp DNA fragment has a PPARr coding region of 302 amino acids from amino acid 204 of PPARr2 to just before the stop codon.
- the vector pDBtrp cut into linear form by restriction enzymes Sal I and Ncol and the PGR fragment containing PPARr GDNA obtained in (1) were simultaneously used for the yeast strain for two-hybrid MaV203 (Invitrogen; Pinn). And transformed by the lithium acetic acid method (G Guthrie, RFink Guide to Yeast Genetics and Molecular Biology, Academic, San Diego, 1991). As a result, homologous recombination occurred in the same yeast cell, and a plasmid (hereinafter abbreviated as pDB-PPARr) was formed in which the PPARr cDNA was inserted into the pDBtrp multicloning site.
- pDB-PPARr a plasmid
- Yeast cells harboring the plasmid are selected by culturing them on a minimal solid synthetic medium (DIFG0) (200/0 agarose) depleted of the plasmid selectable marker tributophan. After processing at room temperature for 30 minutes at room temperature, the plasmid is isolated and purified by the alkaline method (Molecular Cloningj Sambrook, J et al., Cold Spring Harbor Laboratory Press, 1989), and a sequencing kit (Applied Biosystems) ) And a sequencer (ABI 3700 DNA sequencer Applied Biosystems) were used to determine the nucleotide sequence. The PPARr cDNA was inserted with the translational frame matching the coding region of the GAL4 DMA binding region of pDBtII. What you have selected. (3) Yeast two-hybrid screening
- the cells were transformed with 20 jug each of a human kidney GDNA library, a human liver library, or a human skeletal muscle library (and also Clontech's Match Maker cDNA I ibrary), and the cells were transformed into pDB-PPARr and a library.
- the transformants in which both plasmids were introduced were selected by culturing on a solid synthetic minimal medium (DIFG0) (20% agarose) lacking tributophan and leucine, which are the selectable markers for each plasmid. Obtained.
- DIFG0 solid synthetic minimal medium
- the same transformed cell is also a reporter that is expressed when the fusion protein of the GAL4 transcription promoting region binds to the fusion protein of the GAL4 DNA binding region, which was artificially expressed in a single hybrid system.
- Gene / ⁇ ⁇ To select activated cells, remove histidine from the culture medium and further add 3AT (3-AMINO-1, 2, 4-triazole; Sigma), an inhibitor of the enzyme encoded by //, 20 mM The cells were cultured at 30 ° C for 5 days on the minimum solid medium (20% agarose) added.
- a PPARr agonist, GW7282 which strongly induces both main effects and side effects, was added to the medium at a final concentration of 1.5 jwM, and 3AT-resistant, which indicates that a protein that binds to PPARr is expressed in the presence of the agonist Yeast colonies were obtained. After growing these yeast cells on a YPD solid medium for 24 hours with or without the above-mentioned agonist GW7282 at a concentration of 15 M, the binding indicator of a two-hybrid system separate from W is used. The expression of a gene was examined using y3-galactosidase activity as an index.
- Plasmids from Bally were extracted.
- a sequencing kit (Applied Biosystems) using the nucleotide sequence of the gene fragment contained therein as a primer, with the nucleotide sequence represented by SEQ ID NO: 11 (sequence that binds to the GAL4AD region; GenBank Accession No. U29899 derived from Cloning vector pACT2) as a primer And a sequencer (ABI 3700 DNA sequencer Applied Biosystems).
- SEQ ID NO: 11 sequence that binds to the GAL4AD region
- a sequencer (ABI 3700 DNA sequencer Applied Biosystems).
- SRG-1 Smith GL Proc. Natl. Acad. Sci. USA., 20 93 (17) 8884-8888 1996 :
- N-CoR nuclear receptor transcription coupling factor
- Example 3 Detection of ligand-selective interaction between PPARr and EGHLP or A0P2
- the dependence of agonist on the interaction between PPARr and a group of proteins such as EGHLP and A0P2 obtained in Example 2 was described above.
- the S-galactosidase activity of the system was measured as an indicator (Fig. 1; arrowheads in black indicate the compounds whose interaction is greatly changed depending on the concentration difference between the compound with the main action selection and the stripe with the stripes).
- the compound GL-100085 which has a relatively high effect on side effects, similarly induces the binding between PPARr and A0P2 even when the concentration is reduced from 5 / M to 0.
- concentration of PPARr and A0P2 decreased significantly when the concentration was reduced from 5 / iM to 0.5 ⁇ Nl (Fig. 1b).
- Fig. 1b the concentration of PPARr and A0P2 decreased significantly when the concentration was reduced from 5 / iM to 0.5 ⁇ Nl.
- clones # 1, 4, 5, 6, 7 and N-GoR show reduced binding to PPARr even when the concentration of agonist G 262570 or GL-100085 decreases, resulting in the main action of agonist-side effects. No correlation was seen.
- Example 4 Measurement of EGHLP expression level in normal and diabetic model mice Based on the above findings, it was expected that the interaction between EGHLP and PPARr would be involved in the improvement of glucose metabolism, which is the main action via PPARr agonist. Therefore, two types of diabetic model mice KKA y / Ta (lwatsuka et al., Endocrinol. Japon., Vol. 17, pp. 23-35,
- KKAVTa mice and G57BL / KsJ-db / db mice had higher blood sugar and higher body weight than normal mice (KKAVTa mice: blood sugar level 514.2 ⁇ 18.2 mg / dk body weight 49.9 ⁇ 0.7 g, G57BL / KsJ-db / db mouse: blood sugar level 423.7 + 14.1 mg / d and body weight 48.6 ⁇ 0.5 g).
- Blood glucose was collected from the tail vein of the mouse, and the blood glucose was measured using a commercially available kit (Autopack A 'glucose reagent, Boehringer 1. Mannheim) using the glucose oxidase method. These four types of mice were anesthetized with getyl ether, and epididymal fat and pectoralis were removed. Immediately after extraction, they were frozen in liquid nitrogen and stored at -80 ° C.
- RNA extraction The tissue was crushed using a freeze press crusher GRY0-PRESS GP-100 (Microtech, Nichion). The RNA extraction reagent IS0GEN (Futtsubon Gene) was added, and homogenized using a homogenizer ULTRA-TURRAX T-8 (IKA LABORTECH IK). RNA was extracted from these samples according to the protocol attached to the manufacturer. This was treated with DNase (Futtsubon Gene) to degrade the contaminating DNA. Thereafter, phenol black port Holm process, ethanol precipitated, and dissolved in RNase-free H 2 0. MRNA was extracted using QuickPrep Micro mRNA Purification kit (Amersham) according to the attached protocol.
- Affilimetrics DNA array (GeneGhip U74) consists of three subarrays B and G. According to the above protocol of Affymetrix, the labeled GRNA was hybridized to the DNA array, washed, and the fluorescence intensity of each probe was measured.
- correction of measured values between arrays The measured values were corrected between samples, and then corrected between subarrays. Inter-sample correction involves calculating the sum of the fluorescence intensities of the genes on a particular subarray between samples, and adding the measured values of each gene in the other arrays to be equal to the array that showed the highest sum of the fluorescence intensities. A constant magnification was applied to each array. For correction between subarrays, calculate the average value of the fluorescence intensity of the AFFX probe for each subarray, and apply a fixed magnification to the measured value of each gene for each subarray so that the average value is the same for subarrays B and C. Was applied.
- Echl is responsible for the changes in blood glucose that occur as a result of the diabetic condition, because three intraperitoneal doses and no change in tissues 7 hours after the first dose, when blood glucose levels returned to normal levels in the short term. It is thought that the increased expression is not one of the causative factors that induces the diabetic condition, rather than the increased expression.
- a gene encoding a chimeric protein obtained by fusing the GDNA encoding the ligand-binding region of the human PPAR r2 to the G-terminal side of the DNA-binding region (amino acids 1-147) of yeast Gal4 is expressed in an animal cell expression vector pZeoSV (Invitrogen).
- the expression plasmid GAL-PPARr integrated into the multicloning site of the company was prepared.
- a gene fragment encoding the DNA binding region of Gal4 was excised from plasmid pGBT9 (Clontech) using restriction enzymes HindIII and SmaI, and inserted into the pZeoSV multicloning site.
- pZeo-DB a DNA fragment encoding the ligand-binding region of PPAR r was excised from the aforementioned plasmid pDB-PPAR r using Kpn I and Sma I.
- the plasmid GAL-PPARr for expression in animal cells was prepared by inserting Kpn at the multi-cloning site of pZeo-DB between Pvu II sites.
- PCR is a DNA polymerase (Pyrobest DNA
- a primer was designed such that a stop codon was not inserted into EGHLP, and the V5 epitope and HIS6 tag derived from the vector were fused to the G-terminal side.
- a reporter construct (REx8- LuGi,; Shimokawa et al., International Publication No. W099 / 04815) (0.8 ⁇ g nowell) in which eight sequences were repeatedly arranged upstream of the luciferase gene was replaced with pcDNA-EGHLP (0.05-0.2 / g well). ) And transiently cotransfected.
- phosphate buffer hereinafter abbreviated as PBS
- PBS phosphate buffer
- a lysing solution (1 OOmM potassium phosphate (pH 7.8), 0.2% Triton X-100) was added to lyse the cells.
- a luciferase substrate solution 100 1 (Pitka Gene) was added to the cell lysate 1Q0iI, and the amount of luminescence for 10 seconds was measured using an AB-2100 type chemiluminescence measuring device (Atokasha).
- Plasmid PGH110 (Amersham Pharmacy Biotech) carrying both the luciferase reporter gene and the ⁇ -galactosidase expression gene was cotransfected into cells, and ; 8-Galactosidase activity was measured and quantified using a galacto-Light Plus TM system (Applied Biosystems). The above luciferase activity was corrected for each well as the transfusion efficiency of the transgene.
- EGHLP / Ech1 has a structure that is predicted to be two kinds of enzyme active regions in the molecule, enol GoA hydrolase and dienol GoA isomerase, which act on fatty acid metabolism (Fiyol GoA isomerase) (Fi lppula A vJ. Biol. Chem., 273: 1: 349-355 1998). It has long been known that inhibitors of fatty acid metabolizing enzymes lower blood glucose levels in diabetic mice (Collier R et al., Horm. Metab. Res., Vol. 25, No. 1: 9-12, 1993).
- EGHLP has a greater inhibitory effect on PPARr activity
- EGHLP when EGHLP is present in excess, it inhibits PPARr-mediated glucose metabolism and produces energy from lipids through its own fatty acid metabolizing enzyme activity. It is thought to be a molecule responsible for the antagonistic regulation of sugar-fat metabolism, which promotes and decreases PPARr activity when it is reduced, and shifts the energy source of the body to glucose metabolism.
- Example 6 Comparison of A0P2 protein level in normal and diabetic model mice Based on the above findings, it was predicted that the interaction between A0P2 and PPARr would be involved in the induction of edema, a side effect via the PPARr agonist. So diabetes model mouse
- G57BL / 6J was analyzed by fluorescent labeling two-dimensional difference electrophoresis ( ⁇ ⁇ et al., Electrophoresis Vol. 18, pp. 2071-2077, 1997, Tonge et al.
- Proteins were identified using mass spectrometry in a group of proteins in which the protein content was more than twice as large in the disease state model mouse.
- the frozen epididymal fat was homogenized in a Tris buffer containing a rare and amphoteric surfactant using a homogenizer ULTRA-TURRAX T-8 (IKA LAB0RTEGHNIK). According to the protocol attached to the manufacturer, supernatants were obtained from these samples by centrifugation to obtain the following samples for two-dimensional electrophoresis.
- Amersham Fulmasia Biotech protocol was followed. The amount of protein contained was determined by measuring the absorbance of each sample, and the amount containing about 50 g of protein was determined from these, and different amounts of fluorescent dyes (Gy3 and Cy5, Amersham Pharmacia Biotech) were used. After labeling, they were mixed and subjected to first-dimensional isoelectric focusing using an IPG strip (Amersham Pharmacia Biotech).
- the IPG strip Prior to the second dimension of electrophoresis, the IPG strip was Equilibration was carried out with a tris buffer solution containing peptidol, glycerol and dithiothreitol, and further equilibration was carried out with a tris buffer solution containing perea, sodium dodecyl sulfate, glycerol and dithiothreitol in which odoacetamide was dissolved.
- the second dimension electrophoresis was performed using sodium dodecyl sulfate polyacrylamide electrophoresis.
- the gel after completion of two-dimensional electrophoresis is used for each two-dimensional electrophoresis image using excitation and detection wavelengths specific to each fluorescent dye.
- the two electrophoresis images were quantified using analysis software (Amersham Pharmacia Biotech) to identify spots in which the protein content of the pathological model animal showed a two-fold or more difference, and to use a spot picking device. (Amersham Pharmacia Biotech Co., Ltd.), and the protein was digested with trypsin using an in-gel enzyme digestion method (Schevchenko et al., Analytical Chemistry, Vol. 68, pp. 850-858, 1996). The fragments were fragmented, and the peptide mixture was recovered from the gel.
- the obtained peptide mixture was subjected to a capillary reverse phase liquid chromatography one-strength ram (0.075 mm in diameter, 150 countries in length, Elcie Packing Co.) at a flow rate of about 200 nL / min in the presence of 0.2% formic acid.
- the respective peptides were separated by an acetonitrile gradient elution method.
- a quadrupole ion trap mass spectrometer (SAMMOQUEST) with an electrospray ion source directly connected to a liquid mouth chromatograph (Microme's BioResource) automatically converts the molecular ions of each peptide.
- the product ion spectrum of each peptide was obtained using the method of selecting and measuring the product ion spectrum.
- KKAVTa mouse epididymal fat a double-fold increase in protein content compared to normal individuals, was analyzed using Mascot analysis software using the public protein database MSDB (release 20010401).
- MSDB public protein database
- the results of a search and collation showed that the mouse A0P2 protein (A0P2 / 1 -Cys Prx / nonse l en i um l utath i one perox i dase; GenBank access ⁇ on numbers AF004670, AF093852, Y12883)
- GenBank access ⁇ on numbers AF004670, AF093852, Y12883 The partial amino acid sequences of the strength sites were consistent, indicating that the protein was a mouse A0P2 protein. This increases A0P2 protein content in diabetic conditions It became clear.
- the human cDNA library (Clontech) was subjected to the PGR method (DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo) at 98 ° C (1 minute). After that, a cycle of 98 ° C (5 seconds), Z55 ° C (30 seconds), 72 ° C (3 minutes) was repeated 35 times), and A0P2 was encoded. Amplification was detected by agarose gel electrophoresis. As a result, A0P2 was remarkably expressed in fat, muscle, liver, kidney and other hearts where PPAR r acts among major organs. It was confirmed that it was.
- PGR method DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo) at 98 ° C (1 minute). After that, a cycle of 98 ° C (5 seconds), Z55 ° C (30 seconds), 72 ° C (3 minutes) was repeated 35 times), and A0P2 was encoded. Amplification was detected by agarose gel electrophoresis. As a result, A0P
- Example 8 Detection of regulatory effect of A0P2 on ligand-dependent transcription-inducing ability of PPARr From the above results, A0P2 interacts with PPARr via ligand to be involved in the induction of edema, and its expression is enhanced. Has been shown to be associated with the pathology of diabetes. The effect of A0P2 on the transcription-inducing activity of PPAR ⁇ was investigated using a reporter assay using cultured cells C0S-1.
- the human kidney cDNA library (Clontech) was subjected to the PGR method (DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo) at 98 ° C (1 minute). Thereafter, a cycle of 98 ° C (5 seconds), Z55 ° C (30 seconds), and 72 ° C (3 minutes) was repeated 35 times) to obtain a cDNA fragment containing 673 bp encoding the full-length region of A0P2.
- PGR method DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo) at 98 ° C (1 minute). Thereafter, a cycle of 98 ° C (5 seconds), Z55 ° C (30 seconds), and 72 ° C (3 minutes) was repeated 35 times) to obtain a cDNA fragment containing 673 bp encoding the full-length region of A0P2.
- W099 / 04815) (0.8 g well) was transiently cotransfected with pcDNA-A0P2 (0.05-0.2 g well).
- pcDNA-A0P2 0.05-0.2 g well.
- PBS phosphate buffer
- Cells were lysed by adding a cell lysate oOmM potassium phosphate (pH 7.8), 0.20 / 0 Triton X-100).
- a luciferase substrate solution 100 I (Pitka Gene) was added to the cell lysate 100 I, and the amount of luminescence for 10 seconds was measured using an AB-2100 type chemiluminescence measuring device (Aato).
- Plasmid pGH1 10 (Amersham Pharmacia Biotech Inc.), which has a ⁇ -galactosidase expression gene at the same time as the luciferase reporter gene 0.4 Co-transfected 0.4 ⁇ g wells into cells to produce 1-galactosidase activity
- the j8-galactosidase activity was measured using a detection kit Galacto-Light Plus TM system (Applied Biosystems) and quantified. This was used as the transfection efficiency of the transgene, and the luciferase activity described above was corrected for each cell.
- A0P2 is expressed in tissues including the kidney and A0P2 protein level is increased in the disease state in diabetic model mice.
- A0P2 has a peroxidase-like sequence in the molecule due to homology of the amino acid sequence, it is called antioxidant protein 2 (antioxidant protein 2) (GenBank accession number XI001415).
- Physiological activity is independent of calcium (Aci idi G ca IG i utn-independent phospho lipase A2; Kim TS et al., ⁇ Biol. Ghem., 272 Vol. 16 No. 10981 1997)
- the locus of the Aop2 protein has been reported as a causative gene for polycystic nephropathy (LTM / Aop2; lakoubova OA, et al. Genomics 42: 3, 474-478, 1997).
- A0P2 has an action different from the molecular function expected from its amino acid sequence structure, and its original physiological function has not been determined.
- the discovery by the present inventors that A0P2 binds to PPARy in a ligand-dependent manner and functions as a transcription coupling factor thereof is a new finding in the function of the molecule. By using this A0P2, it is possible to find and remove those that cause edema from agonists of PPAR r.
- the interaction between EGHLP and PPARr detectable by the reporter Atssei system in Example 5 and the suppression of the ligand-dependent transcription promoting ability of PPARr by EGHLP can be achieved by screening compounds that inhibit this.
- screening compounds that inhibit this it is possible to screen for a novel antidiabetic drug that improves glucose metabolism and contributes to the recovery of diabetes.
- screening for a substance that does not cause the interaction between A0P2 and PPARr which can be detected by the reporter Atssei system in Example 8, and that does not increase the ligand-dependent transcription promoting ability of PPARr by A0P2.
- a therapeutic agent for diabetes that contributes to the recovery of diabetes without causing edema as a side effect.
- test compound can be screened using the same reporter activity measuring system as in Examples 5 and 8, the following reporter assay system was constructed to efficiently screen a large amount of the test compound more efficiently.
- the cells were cotransfected with GAL-PPAR r (0.15 gZ well) and RE x 8- Luci (0.8 jug well) together with pcDNA-EGHLP (0.15 g well) by the calcium phosphate method. Eclected. After adding the test compound (10-1.0 iM) to the culture medium under the condition that GW7282 at a final concentration of 0.1 M was added as a PPAR ragonist, the cells were cultured for 48 hours. After washing the cells with PBS, the cells were lysed by adding 0.4 ml of cell lysate per well.
- the criterion for screening for a substance that inhibits the suppression of PPARr transcription induction by EGHLP is preferably 10 / M or less, more preferably the following, in terms of inhibitory activity intensity (IG 50 ).
- IG 50 inhibitory activity intensity
- the compound GI-262570 described above partially inhibited the ligand (0.1 M GW7282) -dependent suppression of the ability to induce PPAR ⁇ transcription by EGHLP at 10 / M (FIG. 6a).
- the compound GL-100085 does not inhibit the suppression of the transcriptional inducibility even at 10 M, and GI-262570 has high specificity for the main action of PPAR and is actually selected as a compound with a low main action of GL-100085. I was able to.
- each test compound (10-1.0 / M) selected in the same screening system was used alone here, and pcDNA-EGHLP of the same screening system was converted to pcDNA-A0P2 (0.15 gZ (Pell r) was added to the constructed screening system, and assayed for the presence of test compound-dependent enhancement of APAR2's ability to induce PPARr transcription by A0P2 by measuring luciferase activity corrected as described above.
- the above-mentioned compounds GW7282 and GL-100085 increased the transcription induction ability of PPAR ⁇ by about 4-5 times or 4-6 times in the presence of A0P2 at 1.0-10 M depending on the presence thereof. Confirmed to promote.
- the PGR method (Pyrobest DMA polymerase; Takara Shuzo Co., Ltd.) was performed from the human cDNA library (Clontech) at 98 ° C. (1 minute). A cycle of 98 ° C (5 seconds), 55 ° C (30 seconds), 72 ° C (3 minutes) was repeated 35 times), and the amplification of the GDNA fragment encoding FLJ13111 was detected by agarose gel electrophoresis. .
- FLJ13111 was remarkably expressed in the major organs of muscle and liver where PPAR ⁇ acts, as well as in mammary gland, lung, placenta, ovary, lymphocytes, and leukocytes, but PPARr ligand induced edema. Little expression was seen in the affected kidney. This confirmed that FLJ13111 was a transcriptional coactivator of PPARr from the expression site.
- PCR method DNA polymerase (Pyrobest DNA) was performed from a human liver GDNA library (Clontech).
- FLJ13111 is a protein whose function is unknown, and its target sequence (N-glycoylase) is a nuclear target sequence that suggests the presence in the cell nucleus from its amino acid sequence. There was no information suggesting the molecular function from the amino acid sequence structure except for the presence of e) for i ons.
- the discovery by the present inventor that FLJ13111 binds to PPAR r in a ligand-dependent manner and functions as a transcription coupling factor thereof is a new finding in the function of the molecule. By using this FLJ13111, it is possible to discover agonists selective for the main action of PPARr.
- Screening of compounds that promote the action of FLJ13111 to enhance the ligand-dependent transcription promoting ability of PPAR r improves the glucose metabolism by screening for compounds that promote the recovery of diabetes mellitus. .
- screening for a substance that inhibits the interaction between A0P2 and PPARa that can be detected by the reporter Atssey system in Example 8 and enhances the ability of A0P2 to promote the ligand-dependent transcriptional promotion of PPARr.
- the test compound was screened using the same reporter activity measurement system as in Example 11.
- GAL-PPAR r (0.15 g / ⁇ ; L)
- reporter construct (REx8- Luci; 0.8 jug / ⁇ )
- plasmid PGH110 0.4 ig / ⁇ ) containing the gene expressing S-galactosidase were converted to pcDNA- It was transiently cotransfected into C0S-1 cells with FLJ13111 (0.1 ⁇ g / well).
- test compound After adding the test compound at a final concentration of 10-0.1; uM to the medium and culturing for 48 hours, the luciferase activity and) 8-galactosidase activity were measured to quantify the PPAR activation.
- Screening was performed using the expression of FLJ13111 expression to promote PPAR ⁇ transcription induction (corrected ratio of luciferase activity) as an index.
- the criteria for screening for a substance that promotes the ability of FLJ13111 to induce PPARr transcription was preferably 10 iM or less, more preferably 1.0 / iM or less at the effective concentration (ED50).
- oral diglitazone and pioglitazone promoted the ability of FLJ13111 to induce PPAR ⁇ transcription at 1 M.
- the compound GL-100085 does not promote the transcription inducing ability, but rosiglitazone and piodaritazone have high specificity for the main action of PPARr, and GL-100085 can be actually selected as a compound with a low main action. there were.
- the interaction between FLJ13111 protein and PPAR r was expected to be involved in the improvement of glucose metabolism, which is the main effect mediated by PPAR r agonists. Therefore, the expression levels of the messenger RNA (mRNA) of the mouse ortholog gene of the FLJ13111 gene in the muscles of the two types of diabetes model mice, KKAVTa and G57BL / KsJ-db / db described in Example 4 were measured, and a normal individual G57BL / 6J And G57BL / KsJ- + m / + m.
- mRNA messenger RNA
- the gene expression level was corrected by measuring the expression level of the FLJ13111 gene of the present invention and simultaneously measuring the expression level of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene.
- the measurement system includes PRISM TM 7700 Sequence Detection System and SYBR Green PGR Master Mix (Applied Io Systems).
- the expression level of the target gene is determined by detecting and quantifying the amount of fluorescence of the SYBR Green I dye incorporated in the double-stranded DNA amplified by the PGR in real time.
- the measurement was performed according to the following procedure.
- RNA prepared in (1) Muscle of 15- or 12-week-old mouse
- a reverse transcription reaction kit (Advantage TM RT-for- PCR Kit; Clontech) in a 20 l system. After reverse transcription, 180 ⁇ I of sterile water was added and stored at -20 ° C.
- SEQ ID NOs: 20-24 Four oligonucleotides (SEQ ID NOs: 20-24) were designed as primers for PGR described in section (4).
- the combination of SEQ ID NO: 20 and SEQ ID NO: 21 was used for the FLJ13111 gene, and the combination of SEQ ID NO: 22 and SEQ ID NO: 23 was used for the G3PDH gene.
- mice FLJ13111 gene in each sample was corrected by the expression level of G3PDH gene based on the following equation.
- [FLJ13111 corrected expression level] [do1 ⁇ 1311 ⁇ gene expression level (raw data)] Z [G3PDH gene expression level (raw data)]
- the relative levels are shown in FIG. 8 with the expression level of the G57BL / 6J mouse as 100.
- the expression of the FLJ13111 gene was found to be significantly reduced in muscle muscle of a diabetic model mouse. Therefore, decreased expression of FLJ13111 in muscle is thought to cause insulin resistance. From the above, it can be concluded that FLJ13111 is significantly involved in insulin resistance.
- results of this example revealed that the measurement of the expression level of FLJ13111 can diagnose a diabetic condition.
- Luciferase repo overnight The nucleotide was inserted into the multicloning site of pGL3-Basic Vector (Promega), a vector, using restriction enzymes Bgl II and Hind I II, to produce a reporter plasmid named pGL3-FLJ13111p.
- the plasmid was transfected into G0S-1 cells, and the above-described pGL3-Basic Vector not containing the polynucleotide was transfected.
- the cloned polynucleotide contains a promoter sequence that controls the expression of FLJ13111, and this promoter is positively regulated by PPAR r ligands such as pioglitazone that reduces insulin resistance, It indicates that it is negatively controlled by its existence.
- FLJ13111 not only enhances the activity of PPAR r through the ligand, but also synergizes by increasing the expression level of FLJ13111 itself by the ligand of PPAR r, which is known to have the effect of reducing insulin resistance. Thus, it was expected that this was acting to reduce insulin resistance.
- the FLA1311 promoter atsay in this example can be used to screen a PPARr ligand or an insulin sensitizer without using a PPARr protein.
- EGHLP protein binds to PPAR r depending on the presence of the ligand of PPAR r, and suppresses the transcription-inducing activity of PPAR r. Furthermore, since EGHLP is expressed at an increased level in diabetic conditions, it is expected that its overexpression induces insulin resistance through suppression of PPARr activity, which may cause type 2 diabetes. . On the other hand, PPAR ⁇ promotes the differentiation of lipocytes by inducing ligand-dependent transcriptional activity, and as a result, the differentiated lipocytes take up blood glucose, improving glycemic metabolism and improving insulin resistance. It is known to be reduced. Therefore, it was investigated by the following experiment whether or not the overexpression of EGHLP in the cells actually affects the differentiation of adipocytes linked to insulin resistance.
- EGHLP-FLAG stably expressing L1 cells were established.
- cells infected with the pGLNGX vector empty vector were similarly prepared.
- the expression of EGHLP-FLAG in the established cells was confirmed by the Western blot method using an anti-FLAG NI2 antibody (Sigma).
- the above-mentioned lysate of EGHLP-FLAG-expressing cells was added to 10 I of a 2-fold concentration of SDS sample buffer (125 mM Tris-HCl (pH 6.8), 3 ° / 0 sodium laurel sulfate, 20% Glycerin, 0.14M jS-mercaptoethanol, 0.02% bromphenol blue) was added, treated at 100 ° C for 2 minutes, and subjected to 10% SDS polyacrylamide gel electrophoresis to be included in the sample. The isolated proteins were separated.
- SDS sample buffer 125 mM Tris-HCl (pH 6.8), 3 ° / 0 sodium laurel sulfate, 20% Glycerin, 0.14M jS-mercaptoethanol, 0.02% bromphenol blue
- the primary antibody is a monoclonal antibody that recognizes the FLAG epitope fused to the G-terminal of EGHLP
- the cells in the two wells were dissolved in a 40 I 0.1% SDS solution, and 1 ml of a triglyceride measuring reagent (Triglyceride G-Test Co., Wako Pure Chemical Industries) was added, followed by heating at 37 ° C for 10 minutes.
- the absorbance (0D505) of the reaction solution at a wavelength of 505 nm was measured.
- pioglitazone (0.1-3j «M) increased intracellular triglyceride in a dose-dependent manner, and differentiation into adipocytes was observed.
- EGHLP-overexpressing cells EGHLP-infected L1 cells
- the increase in triglyceride induced by pioglitazone (0.1-3 M) was suppressed to 43-57% of that in control cells at any pioglitazone dose. It was done.
- Example 16 Identification of ligand that selectively induces the binding between FLJ13111 and PPARr Screening with the same reporter assay as shown in Example 12 above resulted in promotion of PPARr transcription-inducing activity Compound XF was obtained (FIG. 11). Its titer was found to be almost comparable to 0.1 M for pioglitazone. Furthermore, it was found that the promotion of the transcriptional activation of PPARr by this compound XF was enhanced by the overexpression of FLJ13111 (0.1 g / ⁇ L) as in the case of pioglitazone.
- Edema caused by PPAR r ligand is caused by an increase in circulating plasma volume, which is known to be caused by an increase in the expression of sodium-dynamic rhizome ATPase in renal cells. Therefore, it was investigated whether compound XF affects the expression level of sodium-dynamic rheumatoid ATPase in renal cells linked to the induction of edema.
- I j renal epithelial cells MDGK Specifically, the 37 1. 5x10 5 cells well in 24 well culture play Bok with 10% fetal bovine serum outermost least essential medium DMEM supplemented with (Sigma) (Gibco) The cells were cultured at ° C for 48 hours.
- a solvent dimethyl sulfoxide alone, pioglitazone (final concentration 0.1-10 M) or test compound XF (final concentration 0.1-10 M) was added to the culture solution, and the cells were further cultured for 6 hours.
- the cells were washed once with assay buffer of 1m l (3mM MgS0 4, 3mM Na 2 HP0 4, 10mM Bok squirrel HCl, 250 mM sucrose), 3 H- Uaba Inn (74Bq / i Amersham Biosciences ) And 200 iI of a measurement buffer containing 2 ⁇ -avaine were added, and the mixture was allowed to stand at 37 ° C for 2 hours.
- the binding radioactivity obtained under these conditions was defined as the total binding amount.
- pioglitazone had a significant effect of increasing the expression level of sodium-hydrogen ATPase at 0.1 juM compared to control cells to which only the solvent was added.
- compound XF showed almost the same effect as pioglitazone in the activation of PPARr transcription from the above-mentioned findings, but did not increase the expression of sodium-powered rhizome ATPase even when added at a concentration of Was.
- compound XF which is a FLJ13111-selective PPAR r ligand, is associated with sodium edema It was clarified that the compound did not cause an increase in the expression level of ATPase, indicating that compound XF was not involved in inducing edema.
- Example 18 Measurement of adipocyte differentiation ability by FLJ13111-selective PPARr ligand
- compound XF affects adipocyte differentiation linked to reduction of insulin resistance.
- the survey was conducted in the same manner as described above. Specifically, compound XF (1.0-10.0 M) was added to mouse cultured preadipocytes 3T3-L1 (ATGC) cells, and the amount of triglycerides in the cells on day 7 was used as an index for adipocytes. The degree of differentiation was measured. As a result, it was observed that the addition of compound XF increased the amount of triglyceride by about 20% as compared with the cells to which only the solvent was added.
- yeast two-hybrid screening method performed in the presence of a ligand according to the present invention, a protein that interacts with PPAR in a ligand-dependent manner, which is a useful tool for screening an insulin sensitizer that has a departure from side effects, is screened.
- a protein that interacts with PPAR in a ligand-dependent manner which is a useful tool for screening an insulin sensitizer that has a departure from side effects.
- the main effect is selectively brought about. Compounds that do not cause side effects can be identified and screened.
- the substance selected by the screening system is useful as a candidate substance for an insulin sensitizer.
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CN1303218C (zh) * | 2003-12-19 | 2007-03-07 | 中国科学院上海药物研究所 | 过氧化物酶体增长因子活化受体拮抗剂和激动剂的筛选方法 |
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EP0193256A1 (en) * | 1985-01-19 | 1986-09-03 | Takeda Chemical Industries, Ltd. | Thiazolidinedione derivatives, their production and use |
WO1997031907A1 (en) * | 1996-02-28 | 1997-09-04 | Glaxo Group Limited | Substituted 4-hydroxy-phenylalcanoic acid derivatives with agonist activity to ppar-gamma |
WO1999012534A1 (fr) * | 1997-09-10 | 1999-03-18 | Ono Pharmaceutical Co., Ltd. | Regulateurs des recepteurs actives par le proliferateur des peroxisomes |
EP0930299A1 (en) * | 1996-08-19 | 1999-07-21 | Japan Tobacco Inc. | Propionic acid derivatives and applications thereof |
EP1057896A1 (en) * | 1997-08-27 | 2000-12-06 | Tanabe Seiyaku Co., Ltd. | Methods for identifying or screening agonists for and antagonists to ppar |
JP2001340080A (ja) * | 2000-03-31 | 2001-12-11 | Hochi Oka | 糖尿病網膜症を増悪させる可能性がなく、浮腫惹起作用を有しないインスリン抵抗性改善薬の検索システム |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5847008A (en) * | 1996-02-02 | 1998-12-08 | Merck & Co., Inc. | Method of treating diabetes and related disease states |
US6365365B1 (en) * | 1998-03-20 | 2002-04-02 | The Regents Of The University Of California | Method of determining whether an agent modulates glycosyl sulfotransferase-3 |
-
2003
- 2003-01-22 EP EP03703025A patent/EP1469071A4/en not_active Withdrawn
- 2003-01-22 CA CA002474353A patent/CA2474353A1/en not_active Abandoned
- 2003-01-22 JP JP2003562294A patent/JPWO2003062427A1/ja not_active Withdrawn
- 2003-01-22 WO PCT/JP2003/000546 patent/WO2003062427A1/ja not_active Application Discontinuation
- 2003-01-22 US US10/502,279 patent/US20050084840A1/en not_active Abandoned
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0193256A1 (en) * | 1985-01-19 | 1986-09-03 | Takeda Chemical Industries, Ltd. | Thiazolidinedione derivatives, their production and use |
WO1997031907A1 (en) * | 1996-02-28 | 1997-09-04 | Glaxo Group Limited | Substituted 4-hydroxy-phenylalcanoic acid derivatives with agonist activity to ppar-gamma |
EP0930299A1 (en) * | 1996-08-19 | 1999-07-21 | Japan Tobacco Inc. | Propionic acid derivatives and applications thereof |
EP1057896A1 (en) * | 1997-08-27 | 2000-12-06 | Tanabe Seiyaku Co., Ltd. | Methods for identifying or screening agonists for and antagonists to ppar |
WO1999012534A1 (fr) * | 1997-09-10 | 1999-03-18 | Ono Pharmaceutical Co., Ltd. | Regulateurs des recepteurs actives par le proliferateur des peroxisomes |
JP2001340080A (ja) * | 2000-03-31 | 2001-12-11 | Hochi Oka | 糖尿病網膜症を増悪させる可能性がなく、浮腫惹起作用を有しないインスリン抵抗性改善薬の検索システム |
Non-Patent Citations (2)
Title |
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See also references of EP1469071A4 * |
TREUTER E. ET AL.: "A regulatory role for RIP140 in nuclear receptor activation", MOLECULAR ENDOCRINOLOGY, vol. 12, no. 6, 1998, pages 864 - 881, XP002915088 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1303218C (zh) * | 2003-12-19 | 2007-03-07 | 中国科学院上海药物研究所 | 过氧化物酶体增长因子活化受体拮抗剂和激动剂的筛选方法 |
WO2005093423A2 (en) * | 2004-03-26 | 2005-10-06 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with peroxisome proliferative activated receptor alpha (ppara) |
WO2005093423A3 (en) * | 2004-03-26 | 2006-02-09 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with peroxisome proliferative activated receptor alpha (ppara) |
Also Published As
Publication number | Publication date |
---|---|
EP1469071A1 (en) | 2004-10-20 |
CA2474353A1 (en) | 2003-07-31 |
US20050084840A1 (en) | 2005-04-21 |
JPWO2003062427A1 (ja) | 2005-05-26 |
EP1469071A4 (en) | 2006-02-15 |
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