WO2003056005A1 - Promoteur homologue de sglt humain et utilisation dudit promoteur - Google Patents

Promoteur homologue de sglt humain et utilisation dudit promoteur Download PDF

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Publication number
WO2003056005A1
WO2003056005A1 PCT/JP2002/013651 JP0213651W WO03056005A1 WO 2003056005 A1 WO2003056005 A1 WO 2003056005A1 JP 0213651 W JP0213651 W JP 0213651W WO 03056005 A1 WO03056005 A1 WO 03056005A1
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Prior art keywords
sequence
salt
dna
human sglt
activity
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PCT/JP2002/013651
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English (en)
Japanese (ja)
Inventor
Keiji Iwamoto
Nozomi Katayama
Mihoko Kawamura
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Takeda Chemical Industries, Ltd.
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Priority to AU2002367140A priority Critical patent/AU2002367140A1/en
Publication of WO2003056005A1 publication Critical patent/WO2003056005A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to a novel promoter for gene expression and its use. More specifically, the present invention relates to DNA containing the promoter region of the human SGLT homolog gene, a transformant transformed with the DNA, a method of screening for a compound that promotes or inhibits the activity of the human SGLT homolog promoter, or a salt thereof, and the like. . Background art
  • a membrane protein called a sugar transporter is required on the cell membrane.
  • the glucose transporter is Na + /, an active transporter that transports glucose against a concentration gradient by conjugating with the passive transporter, facilitated diffusion glucose transporter (GLUT) and Na + ion transporter.
  • GLUT facilitated diffusion glucose transporter
  • Na + ion transporter Na + ion transporter.
  • Glucose transporter (SGLT) GLUT has eight isoforms and has a common structure that penetrates a cell membrane with a molecular weight of about 50,000 12 times.
  • SGLT has a common structure that penetrates the cell membrane with a molecular weight of 750,000 14 times.
  • Human SGLT1 is specifically expressed in the small intestine and kidney and has high affinity for glucose and has low transport ability.
  • Human SGLT2 is specifically expressed in kidney and has low affinity for glucose and has high transport ability. It has been known. SGLT plays a role in absorbing glucose from the small intestine and reabsorbing darcos once excreted in urine from the kidney.
  • GLUT2 a passive transporter, has been mainly expressed in 3 cells and hepatocytes. Have been considered. GLUT2 is characterized by a low affinity for glucose and a high maximum transport capacity. It is thought that Teng / 3 cells take up glucose in response to blood glucose levels and, together with dalcokinase, function as a dalcose sensor for glucose-dependent insulin secretion. In hepatocytes, glucose is taken up into cells during postprandial hyperglycemia according to the glucose concentration gradient inside and outside the cell, and glucose produced inside cells by glycogenolysis or gluconeogenesis is released into blood during fasting. It functions as a sugar transporting carrier.
  • the SGLT homolog function By activating the SGLT homolog function, it can be expected that glucose uptake into ⁇ / 3 cells is promoted and blood glucose level-dependent insulin secretion is enhanced. In addition, it is expected that the SGLT function activator does not cause the above-mentioned side effects of the insulin secretagogue (SU agent) currently used.
  • SU agent insulin secretagogue
  • GLUT2 releases glucose from the liver into the blood during fasting
  • the SGLT homolog promotes glucose uptake from the blood to the liver regardless of the glucose concentration gradient inside and outside the cell. Therefore, it can be expected to suppress the release of glucose from the liver into the blood, and to suppress the fasting hyperglycemia observed in diabetic patients without causing side effects such as hypoglycemia.
  • SGLT inhibitors can lower blood sugar by suppressing reabsorption of sugar from the kidneys, and are expected to suppress fat synthesis by suppressing sugar uptake in the liver. I can wait.
  • a human SGLT homolog genomic gene As a result of intensive studies, the present inventors have succeeded in obtaining a human SGLT homolog genomic gene with the aim of establishing a screening method for searching for a human SGLT homolog gene-expressing substance.
  • This gene was subjected to PCR to obtain 2.3 kb DNA upstream of the structural gene encoding the human SGLT homolog, and a plasmid DNA was constructed downstream of which a luciferase gene was ligated as a repo overnight gene.
  • a human SGLT homolog promoter By measuring the luciferase activity in HepG2 cells transformed with the above, a human SGLT homolog promoter could be found in the 2.3 kb DNA upstream of the human SGLT homolog structural gene.
  • a regulator sequence that seems to regulate the expression of the human SGLT homolog.
  • the regulator sequence is a sequence containing a PPRE (Peroxisome Proliferator Response Element);
  • sequence containing the PPRE is a sequence containing the 1334th to 1339th nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 5;
  • the regulator sequence is a sequence containing a HNF4 (Hepatocyte Nuclear Factor 4) binding sequence.
  • HNF4 Hepatocyte Nuclear Factor 4
  • sequence containing the HNF4 binding sequence is a sequence containing the 1720th to 1731rd base sequences of the base sequence represented by SEQ ID NO: 5;
  • the sequence containing the Isll (Islet 1) binding sequence is a sequence containing the 687th to 692th or the 2149th to 2154th base sequence of the base sequence represented by SEQ ID NO: 5.
  • the sequence of the Regile contains an HNF5 (Hepatocyte Nuclear Factor 5) binding sequence.
  • the DNA according to (2) which is a sequence having
  • a method for screening for a diabetic drug or an antihyperlipidemic drug which comprises using the transformant according to the above (15).
  • a promoter or inhibitor of sugar uptake comprising a compound having a promoting or inhibiting activity of human SGLT homolog promoter activity or a salt thereof,
  • an antidiabetic or antihyperlipidemic agent comprising a compound having an activity of promoting or inhibiting human SGLT homolog promoter activity or a salt thereof;
  • the present invention relates to a method for preventing and treating diabetes or hyperlipidemia, which comprises administering a compound having a promoting or inhibiting effect or a salt thereof.
  • FIG. 1 is a drawing showing a hydrophobicity plot of a human SGLT homolog.
  • FIG. 2 is a drawing showing SNPs present in the upstream region of the human SGLT homolog gene.
  • FIG. 3 is a drawing showing a deletion mutant of the upstream region of the human SGLT homolog gene.
  • FIG. 4 is a drawing showing a graph of the value of promoter activity in the upstream region of the human SGLT homolog gene when the activity of sea pansy luciferase is set to 1.
  • Figure 5 shows the upstream region of the human SGLT homologue gene, which is promoted by the addition of dexamethasone 0 to 3 M in HepG2 cells and Huh-7 cells transfected with the human SGLT homologue gene upstream region.
  • 3 is a drawing showing a graph of the value of promoter activity (the amount of light emitted by luciferase) in Example 1.
  • human SGLT homolog containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 14 (hereinafter sometimes referred to as human SGLT homolog) used in the present invention is human ⁇ Cells of warm-blooded animals (eg, guinea pigs, rats, mice, chickens, egrets, pigs, hidges, horses, monkeys, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, kidney cells, bone marrow) Cells, mesangial cells, Langer's cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chon
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 includes about 60% or more, preferably about 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 14 Amino acid sequences having a homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • Examples of human SGLT homologs containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 include, for example, those substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 14
  • a protein having an amino acid sequence of SEQ ID NO: 14 and having substantially the same activity as a human SGLT homolog containing an amino acid sequence represented by SEQ ID NO: 14 is preferred.
  • the activity of substantially the same quality includes, for example, an active transport activity of glucose.
  • Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, the active transport activity of glucose It is preferably equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times), but the degree of these activities, the molecular weight of the protein, etc. May be different.
  • Activity such as glucose active transport activity can be measured according to a known method.
  • the human SGLT homolog used in the present invention includes, for example, 1) 1 or 2 or more (preferably about 1 to 30, preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 14; Amino acid sequence in which the number (1 to 5) amino acids have been deleted, and more preferably 1 or 2 or more (preferably about 1 to 30 amino acids) in the amino acid sequence represented by SEQ ID NO: 14.
  • the so-called mutin such as a protein containing an amino acid sequence in which about 10 or more, more preferably a number (1 to 5) amino acids are substituted with other amino acids, or a protein containing an amino acid sequence combining them is also used. included.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (capilloxy terminus) according to the convention of peptide labeling.
  • the human SGLT homolog used in the present invention including the human SGLT homolog containing the amino acid sequence represented by SEQ ID NO: 14, has a C-terminal lipoxyl group (one COOH), carboxylate (one COO-) Or an amide (1-C ⁇ NH 2 ) or an ester (—COOR).
  • R in the ester for example, methyl, ethyl, n-propyl, Isopropyl, ( ⁇ _ 6 alkyl groups such as n- butyl, cyclopentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, C 6, such as ⁇ - naphthyl Le - 1 2 ⁇ Li Ichiru group, for example, benzyl, C 7 such as phenylene Lou C ⁇ 2 alkyl or ⁇ - naphthylmethyl etc.
  • the human SGLT homolog need, the N-terminal amino acid residue (eg, Mechionin residues)
  • Amino group protecting groups e.g., formyl group, C ⁇ such Arukanoiru such Asechiru groups - such as 6 Ashiru group
  • N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule for example, --OH, --SH, amino group, imidazo , An indole group, a guanidino group, etc.
  • an appropriate protecting group for example, an acyl group such as a 6- alkanoyl group such as a formyl group or an acetyl group
  • a sugar chain is bound. It also includes complex proteins such as so-called glycoproteins.
  • human SGLT homolog used in the present invention include, for example, a human SGLT homolog derived from human kidney and having the amino acid sequence represented by SEQ ID NO: 14.
  • the salts of human SGLT homologs used in the present invention include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals), especially physiologically acceptable salts.
  • physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metals
  • physiologically acceptable salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid,
  • the human SGLT homolog or a salt thereof used in the present invention may be a human SGLT as described above. It can be produced from known cells or tissues by a known protein purification method, or by culturing a transformant containing DNA encoding a human SGLT homolog. Further, it can be produced according to the peptide synthesis method described later.
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining the above chromatography.
  • a commercially available resin for protein synthesis can usually be used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or an amide thereof.
  • the protected amino acids described above various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • the carbodiimides include DCC, ⁇ , ⁇ ′-diisopropyl carbodiimide, ⁇ -ethyl- ⁇ ′-(3-dimethylaminoprolyl) carbodiimide, and the like.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, HOB t, HOOB t), or the protected amino acid may be pre-formed as a symmetric anhydride or HOB t ester or HOOB t ester. It can be added to the resin after activation has taken place.
  • Solvents used for activation of protected amino acids and condensation with resins include proteins It can be appropriately selected from solvents known to be usable for the condensation reaction. For example,
  • Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpiperidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; Sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used.
  • the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • the ninhydrin reaction when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group.
  • unreacted amino acids can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.
  • Examples of the protecting group for the starting amino group include, for example, Z, Boc, t-pentyloxycarbonyl, isopolnyloxycarbonyl, 4-methoxybenzyloxycarbonyl, ⁇ - ⁇ , Br_Z, adamantyl Oxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxy It can be protected by carbonylation, t-butoxycarbonylhydrazide, tritylhydrazide and the like.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cycl
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower-alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydrovinylyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz l, C l 2 - Bz l, 2 - nitrobenzyl, Br-Z, t _ butyl is used.
  • imidazole protecting group for histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Activated carbonyl groups of the raw material include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t)].
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol
  • Phenol cyanomethyl alcohol
  • paranitrophenol H0NB
  • N-hydroxysuccinimide N-hydroxyphthalimide
  • esters with HOB t esters with HOB t
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or the like.
  • the elimination reaction by the acid treatment is generally performed at a temperature of about 120 ° C. to 40 ° C.
  • a cation scavenger such as toluene, dimethyl sulfide, 1,4-butanedithiol or 1,2-ethanedithiol.
  • a cation scavenger such as toluene, dimethyl sulfide, 1,4-butanedithiol or 1,2-ethanedithiol.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
  • the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4-butane described above.
  • dilute sodium hydroxide It is also removed by alkali treatment with a lithium solution or diluted ammonia.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide form of a protein for example, first, after amidating the a; -hydroxyl group of the carboxy terminal amino acid and protecting it, a peptide (protein) chain is added to the amino group side of the desired chain. After lengthening the protein chain, a protein or partial peptide from which only the protecting group for the N-terminal amino group of the peptide chain is removed, and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group are removed, These proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • a desired crude protein After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained.
  • This crude protein can be purified by various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired protein.
  • an ester of the protein for example, a-force After condensing a ropoxyl group with a desired alcohol to form an amino acid ester, an ester of the desired protein can be obtained in the same manner as the amide of a protein.
  • the human SGLT homolog or a salt thereof used in the present invention can be produced according to a known protein synthesis method.
  • any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target protein can be produced by condensing a partial peptide or amino acid capable of constituting the protein used in the present invention with the remaining portion, and removing the protective group when the product has a protective group.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the human SGLT homolog used in the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the human SGLT homolog obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method analogous thereto. Can be converted into a free form or another salt by a method analogous thereto.
  • the DNA encoding the human SGLT homolog used in the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the human SGLT homolog used in the present invention. Further, it may be any of genomic DNA, genomic DNA library, the above-mentioned cDNA derived from cells and tissues, the above-mentioned cDNA library derived from cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT_PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cell'tissue.
  • RT_PCR method Reverse Transcriptase Polymerase Chain Reaction
  • DNA encoding the human SGLT homolog used in the present invention for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 15 or SEQ ID NO: 16, or SEQ ID NO: 15 or SEQ ID NO: A DNA having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by 16 and encoding a protein having substantially the same properties as the human SGLT homolog used in the present invention. Any one may be used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 15 under high stringent conditions include, for example, about 50% or more, preferably about 60%, of the nucleotide sequence represented by SEQ ID NO: 15 Or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95%.
  • DNA containing a base sequence having the above homology is used.
  • Examples of a DNA which can be hybridized with the nucleotide sequence represented by SEQ ID NO: 16 under high stringency conditions include, for example, about 50% or more, preferably about 60% or more of the nucleotide sequence represented by SEQ ID NO: 16 More preferably, DNA containing a nucleotide sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
  • Hybridization is performed according to a known method or a method analogous thereto, for example, a method described in Molecular 'Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be done. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
  • High stringency conditions refer to, for example, conditions where the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. . In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • the DNA encoding the human SGLT homolog containing the amino acid sequence represented by SEQ ID NO: 15 includes a DN containing the nucleotide sequence represented by SEQ ID NO: 15 or SEQ ID NO: 16. A or the like is used.
  • the PCR method is performed by using a synthetic DNA primer having a part of the nucleotide sequence encoding the human SGLT homolog of the present invention.
  • Hybridization can be selected by the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). And so on. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be converted using PCR or a known kit such as Mutan TM -super Express Km (Takara Shuzo Co., Ltd.) or Mutan TM -K (Takara Shuzo Co., Ltd.). It can be carried out according to a known method such as the gapped duplex method or the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned human SGLT homolog can be used as it is, or can be digested with a restriction enzyme or added with a linker if desired.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation termination codon at its 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the DNA containing the promoter overnight region of the human SGLT homolog of the present invention may contain a regulator sequence described below, and may be any MA having human SGLT homolog promoter activity. Good.
  • any nucleotide sequence may be used as long as it contains the nucleotide sequence represented by the first to second nucleotides of SEQ ID NO: 5 or a part thereof.
  • human or other mammalian cells eg, hepatocytes, spleen cells, nerve cells, glial cells, kidney cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts , Fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obese cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes Spheres, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, or stromal cells, or precursors of these cells, stem cells, or cancer cells), or any of these cells Tissues, for example, brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla
  • the recombinant DNA containing the promoter region of the human SGLT homolog of the present invention is specifically described below. Can be obtained as follows.
  • a human gene library integrated into EMBL3 vector is screened by a known method, and the ⁇ phage that reacts with this probe is screened.
  • DNA is extracted from this phage clone, a restriction enzyme map of the incorporated human gene is prepared, and a DNA fragment obtained by digestion with a restriction enzyme that reacts with a probe at the uppermost stream of the cDNA is not particularly limited. Natl. Acad. Sci. USA, 84, 8573-8577), retroviral vectors (Cone, RD and Mulligan, RC (1984) Proc. Natl.
  • the obtained DNA can be used as it is depending on the purpose, or digested with a restriction enzyme, if desired, or added with a linker.
  • a detectable structural gene may be ligated downstream of the promoter.
  • Various reporter genes are used as the structural gene linked downstream of the promoter region.
  • the repo overnight gene in addition to the luciferase gene, CAT (Chloramphenicol acetyl transferase) gene, and al riphosphatase gene, the j8-galactosidase gene is widely used. Any other structural gene can be used as long as it has a method for detecting its gene product.
  • an appropriate restriction site downstream of the promoter region is used. Then, the above-mentioned structural genes may be ligated in such a direction as to be transcribed correctly.
  • Examples of the host transformed with the recombinant vector include, for example, Escherichia sp. Bacteria, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
  • Escherichia examples include Escherichia coli K12, DH1 [Processings of the National Academy of Sciences, Obs-Sciences of Ob-The-Usc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM 103 [Nucleic Acids Research, Vol. 9, 309 (198 1)], J 109, JA 2 21 [Journal of Molecular Biology, 120, 517 (1978)], HB 10 1 [Journal of Molecular Biology, 41, 459 (1969) )] And C600 [Genetics, 39, 440 (1954)].
  • Bacillus subtilis Bacillus subtilis M 111 (Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry). 95, 87 (1 98 '4)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces, Schizosaccharomyces pombe NCY CI 913 , NC YC 2036, Pichia pastoris, etc. are used.
  • insect cells for example, when the virus is AcNPV, a cell line derived from the larvae of night moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from Trichoplusia ni midgut, Trichoplusia ni Egg-derived High Five TM cells, Mamestra brassicae-derived cells, Estigmena acrea-derived cells, and the like are used.
  • Sf cell a cell line derived from silkworm (Bombyxmori N; BmN cell) is used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like are used.
  • insects for example, silkworm larvae and the like are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CH ⁇ (dhfr-) cell).
  • Mouse L cells Mouse AtT-20, mouse myeloma cells, rat GH3, mouse fibroblasts 3T3-L1, human liver cancer cells HepG2 (hereinafter abbreviated as HepG2 cells), Human osteosarcoma cells MG-63 (hereinafter abbreviated as MG-63 cells), human FL cells, white adipocytes, egg cells, ES cells (Evans, MJ and Kaufman, K. ⁇ . (1981) Nature, 292, 154), and cells that have been induced to differentiate under appropriate differentiation conditions are used. Among them, animal cells, especially white fat cells, can be used. Egg cells or ES cells (Evans, MJ and Kaufman, K. ⁇ . (1981) ature., 292, 154) are also used as a step in the transfer of DNA into animal individuals.
  • Transformation methods for these cells include the calcium phosphate method (Graham et al. (1973) Virology, 52, 456), the electroporation method (Ishizaki et al. (1986) Cell Science, 5, 577), and the microinjection method. Are used.
  • biotechnology To transform insect cells or insects, for example, biotechnology
  • the transformant is cultured in the presence of a specific compound, and by measuring and comparing the amount of the gene product in the culture, the ability of the compound to control the promoter overnight activity can be known.
  • the transformant is cultured by a method known per se.
  • a liquid medium is suitable as a medium for culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources, inorganic substances and others. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • nitrogen source examples include ammonium salts, nitrates, corn chip, liqueur, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • a medium for cultivating a bacterium belonging to the genus Escherichia for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experimen, in Molecular Genetics (Journal of Experiments in Molecular Genetics) ), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as, for example, 33-indolyl acrylic acid can be added to make the promo work efficiently. If the host is a bacterium of the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours.
  • the cultivation is usually performed at about 30 to 4 for about 6 to 24 hours.
  • the culture medium used was Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used. It is preferable to adjust ⁇ of the culture medium to about 6., 2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal calf serum [Seience, 122, 501 (1952)] , DMEM medium (Virology, 8, 396 (1959)), RPM I 1640 medium (Journal of the American Medical Associatation) ion) 199, 519 (1 967)], 199 Medium [Proceeding of the Society for the Biological Medicine], 73 , 1 (1950)].
  • the pH is about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • a transcription control factor of a human SGLT homolog may be used. Any sequence can be used as long as it can bind.
  • a PPRE Peroxisome Proliferator Response Element
  • PPRE Peroxisome Proliferator Response Element
  • DNA of the present invention may contain the Regiyure Isseki one sequence may contain a plurality of said regulator aerator sequence.
  • the nucleotide sequence containing a part of the nucleotide sequence of the 1st to 2254th nucleotides in the nucleotide sequence represented by SEQ ID NO: 5 is any nucleotide sequence containing the above-mentioned all-over-one sequence. More specifically, it may be, for example, the 1806th to 2254th base sequence of the base sequence represented by SEQ ID NO: 5, or the 958th to 2254th base sequence of the base sequence represented by SEQ ID NO: 5. And the like.
  • nucleotide sequence represented by SEQ ID NO: 5 (1) the 698th C is converted to T, (2) the 824th A (3) a nucleotide sequence having a nucleotide sequence in which C at position 698 has been converted to T and nucleotide 824 at position A has been converted to T (SEQ ID NOS: 8, 9, and 10) Or a part thereof can also be used as the promoter region of the present invention.
  • the DNA of the present invention is a DNA containing the promoter region of a human SGLT homolog, a compound that promotes or inhibits the activity of the human SGLT homolog promoter (eg, (A compound that promotes or inhibits) or a salt thereof.
  • a compound that promotes or inhibits the activity of the human SGLT homolog promoter eg, (A compound that promotes or inhibits) or a salt thereof.
  • the screening method, the screening kit, the screening method, and a compound or a salt thereof that promotes or inhibits the activity of the human SGLT homolog promoter obtained using the screening kit will be specifically described.
  • a method for screening for a compound that promotes or inhibits the activity of the human SGLT homolog promoter eg, a compound that promotes or inhibits sugar uptake
  • a transformant transformed with the above-described DNA of the present invention Is useful for searching for or determining a compound or a salt thereof that promotes or inhibits the activity of the human SGLT homolog promoter of the present invention.
  • the transformant of the present invention is contacted with a test compound. And comparing a transformant not containing the human SGLT homologous promoter region of the present invention with a test compound and measuring the expression level of the polypeptide.
  • test compound examples include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • polypeptide to be expressed a polypeptide encoded by the above structural gene (preferably, a repo overnight gene) or the like is used.
  • Examples of a method for measuring the expression level of a polypeptide include measuring luciferase activity by a method according to the method described in Brasier, A.R. et al. (1989) Biotechniques vol. 7, 1116-1122.
  • a screening kit used to screen for compounds that promote or inhibit the activity of the human SGLT homolog promoter for example, compounds that promote or inhibit sugar uptake
  • salts thereof for example, compounds that promote or inhibit sugar uptake
  • kits for determining a compound or a salt thereof that promotes or inhibits the activity of the human SGLT homolog promoter of the present invention is characterized by using the above-mentioned transformant.
  • kits for determining a compound or its salt that promote or inhibit include the following.
  • Dulbbecco's modified Eagle's MEM (manufactured by Gibco) supplemented with 5% of egosum serum (manufactured by Gibco).
  • PGV-B2 (Nitsuho Nishi) plasmid DNA having a human SGLT homolog promoter sequence and a structural gene (eg, luciferase gene) inserted downstream of the human SGLT homolog promoter according to the present invention 4.t main cell line
  • HepG2 cells human hepatoma cell line: ATCC HB 8065
  • Huh-7 cells human hepatoma cell line
  • a host cell strain was seeded by lxlO 5 / well in 96-well microplate, over ⁇ 37 ° (:, cultured in 5% C0 2 hatching eggs device.
  • the plasmid for measuring overnight activity of the human SGLT homolog promoter of the present invention is introduced into cells at 1 g / well. After the introduction, the test compound in 1 hour was added in O.Lml / well, for 48 hours at 37 ° C, 5% C0 2 incubator.
  • a compound that promotes or inhibits human SGLT homolog promoter activity for example, a compound that promotes or inhibits sugar uptake
  • a salt thereof obtained by using the screening method of (1) or the screening kit of (2).
  • the compound when it reduces or inhibits the promoter activity, it can be used as an antihyperlipidemic agent or the like because it inhibits lipid production.
  • a pharmaceutically acceptable salt or the like is used as a salt of the compound obtained by using the above-described screening method or screening kit.
  • examples include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
  • the salt with an inorganic base include an alkali metal salt such as a sodium salt and a potassium salt, an alkaline earth metal salt such as a calcium salt and a magnesium salt, and an aluminum salt and an ammonium salt.
  • Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-alutidine, ethanolamine, genoaluminamine, triethanolamine, cyclohexylamine, dicyclohexylamine. Salts with min, N, N, dibenzylethylenediamine and the like are included.
  • salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like.
  • Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid And the like.
  • salts with a basic amino acid include, for example, salts with arginine, lysine, or oltinine
  • salts with the acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like. can give.
  • the compound or a salt thereof When used as a prophylactic and / or therapeutic agent for the above-mentioned diseases, it can be formulated according to conventional means.
  • the compound or a salt thereof may be orally administered as a tablet, capsule, elixir, microcapsule or the like, if necessary, coated with sugar or water or another pharmaceutically acceptable liquid. It can be used parenterally in the form of injections, such as sterile solutions or suspensions.
  • the compound can be formulated in a unit dosage form required for generally accepted formulation practice with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be manufactured by mixing. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid Leavening agents such as, for example, lubricating agents such as magnesium stearate, sucrose, lactose or saccharin And sweeteners such as peppermint, cacao oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 (TM), HCO-50) You may.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • the prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, for example, against human mammals (eg rats, mice, egrets, sheep, pigs, pigs, cats, dogs, dogs, etc.). Can be administered.
  • human mammals eg rats, mice, egrets, sheep, pigs, pigs, cats, dogs, dogs, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for an adult (as 60 kg), the dose is about 0.1 per day. 100100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • it is usually one dose for adults (60 kg).
  • the dose can be administered in terms of 60 kg.
  • DNA Deoxylipo nucleic acid
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • Example 1 shows the nucleotide sequence of Primer XI used in Example 1.
  • Example 1 shows the nucleotide sequence of the DNA of the upstream region of 8 bp from 2261 bp upstream of the translation start point of the human SGLT homologue gene obtained in Example 1.
  • Example 3 shows the nucleotide sequence of a primer for P1 mutation introduction used in Example 2.
  • Example 3 shows the nucleotide sequence of a primer for P2 mutation introduction used in Example 2.
  • Example 3 shows the nucleotide sequence of DNA having a base substitution of P2 obtained in Example 2.
  • [SEQ ID NO: 9] 3 shows the nucleotide sequence of DNA having a P1 base substitution obtained in Example 2.
  • FIG. 3 shows the nucleotide sequence of DNA having both P1 and P2 base substitutions obtained in Example 2.
  • FIG. 2 shows the amino acid sequence of a human SGLT homolog protein.
  • Fig. 3 shows the nucleotide sequence of DNA encoding a human SGLT homolog protein containing a 3 'untranslated region (2026-3140).
  • [SEQ ID NO: 22] 8 shows the base sequence of 8 used in Reference Example 2.
  • the transformant Escherichia coli XL / Blue / pTB2254 obtained in Example 1 described below was used from December 6, 2001 (Heisei 13), Tsukuba, Ibaraki Pref. 6 (Postal code 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) Patent No. FERM BP-7178, 2001 No. 1 January 20 Osaka, Osaka, Japan It has been deposited with the Fermentation Research Institute (IFO) under the number 7-85 (zip code 532-8686), 2-3-1 Jusanhoncho, Yodogawa-ku, Ichikawa.
  • IFO Fermentation Research Institute
  • the transformant, Escherichia coli XL BIue / pTB2255, obtained in Example 2 has been used since December 6, 2001 (Heisei 13), 1-1 Tsukuba, Higashi 1-chome, 1 Chuo No. 6 (Ibaraki Prefecture) Submitted to the National Institute of Advanced Industrial Science and Technology (AIST) with the postal code 305-8566) as the accession number FERM BP-78 19, 2001. 1 January 20 Osaka, Osaka -Fermentation Research Institute (IF 0) of Jusanhoncho, Yodogawa-ku, Yokohama, 2-1-7-85 (zip code 532-8686) Deposit No. IF # 16730.
  • IF 0 Osaka -Fermentation Research Institute
  • the transformant Escherichia coli XU-Blue / pTB2256 obtained in Example 2 was obtained from December 6, 2001, at 1-1, Tsukuba, Higashi 1-chome, Chuo 6 Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) with a zip code of 305-8566) as the deposit number FERM BP-7820 as a deposit number of FERM BP-7820 from January 20, 2001. It has been deposited with the Fermentation Research Institute (IF ⁇ ) of the 2-7-1 Mihonmachi 7-85 (zip code 532-8686) under the accession number IF ⁇ 1673-1.
  • IF ⁇ Fermentation Research Institute
  • Example 2 The transformant Escherichia coli DH5Q! / PTB2257 obtained in Example 2 was obtained from December 19, 2001 at Tsukuba East 1-chome 1 Chuo No. 6 Submitted to the National Institute of Advanced Industrial Science and Technology (AIST) with a zip code of 305—8566) as a deposit number FERM BP—7832 as a deposit number FERM BP—7832. It has been deposited with the Fermentation Research Institute (IFO) of Mihonmachi 2-1-7-85 (zip code 532-8686) under the accession number IF ⁇ 16732.
  • IFO Fermentation Research Institute
  • PCR reaction was performed using two primers, Primer 3 (SEQ ID NO: 17) and Primer 4 (SEQ ID NO: 18).
  • the composition of the reaction solution in the reaction was as follows, using the above cDNA 1 ⁇ 1 as type I, 11 volumes of Pfu Turbo DNA Polymerase (STRATAGENE), primer 1 (SEQ ID NO: 17) and primer 4 (SEQ ID NO: 18) was added to each of 0.5 M, dNTPs was added to 200 ⁇ l, and the buffer attached to the enzyme was added to 51 to make a liquid volume of 501.
  • PCR reaction is performed at 94 ° C A cycle of 20 seconds, 60 ° C for 30 seconds, and 72 minutes for 2 minutes was repeated 35 times, and a final extension reaction for 72 minutes was performed. Further, the PCR reaction product was designated as type ⁇ , and a PCR reaction was performed using two primers, primer 5 (SEQ ID NO: 19) and primer 6 (SEQ ID NO: 20).
  • the composition of the reaction solution was prepared by using the above PCR reaction product (1 zl) as type III, 1 ⁇ 1 amount of Hu Turbo DNA Polymerase (STRATAGENE), primer 5 (SEQ ID NO: 19) and primer 16 ( SEQ ID NO: 20) was added to each of 0.5 iM, dNTPs was added to 200 M, and buffer attached to the enzyme was added to 5 ⁇ 1, to obtain a liquid volume of 501.
  • PCR reaction repeat the cycle at 94 ° C for 1 minute, 96 ° C for 20 seconds, 60 hours, 30 seconds, 72 ° C for 2 minutes 35 times, and finally perform the extension reaction at 72 ° C for 7 minutes.
  • STRATAGENE Hu Turbo DNA Polymerase
  • the PCR reaction product and the plasmid vector PME18S were digested with restriction enzymes EcoRI and Spel 37 overnight. 1% agarose gel electrophoresis to cut out 2 Kbp DNA fragment (SGLT homolog) and 3 Kbp DNA fragment (pME18S)
  • the SGLT homolog was subcloned into PME18S according to the prescription of the company. This was introduced into E. coli DH5Q !, and clones having cDNA were selected on LB agar medium containing ampicillin.
  • a cDNA sequence SEQ ID NO: 15
  • SEQ ID NO: 14 novel NaV Dalco-Stranspo overnight proteins containing the amino acid sequence (SEQ ID NO: 14) were designated as human SGLT homologs.
  • the transformant was named Escherichia coli DH5 ⁇ / ⁇ 2193.
  • a cDNA sequence (SEQ ID NO: 16) encoding the SGLT homolog protein containing The transformant was named Escherichia coli DH5 / TKD-l.
  • Example 1 Cloning of Human SGLT Homolog Gene Upstream Region and Determination of Nucleotide Sequence
  • the human genomic gene (CL0NTECH) was used as type III, and two primers 1 (SEQ ID NO: 1) and primer 1 2 (SEQ ID NO: 2) were used. PCR reaction was performed. In the reaction, the genomic DNA 11 was used as type III, the amount of Pfu Turbo DNA Polymerase (STRATAGENE) 11, primer 1 (SEQ ID NO: 1) was 0.5 M, and the 2 (SEQ ID NO: 2) was added at 0.5 ⁇ , dNTPs at 200 ⁇ , and the buffer attached to the enzyme to give a volume of 501.
  • STRATAGENE Pfu Turbo DNA Polymerase
  • PCR reaction a cycle of 94 ⁇ 1 minute, 96 ⁇ 20 seconds, 65 ⁇ 30 seconds, 72 ° C ⁇ 4 minutes was repeated 40 times, and finally an extension reaction at 72 ° C ⁇ 7 minutes was performed. Further, using the PCR reaction product as type III, a PCR reaction was performed using two primers, Primer-1 K1 (SEQ ID NO: 3) and Primer-1 XI (SEQ ID NO: 4).
  • the composition of the reaction solution was prepared using the above PCR reaction product 11 as type III, 11 amounts of Pfu Turbo DNA Polymerase (STRATAGENE), primer 1 K1 (SEQ ID NO: 3) and primer XI (SEQ ID NO: 4) was added to each of 0.5 M, dTs was 200 M, and the buffer attached to the enzyme was 51 to make a liquid volume of 501.
  • the PCR reaction is repeated at 94 ° C for 1 minute, followed by 40 cycles of 96 ° C for 20 seconds, 65 ⁇ 30 seconds, 72 ° C for 3 minutes, and finally 72 ° C for 7 minutes. The reaction was performed.
  • the PCR reaction product and firefly and luciferase expression plasmid vector PGV-B2 were digested with restriction enzymes KpnI (10U) and Xhol (10U) at 37 ° C. overnight. Perform 1% agarose gel electrophoresis, cut out a 2.3 Kbp DNA fragment (upstream region of the human SGLT homolog gene) and a 4.8 Kbp DNA fragment (pGV-B2), and convert the DNA using a gel extraction kit (Qiagen). It was extracted and the human SGLT homolog gene upstream region was subcloned into PGV-B2 according to the recipe of the Reigeshon Kit (Takara Shuzo).
  • a search of Celela's SNP database revealed two SNPs in the upstream region of the human SGLT homolog gene (Figure 2).
  • C698 (translation start point 1564 bp upstream) T was named SNP_P1
  • A824 (translation start point 1438 bp upstream) T was named SNP-P2.
  • PGV-B2-human SGLT homolog gene upstream region PGV2254 DNA using the QuickChange XL Site-Directed Mutagenesis Kit (STRATAGENE).
  • PTB2254 10ng, reaction buffer 51, primer for PI mutagenesis (SEQ ID NO: 6) or primer for P2 mutagenesis (SEQ ID NO: 7) 125ng, dNTP mix 1 ⁇ I, distilled water into QuickSolution 31 This was added to make 501, and 11 of Pfu Turbo DNA Polymerase (2.5 U / 1) was added.
  • the PCR reaction was repeated at 95 ° C for 1 minute, followed by a cycle of 95 ° C for 50 seconds, 50 seconds and 68/12 minutes 18 times, and finally an extension reaction at 68 ° C for 7 minutes was performed.
  • Restriction enzyme Dpnl (1OU) was added to the PCR reaction product and reacted at 37 ° C. for 1 hour to cleave the methylated parent strand DNA. This was introduced into Escherichia coli XL-1 Blue, and a clone having the plasmid was selected on LB agar medium containing ampicillin. The sequence of each clone was analyzed to obtain a DNA sequence containing P2 base substitution (SEQ ID NO: 8).
  • the transformant was named Escherichiacoli XL1-Blue / pTB2255.
  • a DNA sequence containing the base substitution of P1 (SEQ ID NO: 9) was obtained.
  • the transformant was named Escherichia coli XLl_Blue / pTB2256.
  • a DNA sequence (SEQ ID NO: 10) containing both PI and P2 base substitutions was obtained.
  • the transformant was named Escherichiacoli DH5o; / pTB2257.
  • the human SGLT homolog gene upstream region (PTB2254) DNA was type III, and two primers, primer K2 (SEQ ID NO: 11) and primer XI (SEQ ID NO: 4), primer K3 (SEQ ID NO: 12) and PCR using primer XI (SEQ ID NO: 4), primer K1 (SEQ ID NO: 3) and primer X2 (SEQ ID NO: 13), primer K2 (SEQ ID NO: 11) and primer X2 (SEQ ID NO: 13) The reaction was performed.
  • the composition of the reaction solution used in the reaction was as follows: PTB2254 DNA lOng was used as type II, 11 volumes of Pfu Turbo DNA Polymerase (STRATAGENE), 0.5 M of each primer set, 200 M of dNTPs, and enzyme. 51 of the attached buffer was added to make a liquid volume of 50 ⁇ 1.
  • the PCR reaction is After 94 ° C for 1 minute, a cycle of 96.20 seconds, 65 ° C for 30 seconds, 72 ° C for 3 minutes was repeated 40 times, and finally an extension reaction at 72 ° C for 7 minutes was performed.
  • the PCR reaction product and the luciferase expression plasmid vector pGV-B2 were digested overnight with restriction enzymes KpnI (10U) and XhoI (lOU) at 37 ° C.
  • Promoter-less firefly Luciferase expression plasmid vector PGV-B2 (Futtsu Gene), SV40 virus early enhancer / promo + firefly Luciferase expression plasmid vector PGV-C2 (Futtsu Gene), human SGLT homolog gene upstream region + repo overnight plasmid K1X1 (CA), K1XKCT), KlXl (TA), KIXKTT), K2X1, 3X1, K1X2, K2X2
  • Each of pRL-TK as a control for internal standardization (Expression of sea pansy luciferase downstream of the thymidine kinase promoter of simple herpes virus, Nippon Gene) 0.5 g and FuGENE 6 (Roche) 31 with Opt i-MEM (Gibco_BRL) After adding to Step 1 and allowing to stand at room temperature for 15 minutes, add 10 I / wel 1 of each sample 4 wel 1 to human
  • Promoter activity was measured using Pitka Gene Dual 'Sea Pansy (Futtsu Gene). HepG2 cells were washed twice with PBS (200 ⁇ 1), the attached cell lysing agent was added to each well, and shaken at room temperature for 15 minutes. The cell lysate was transferred to a 96-well fluoro black plate (Dainippon Pharmaceutical Co., Ltd.). LuminoskanRS (Labsystems) was used to measure the amount of luminescence by Luciferase. E Tal 'luciferase activity was determined by adding 50 ⁇ l of each of the pikacadin luminescence reagents 11 to each well, and measuring the amount of luminescence for 5 seconds 1 second after the measurement delay time.
  • the luminescence reagent was added to each well, and the luminescence was measured for 1 second and 5 seconds after the measurement delay time.
  • the overnight activity of the promoter was shown as the ratio of the activity of the luciferase in each well to the activity of the luciferase in the pansy (Fig. 4). It was found that the X1K3 region is essential for the promoter activity of the human SGLT homolog, and that the presence of the K1K3 region further enhances the promoter activity. SNP showed no difference in promoter activity among the CA, CT, and TA types, but the TT type was found to have reduced activity.
  • Human SGLT homolog gene upstream region + repo overnight plasmid pTB2254 0.5 g and FuGENE 6 (Roche) 1.51 were added to Opti-MEM (Gibco_BRL) 501 and left at room temperature for 15 minutes.
  • Cells, Huh-7 cells (1 X 10 5 cells / 0.2 ml 10% FBS-supplemented DMEM medium / 48 well plate) by adding 4 zs 1 / wel 1 of each sample 10 z 1 / wel 1 and dexamethasone (Wako) was added at 0 to 3 M and cultured for 3 days. Promoter activity was measured using the Pizza Gene Luciferase assay system (Nippon Gene).
  • HepG2 and Huh-7 cells were washed twice with PBS (2001), and the attached cell lysing agent was added to each well, and shaken at room temperature for 15 minutes.
  • the cell lysate was transferred to a 96-well fluoro black plate (Dainippon Pharmaceutical Co., Ltd.) with 10 xl of each well.
  • the luminescence by luciferase was measured using a 1420 ARVO SX multilabel counter (Wallac), adding a Pitka Gene luminescence reagent to each well, and measuring the luminescence for 10 seconds (Fig. 5).
  • Dexamethasone was found to promote the activity of the upstream region of the human SGLT homolog gene.
  • the human SGLT homolog promoter of the present invention contains a sequence of regulae, it usually has an activity that reflects the expression mode of the human SGLT homolog that is closer to the human body as compared to those that do not contain them. Therefore, it can be used as a mouth motor to be incorporated into a vector used for treatment of human diseases, setting of a drug screening system, and the like under conditions closer to the human body.

Abstract

La présente invention concerne un ADN ayant une région promotrice contenant une séquence régulatrice homologue de SGLT humaine, un transformant transformé par cet ADN, un procédé de criblage d'un composé ou de son sel à action de promotion ou d'inhibition de l'activité de promoteur homologue de SGLT humain reposant sur l'utilisation du transformant susmentionné, un procédé de criblage d'un médicament contre le diabète ou d'un médicament contre l'hyperlipidémie reposant sur l'utilisation du transformant susmentionné, un kit de criblage d'un composé ou de son sel ayant une action de promotion ou d'inhibition de l'activité de promoteur homologue de SGLT humain à l'aide dudit transformant, des compositions médicinales contenant un composé ou son sel ayant une action de promotion ou d'inhibition de l'activité du promoteur homologue de SGLT humain, obtenues à l'aide du transformant susmentionné qui est lui-même obtenu à l'aide du procédé ou du kit de criblage susmentionnés, etc.
PCT/JP2002/013651 2001-12-27 2002-12-26 Promoteur homologue de sglt humain et utilisation dudit promoteur WO2003056005A1 (fr)

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US9308204B2 (en) 2009-11-02 2016-04-12 Pfizer Inc. Dioxa-bicyclo[3.2.1]octane-2,3,4-triol derivatives

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US9439901B2 (en) 2009-11-02 2016-09-13 Pfizer Inc. Dioxa-bicyclo[3.2.1]octane-2,3,4-triol derivatives
US9439902B2 (en) 2009-11-02 2016-09-13 Pfizer Inc. Dioxa-bicyclo[3.2.1]octane-2,3,4-triol derivatives

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