WO2005073398A1 - Methode d'analyse de la capacite a inhiber l'augmentation du niveau de graisse - Google Patents

Methode d'analyse de la capacite a inhiber l'augmentation du niveau de graisse Download PDF

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WO2005073398A1
WO2005073398A1 PCT/JP2004/000976 JP2004000976W WO2005073398A1 WO 2005073398 A1 WO2005073398 A1 WO 2005073398A1 JP 2004000976 W JP2004000976 W JP 2004000976W WO 2005073398 A1 WO2005073398 A1 WO 2005073398A1
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amino acid
acid sequence
seq
dna
protein
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PCT/JP2004/000976
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English (en)
Japanese (ja)
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Yasushi Hiramine
Syunsuke Takasuga
Hiroko Murakami
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Sumitomo Chemical Company, Limited
Dainippon Sumitomo Pharma Co., Ltd.
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Priority to PCT/JP2004/000976 priority Critical patent/WO2005073398A1/fr
Publication of WO2005073398A1 publication Critical patent/WO2005073398A1/fr

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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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for testing the ability to suppress an increase in fat mass, and the like.
  • Fat in the body can be broadly classified into fat, which is accumulated finely in cells of body tissues, and fat, which is present in blood.
  • the former fat is used for (1) subcutaneous adipose tissue such as the abdomen, thigh, buttocks or chest, (2) fat tissue or intestine in contact with the kidney or epididymis, etc. It accumulates in various tissues such as membrane adipose tissue or abdominal adipose tissue such as omental adipose tissue, (3) adipose tissue contained in the liver, or (4) pleural adipose tissue.
  • abdominal adipose tissue especially, adipose tissue existing around blood vessels flowing into the portal vein such as mesenteric adipose tissue and omental adipose tissue
  • metabolic diseases such as impaired glucose tolerance, diabetes, hyperlipidemia, hypertension, and atherosclerosis
  • cardiovascular disorders such as coronary artery disease, angina pectoris, and myocardial infarction, etc. has been revealed.
  • Fat in the blood is the release of fat synthesized in the cells into the blood. Specifically, the fat in the blood forms complex particles (lipoproteins) with a protein core called apoprotein. Circulating.
  • TMGAT monoacylglycerol acyltransferase
  • the present inventors have conducted intensive studies in such a situation, and found that a substance having a monoacylglycerose / retransferase (MGAT) P and harmful activity is capable of accumulating fat in adipocytes and liposome in the small intestine and liver. It has an effect of suppressing protein synthesis and has been found to be effective as a substance that suppresses an increase in fat mass.
  • MGAT monoacylglycerose / retransferase
  • a method for searching for a fat mass increase inhibiting substance which comprises selecting a substance having a fat mass increase inhibiting ability based on the fat mass increasing inhibiting ability tested by the assay method described in Item 1;
  • An agent for suppressing an increase in fat mass characterized in that the substance selected by the search method described in Item 2 or a pharmaceutically acceptable salt thereof is used as an active ingredient;
  • An agent for suppressing an increase in fat mass characterized by comprising a substance having a monoacylglycerose lucyltransferase inhibiting ability or a pharmaceutically acceptable salt thereof as an active ingredient;
  • (b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, 15 or 17, comprising one or more amino acids deleted, added or substituted, and a monosaccharide Protein with lurasyltransferase activity
  • (c-1) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monoglycylglyceryltransferase activity
  • (d-1) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18
  • (e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with a DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18, and Protein with lucyltransferase activity
  • (f-1) consists of a DNA consisting of a base sequence complementary to a DNA having the base sequence of SEQ ID NO: 14, 16 or 18, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions , And a protein having a monoacylglycerol acyltransferase activity ⁇
  • a method for searching for a fat accumulation inhibiting substance which comprises selecting a substance having a fat accumulation inhibiting ability based on the fat accumulation inhibiting ability tested by the assay method described in Item 7; 9.
  • a fat accumulation inhibitor characterized by comprising, as an active ingredient, a substance selected by the search method described in Item 8 or a pharmaceutically acceptable salt thereof;
  • a fat accumulation inhibitor comprising a substance having a monoacylglycerolacyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient;
  • a method for searching for a lipoprotein synthesis-inhibiting substance which comprises selecting a substance having lipoprotein synthesis-inhibiting ability based on the lipoprotein synthesis-inhibiting ability tested by the assay method described in Item 11;
  • a lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance selected by the search method described in Item 12 or a pharmaceutically acceptable salt thereof;
  • a lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance having an ability to inhibit monoacylglycerol luasyltransferase or a pharmaceutically acceptable salt thereof;
  • the substance having the ability to inhibit monoacylglycerolacyltransferase is N- [2- (4-benzinole-1-ethyl / lephenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [ [15]
  • a protein characterized by having one of the following amino acid sequences
  • amino acid sequence of a protein having monoasinoglycerol transferase activity is the amino acid sequence of a protein having monoasinoglycerol transferase activity
  • (b-1) an amino acid sequence represented by SEQ ID NO: 13, 15 or 17, wherein one or more amino acids are deleted, added or substituted, and Glycerol acyltransferase activity
  • (c-1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monosylglycerose lucyltransferase activity;
  • (d-1) an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18
  • (e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18; Amino acid sequence of proteins having oral-lucyltransferase activity
  • (f-1) a DNA having the base sequence of SEQ ID NO: 14, 16 or 18; Amino acid sequence of a protein comprising a DNA consisting of a complementary nucleotide sequence and an amino acid sequence encoded by DNA that hybridizes under stringent conditions and having monoacylglycerol lucyltransferase activity
  • a gene characterized by having a nucleotide sequence encoding any of the following amino acid sequences;
  • (b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17, wherein one or several or more amino acids are deleted, added or substituted, and Amino acid sequence of protein having monoacylglycerol luacyltransferase activity
  • (c-1) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NOS: 13, 15, or 17, wherein Amino acid sequence of a protein having
  • (e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with the DNA having the nucleotide sequence represented by SEQ ID NO: 14, 16, or 18, and Amino acid sequence of protein having activity of silglycerol asinotransferase
  • (f-1) an amino acid sequence encoded by a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18, and a DNA that hybridizes under stringent conditions.
  • Amino acid sequence of a protein having monoacylglycerol lucinoletransferase activity is an amino acid sequence encoded by a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18, and a DNA that hybridizes under stringent conditions.
  • a catalyst for the following reaction comprising the protein according to item 17.
  • Figure 1 Using the mouse AT12 sequence as a probe, Northern hybridization was performed on mouse Adu1tNormal1Tissue mRNA NorthernBlot. From left lane, brain, heart, kidney, liver, lung. Tissue RNA of skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus.
  • Figure 2 Using the mouse AT14 sequence as a probe, northern predication was performed on mouse Adu1tNormal1Tissue mRNA NorthernBlot. From left lane, brain, heart, kidney, liver, lung. Tissue RNA of skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus.
  • Figure 4 Using the human AT14 sequence as a prop, Northern hybridization was performed on human AdlntNoRmTlssueTotalRNANorthernBlOT. Starting from the left lane, they are tissue RNA from the esophagus, stomach, small intestine, colon, ova, placenta, bladder, and fat. On the left of the figure, the gender (male-M, female-F) and age of the specimen from each tissue are shown.
  • FIG. 5 The mice were divided into two groups (group of 8), the first group was administered only olive oil and the second group was administered olive oil and Compound A. Approximately 101 blood samples were collected from the tail vein every hour from immediately after administration to 6 hours, and the amount of triglyceride in the blood was determined. The quantification results are shown in a graph in which the vertical axis plots the blood triglyceride concentration (mg / dL) and the horizontal axis plots time.
  • Figure 6 Divide mice into 3 groups (8 / group), group 1 only olive oil, group 2 olive oil + compound A: 1 Omg / kg, group 3 olive About 10 t1 of blood was collected from the tail vein before administration (Ohr) and 3 hours (3 hr) after administration for each group, and trig in the blood was administered. The amount of reseride was determined The measured triglyceride concentration (mg / dL) is shown on the vertical axis.
  • fat refers to fat contained in any cell, tissue or body fluid in a living body of a mammal, and specifically refers to a glycerol fatty acid ester such as triglyceride.
  • fat mass increase suppression ability is a concept that includes both fat accumulation suppression ability and fat synthesis suppression ability, and specifically, the ability to suppress fat accumulation in fat cells, adipose tissue or hepatocytes, or It means the ability to suppress fat synthesis in the small intestine or liver.
  • Adipose tissue refers to any adipose tissue of a mammal. Subcutaneous adipose tissue such as the side, thigh, buttocks, or chest; (2) adipose tissue in contact with the kidney or epididymis, mesenteric adipose tissue, or abdominal adipose tissue such as the omentum adipose tissue; 3) Adipose tissue contained in the liver, or (4) Intrathoracic adipose tissue. Fat cells refer to the individual cells that make up adipose tissue.
  • glycerol fatty acid esters such as triglyceride accumulate as fat.
  • fats in blood exist in the form of lipoproteins in which glycerol fatty acid esters such as triglycerides form complexes with apoproteins.
  • the lipoproteins are classified according to their densities into chylomicron, VLDL (very low density lipoprotein), LDL (low density lipoprotein), HDL (high density lipoprotein) and the like.
  • the tissues supplying triglyceride to the blood are the small intestine and the liver.
  • the small intestine controls triglyceride absorption from food, and the liver regulates the amount of triglyceride in the fluid by collecting and re-secreting lipoproteins in the blood.
  • tridaliseride in food is decomposed into monoacylglycerol and fatty acids in the intestinal tract by the action of knee lipase, then absorbed by small intestinal epithelial cells, and recombined into triglyceride in small intestinal cells .
  • the resynthesized triglyceride is incorporated into chylomicron, a type of lipoprotein, in small intestinal cells and secreted into the blood.
  • Chylomicron is converted to particles called chiral micron remnants while supplying triglyceride to peripheral tissues, and is eventually absorbed by the liver.
  • triglyceride synthesis is performed using fatty acids in liver cells, and the synthesized tridalicelide is incorporated into VLDL, a type of lipoprotein, and secreted into the blood.
  • VLDL like chylomicron, circulates through the body while supplying triglyceride to peripheral tissues, and is finally recovered in the liver in the form of LDL.
  • the above-mentioned ability to inhibit fat synthesis preferably refers to the ability to inhibit lipoprotein synthesis in the small intestine or liver.
  • the monoacylglycerol lysyltransferase used in the assay method of the present invention is an enzyme that catalyzes the following reaction (hereinafter sometimes referred to as the present enzyme):
  • the activity of such an enzyme can be measured by the following method, for example, the method of S.C. Jamdar et al. (Arch. Biochem. Biophys. 1992; 296 (2): 419-25).
  • reaction buffer 24 mM Tris-hydrochloric acid (H7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL ⁇ serum albumin, lmM DTT , 0.5 m
  • monoacylglycerolacyltransferase derived from mammals such as humans, mice, and rats is preferred.
  • a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 3 or 11,
  • b1 an amino acid sequence S represented by SEQ ID NO: 1 (single letter code:
  • a protein having a transferase activity ( ⁇ 3) a DNA having a nucleotide sequence complementary to D ⁇ having a nucleotide sequence represented by SEQ ID NO: 12, and an amino acid encoded by a DNA that hybridizes under stringent conditions (F4) a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence shown in SEQ ID NO: 14, and a protein having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 14.
  • Consists of an amino acid sequence encoded by DNA that hybridizes with A protein having a transferase activity (f5) an amino acid encoded by a DNA having a base sequence complementary to a DNA having a base sequence represented by SEQ ID NO: 16, and a DNA hybridizing under stringent conditions (F6) a DNA having a base sequence complementary to a DNA having a base sequence represented by SEQ ID NO: 18, and a protein having a monosaccharide glycerol acyltransferase activity;
  • a protein consisting of an amino acid sequence encoded by a DNA hybridizing with the above and having a monoacylglycerol lucyltransferase activity can also be mentioned as a preferable example.
  • sequence identity of ⁇ 80% or more in (e 6) includes, for example, a protein having an amino acid sequence represented by SEQ ID NO: 1, 3, 11, 13, 15, or 17 It includes mutations that occur naturally due to processing, species differences, individual differences, differences between tissues, etc. of the organism from which the protein is derived, and artificial amino acid mutations.
  • amino acid Also referred to as modification
  • Examples of the site-directed mutagenesis method include a method using amber mutation (gapped 'duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), and a method using PCR using mutagenesis primers. And the like.
  • the number of amino acids modified as described above is at least one residue, specifically, one or several, or more. The number of such modifications may be within a range in which monoacylglycerol transacylase activity can be found.
  • deletions, additions or substitutions modifications relating to amino acid substitutions are particularly preferred.
  • the substitution is more preferably an amino acid having similar properties such as hydrophobicity, charge, p, and steric structure.
  • substitutions include, for example, 1 glycine, alanine; 2 parin, isoleucine, leucine; 3 aspartic acid, glutamic acid, asparagine, glutamine; 2 serine, threoyun; 2 lysine, arginine; Substitution within the group of Yun, Tyrosine.
  • sequence identity 14 refers to sequence identity and homology between two DNAs or two proteins.
  • the “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared.
  • the DNA or protein to be compared may have an addition or deletion (for example, a gap or the like) in the optimal alignment of the two sequences.
  • Such sequence identity can be calculated, for example, by creating an alignment using ClustalWalgorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. Can be.
  • the sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENET II, or a public database.
  • the public database is generally available, for example, at homepage address http: ⁇ ww.ddbj.nig.ac.jp.
  • the sequence identity in the present invention may be 80% or more, preferably 90% or more, and more preferably 95% or more.
  • F (f 1).
  • F 2 (f 3), (f 4), (f 5), (f 6) “Hybridize under stringent conditions” The hybridization used in, for example, Sambrook J "., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, published by Cold Spring Harper Laboratory (Cold Spring)
  • Stringent conditions refers to, for example, a solution containing 6XSSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10XSSC) and a solution containing 50% formamide at 45 ° C. After forming a hybrid at, conditions such as washing at 50 ° C with 2X SSC (Molecular Biology, John Wiley & Sons,
  • the salt concentration in the washing step ranges from 50 ° C (low stringency conditions) at 2 XSSC to 50 at 0.2 XSSC. You can select from conditions up to C (high stringency conditions).
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C (high stringency conditions). It is also possible to change both the salt concentration and.
  • the present enzyme is in the form of an enzyme sample containing the present enzyme, for example, a microsomal fraction
  • the present enzyme is prepared by a normal cell fractionation method using an adipose tissue containing the present enzyme as a biomaterial. do it.
  • the method described in Wang Fang Cao et al. can be mentioned.
  • a gene consisting of a nucleotide sequence encoding the amino acid sequence of the present enzyme for example, (I) SEQ ID NO: 1, 3 , A nucleotide sequence encoding the amino acid sequence represented by 11, 13, 15 or 17; (II) a nucleotide sequence represented by SEQ ID NO: 2, 4, 12, 14, 16 or 18;
  • the offspring can be prepared using conventional genetic engineering techniques (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Springno, One Bar). Laboratory acquisition (method described in Cold Spring Harbor Laboratory press). Next, using the obtained present gene, the present enzyme is produced and obtained according to a usual genetic engineering method. Thus, the present enzyme can be prepared.
  • a culture obtained by preparing a plasmid capable of expressing the present gene in a host cell introducing it into a host cell, transforming the cell, and culturing the transformed host cell (transformant).
  • the enzyme may be obtained from a product.
  • the above-mentioned plasmid contains, for example, genetic information that can be replicated in a host cell and can be propagated autonomously, is easily isolated and purified from the host cell, and can function in the host cell
  • an expression vector having a simple promoter and a detectable marker into which a gene comprising a base sequence encoding the amino acid sequence of the present enzyme has been introduced.
  • Various types of expression vectors are commercially available.
  • vectors used for expression in mammalian cells include the SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (HSV promoter), actin gene promoter ⁇ ", aP
  • CMV promoter cytomegalovirus promoter
  • HSV promoter Raus Sarcoma Virus promoter
  • actin gene promoter ⁇ " actin gene promoter ⁇ "
  • aP This is an effort vector containing a promoter such as a two-gene promoter, which is commercially available from Toyobo, Takara Shuzo, or the like.
  • Examples of the host cell include a prokaryotic or eukaryotic microbial cell, an insect cell, a mammalian cell, and the like.
  • a prokaryotic or eukaryotic microbial cell an insect cell
  • a mammalian cell and the like.
  • mammalian cells and the like can be preferably mentioned from the viewpoint that the original structure of the present enzyme can be expected to be maintained.
  • the plasmid obtained as described above can be introduced into the host cell by a conventional genetic engineering method.
  • the transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells.
  • culture is performed in a medium containing an appropriate carbon source, a nitrogen source, micronutrients such as vitamins, and cell growth factors such as blood serum.
  • the culture method may be any of solid culture and liquid culture, and preferably includes liquid culture such as aeration and agitation culture. This enzyme may be obtained by combining methods generally used for isolation and purification of general proteins.
  • transformants obtained by culturing tins are collected by centrifugation or the like, and the transformants are crushed or dissolved, proteins are solubilized if necessary, and various types of ion exchange, hydrophobicity, gel filtration, etc. are performed.
  • Purification may be performed by using the steps using chromatography alone or in combination.
  • affinity purification may be performed by adding an amino acid sequence suitable for affinity purification to the N-terminus or C-terminus of the present enzyme by ordinary genetic engineering techniques. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.
  • the assay method of the present invention comprises: (1) a first step of measuring the activity of monoacylglycerol acyltransferase in a contact system between monoacylglycerol acyltransferase and a test substance; and (2) a first step.
  • the fat of the test substance can be calculated. It is simple and suitable for primary screening, etc., because the ability to suppress the amount of B intensification can be evaluated.
  • a divalent metal cation such as magnesium ion, dithiothreitol (hereinafter referred to as DTT), etc.
  • DTT dithiothreitol
  • the added concentration is, for example, 2 mM or more, and preferably 5 niM to 10 mM.
  • the added concentration is, for example, 50 ⁇ M or more, and preferably 0.2 mM to 2 mM.
  • PC phosphatidicolin
  • PS phosphatidinoreserin
  • the first step of the assay method of the present invention comprises: (A) two kinds of substrates (for example, 2 or 1-mono A form of contacting the test substance with a test substance, such as a glycylglycerol (preferably, 2-monoacinoreglycerol) and acetyl-CoA); and (B) two types of substrates (for example, 2 or 1-Monoacylglycerol and / or acyl-CoA) and the test substance are in contact with the enzyme (i.e., the test substance is used as the other of the above two substrates). When handling), either of the forms.
  • substrates for example, 2 or 1-mono A form of contacting the test substance with a test substance, such as a glycylglycerol (preferably, 2-monoacinoreglycerol) and acetyl-CoA
  • B two types of substrates
  • the test substance for example, 2 or 1-Monoacylglycerol and / or acyl
  • the order in which the enzyme and the test substance are brought into contact with the enzyme is as follows: (a) Contact the enzyme with the test substance first, incubate for a certain period of time, A form in which one kind of substrate is added, (b) a form in which the enzyme, the test substance and two or one kind of substrate are contacted at the same time, and (c) a form in which this enzyme and two kinds or one kind of substrate are used. Any of the following forms may be used: first, contact, incubation for a certain period of time, and then addition of a test substance, but the form (a) is preferred.
  • the contact time between the present enzyme and the test substance may be, for example, 1 minute or more, preferably 1 minute to 1 hour.
  • the heat retention temperature in the contact system for example, 0 ° C to 70 ° C, preferably 4 ° C to 40 ° C can be mentioned.
  • the heat retaining temperature in the contact system for example, 0 ° C to 70 ° C, preferably 4 ° C to 40 ° C can be mentioned.
  • the concentration of the test substance in the first step of the assay method of the present invention is, for example, 1 ⁇ to 10 mM, and preferably 0.01 / zM to 5 mM.
  • the concentration of the substrate is, for example, 1 nM to 1 mM, preferably 5 ⁇ M to 100 M.
  • the form of the enzyme to be brought into contact with the test substance in the first step of the assay method of the present invention may be (A) a purified product or a crudely purified product of the enzyme, or (B) an intracellular form.
  • a test substance with a transformed cell (hereinafter, sometimes referred to as a transformed cell) into which a gene consisting of a nucleotide sequence encoding the amino acid sequence of the present enzyme is introduced. Or the like).
  • the cells may be cells separated from the tissue or cells forming a group having the same functional form.
  • the concentration of the enzyme to be brought into contact with the test substance is, for example, 1 ng / m 1 or more, and preferably 10 ng / m 1 or more.
  • the concentration of the present enzyme to be brought into contact with the test substance is, for example, 1 ⁇ 10 3 cells or more, preferably 1 ⁇ 10 4 cells or more, as the concentration of the transformed cells. l or more.
  • This transformed cell can be prepared as follows.
  • Plasmids are prepared by inserting the present gene into a vector that can be used in cells into which the present gene is to be introduced in such a manner that it can be expressed and connected to a promoter, using ordinary genetic engineering techniques.
  • the promoter used here may be any promoter that can function in the cell into which the present gene is to be introduced.
  • the cell is an animal cell, the SV40 virus promoter, the cytomegalovirus promoter-1 (CMV Promoter, Raus Sarcoma Virus promoter (RSV promoter), J3 actin gene promoter, aP2 gene promoter and the like.
  • a commercially available vector containing such a promoter upstream of the multiple cloning site may be used.
  • the plasmid is introduced into cells.
  • the method of introduction into cells include the canoleic acid phosphate method, the electroporation method, the DAE dextran method, and the Myseno 1 ⁇ synthesis method.
  • the calcium phosphate method is described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., and the electroporation introduction method and the DEAE dextran method are Ting, AT et al. , EMBO J., 15,
  • Examples of the method described in 6189-6196 and the method for producing miseno include the method described in Hawkins, C. J. et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790.
  • a micelle formation method When a micelle formation method is used, a commercially available reagent such as lipofectamine (manufactured by Gibco) or Fugene (manufactured by Behringer) may be used.
  • the transformed cells can be obtained by culturing the cells into which the plasmid has been introduced, for example, by using a selection marker gene previously contained in the vector and culturing them in a medium under selection conditions according to the selection marker gene. Can be selected. The selection may be continued to obtain the present transformed cell which has become a stable transformant in which the present gene has been introduced into the chromosome.
  • the introduced gene is located on the chromosome
  • the presence of the present gene in genomic DNA may be detected and confirmed using a method such as Southern hybridization using DNA having a partial base sequence as a probe.
  • a method for measuring the activity of monoacylglycerol lucyltransferase includes, for example, a method in which a mixed solution containing the present enzyme, a test substance, and a substrate is fixed. After reacting for a period of time, a method of analyzing the amount of reduction of the raw materials such as substrates in the reaction or a method of analyzing the amount of addition of the product in the reaction can be exemplified. These methods may be any known activity measuring method. Specific examples include the method described in Arch. Biochem. Biophys. 1992; 296 (2): 419-25 described above.
  • the reaction temperature at the time of activity measurement in the first step of the assay method of the present invention may be, for example, 15 ° C to 70 ° C, and preferably 20 ° C to 40 ° C.
  • the reaction time is, for example, 1 minute or more, preferably 15 minutes to 1 hour.
  • the reaction pH is, for example, from 6.0 to 9.5, preferably ⁇ 7.0 to 8.0.
  • the reaction solution is separated by, for example, HPLC, thin-layer chromatography, paper chromatography, or the like, and then the raw material or the product is separated.
  • a method such as detecting the ultraviolet absorption or radioactivity of the product [4 (when the raw material previously labeled with a radioisotope is used)), or measuring the ultraviolet absorption or radioactivity, etc. If it is necessary to remove the protein from the reaction solution during the above analysis, the protein may be removed from the reaction solution by the method described in, for example, Methods Enzyraol. 280, 211-221. Should be removed.
  • the ability of the IB substance to suppress an increase in fat mass is evaluated. That is, when the activity measured as described above is lower than the activity in the control, it may be evaluated that the substance has an ability to suppress an increase in fat mass. In this manner, the test for the ability of the test substance to inhibit the increase in fat mass (the test method of the present invention) can be performed.
  • the fat mass increase suppression ability of the other test substance may be evaluated based on the fat mass increase suppression ability of at least one substance (for example, a reference substance). Of course, both may evaluate.
  • the first step of the assay method of the present invention comprises the step (A) wherein the two substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol)) are used.
  • the two substrates for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol)
  • the inhibition rate should be determined according to the following formula:
  • Inhibition rate (%) ⁇ control (negative control) value-measured (test substance) value ⁇
  • the fat mass increase suppression ability may be evaluated based on the calculated inhibition rate. That is, the inhibition rates of the various test substances calculated as described above are compared, and as a result, the test substance having a high inhibition rate has a higher P than the test substance having a low damage rate. What is necessary is just to evaluate that it has the capacity
  • the first step of the assay method of the present invention comprises the step (A) wherein the two kinds of substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol) and asinole-CoA) are used. And the test substance are in contact with this enzyme, and the control (reference substance) value is measured when a substance (reference substance) having the ability to suppress fat mass increase is used as a control. (Test substance) By comparing the values, the ability to suppress the increase in fat mass may be evaluated. In this case, if the measured (test substance) value is lower than the control (reference substance) value, the test substance is evaluated as having a higher ability to suppress the increase in fat mass than the reference substance. 4 000976
  • the first step of the assay method of the present invention may comprise the step (B) of one of the two substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol))
  • the test substance is in the form of contact with the enzyme (i.e., diasyl-CoA) and the test substance (that is, when the test substance is treated as the other of the two substrates)
  • a substance having a fat mass increase suppressing ability may be used as a control, and the fat mass increase suppressing ability may be evaluated by comparing the control (reference substance) value and the measured (test substance) value. In this case, if the measured (test substance) value is higher than the control (reference substance) value, the test substance is superior to the reference substance as a substrate for the enzyme. This means that this enzyme is superior to the reference substance as a competitive inhibitor of this enzyme.
  • a substance having the ability to inhibit the increase in fat mass may be selected based on the ability to inhibit the increase in fat mass, which has been assayed by the assay method of the present invention (the present invention search method).
  • the inhibition rate which is an index for evaluating the ability of the test substance to suppress the increase in fat mass
  • the inhibition rate is a statistically significant value.
  • a substance having an inhibition rate of 30% or more in the above formula more preferably a substance having an inhibition rate of 50% or more, is selected as a substance having an ability to suppress an increase in fat mass.
  • the measured (test substance) value is higher than the control (reference substance) value.
  • Substances exhibiting low values are selected as substances having the ability to suppress fat addition.
  • a substance whose measured (test substance) value is higher than the control (reference substance) value is selected as a substance having an ability to suppress the increase in fat amount.
  • the substance may be any substance such as a low molecular weight compound, a protein or a peptide as long as it has an ability to suppress an increase in fat mass.
  • the substance selected by the search method of the present invention has an ability to suppress an increase in fat mass, and may be used as an active ingredient of a fat mass increase inhibitor.
  • Examples of such a substance include, for example, ⁇ — [2 -— (4-benzyl-2-ethyl) Phenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidin-1 7-amine.
  • the monoacylglycerol lysyltransferase inhibitor (hereinafter referred to as P and harmful agent) selected by the search method of the present invention has the ability to suppress the increase in the amount of fat, as described in Examples in the present specification. Alternatively, it can be confirmed by a known method such as the method described in WO 00/47475.
  • the fact that the inhibitor selected by the search method of the present invention has the ability to inhibit lipoprotein synthesis can be confirmed by the following known method.
  • cells having lipoprotein synthesis ability are cultured in a test tube in the presence of a substance having an ability to inhibit monoacylglycerol lucyltransferase (hereinafter sometimes referred to as the present inhibitor), and the amount of triglyceride is determined. Is measured and compared with the amount of triglyceride in the absence of the present inhibitor.
  • the present inhibitor a substance having an ability to inhibit monoacylglycerol lucyltransferase
  • the present inhibitor a substance having an ability to inhibit monoacylglycerol lucyltransferase
  • the preparation of liver cells can be performed, for example, by the method described in Endocrinology, Jonathan R. Seckl et al., P4754-4761, 1995, that is, a method of preparing liver cells by treating an isolated liver with collagenase, or a method of preparing liver cells.
  • fatty acids As a method for quantifying the amount of triglyceride in the above liver and small intestine cells, fatty acids Alternatively, there is a method in which glycerol is added to cells and cultured for a certain period of time, and the amount of triglyceride contained in the supernatant fraction or cells is quantified.
  • a method using a radiolabel There are two methods for quantifying triglyceride: a method using a radiolabel, and a method for directly quantifying tridarylide without using a radiolabel. Examples of the former method include the method described in Journal of Lipid Research, Joan A. Higgins et al., Pl728-1739, 2000,
  • the evaluation of the ability of the inhibitor to inhibit lipoprotein synthesis can be carried out by allowing the above-mentioned tridaliceride synthesis to be carried out in contact with the inhibitor and comparing it with the ability of synthesizing triglyceride in the absence of P.
  • the VLDL and chylomicron can be isolated by methods well known to those skilled in the art, such as centrifugation.
  • the ability to inhibit protein synthesis can also be confirmed.
  • blood triglyceride concentration was reduced by fasting, and a method of measuring an increase in blood triglyceride concentration when lipid was administered thereto was used (International Journal of Obesity, K. Cianflone et al., P705- 713, 2001).
  • a fat mass increase inhibitor (namely, the fat mass increase inhibitor of the present invention) characterized by comprising a substance having a monoglycol glycerol lusyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient.
  • An effective amount thereof can be orally or parenterally administered to mammals such as humans.
  • the fat mass increase inhibitor of the present invention can be used in a usual form such as a tablet, a capsule, a syrup, a suspension and the like.
  • the present invention can be used in the form of ordinary liquids such as solutions, emulsions, suspensions and the like.
  • Examples of the method for parenterally administering the above-mentioned fat-increasing inhibitor of the present invention in the form of injection include a method of injection, a method of rectum administration in the form of suppositories, and the like.
  • the above-mentioned suitable dosage form is a substance having a monoacylglycerol lucyltransferase inhibitory activity or an acceptable pharmaceutically acceptable carrier, excipient, binder, stabilizer, diluent or the like. It can be produced by blending a salt.
  • an acceptable buffer, solubilizing agent, isotonic agent and the like can be added.
  • the dosage varies depending on the age, sex, body weight, degree of disease, type of fat-increasing U-suppressant, dosage form, etc., of the mammal to be administered.
  • About 1 mg to about 2 g, preferably about 5 mg to about 1 g, of the active ingredient may be administered per injection.In the case of injection, about 0.1 mg to about 50 mg of the active ingredient is used for an adult. 0 mg may be administered.
  • the above-mentioned dose can be administered once or in several divided doses.
  • Examples of the disease to which the fat mass increase inhibitor of the present invention can be applied include metabolic diseases such as decreased glucose tolerance due to insulin resistance, type II diabetes, hyperlipidemia, hypertension, and atherosclerosis, coronary artery disease, and stenosis. Diseases such as cardiovascular disorders such as heart disease and myocardial infarction can be mentioned.
  • a pharmacologically effective amount of a substance having an ability to inhibit monoacylglycerol acyltransferase for inhibiting the activity of monoacylglycerol acyltransferase is administered to the living body.
  • an increase in fat mass can be suppressed.
  • the use of the monoacylglycerol transferase gene as a reagent to provide the enzyme is also ⁇ .
  • the former include the use of monoacylglycerol acyltransferase associated with the assay method of the present invention, the search method of the present invention, and the like.
  • Another example is the regulation of the expression of monoacylglycerol lucyltransferase to reduce fat content. It can be used as a reagent for providing an index for evaluating the ability to suppress the increase in fat mass when suppressing the increase. More specifically, use as a primer or probe in known methods for specifically detecting a specific gene, such as the Northern blot method, RT-PCR method, in situ hybridization method, and DNA chip. And the like.
  • mesenteric fatty thread II Thirty 14-week-old male Wistar rats (three SLC) were sacrificed and laparotomized to remove adipose tissue attached to the mesentery (hereinafter referred to as mesenteric fatty thread II).
  • EDTA'4Na ImM tetrasodium ethylenediaminetetraacetate
  • DTT ImM dithiothreitol
  • the middle layer that did not contain atCake) and the lower layer (H) was separated.
  • the separated intermediate layer was further centrifuged at 4 ° C. at 16,000 g ⁇ 15 minutes.
  • the resulting supernatant was centrifuged at 105,000 g ⁇ 60 minutes at 4 ° C. to collect a precipitate (microsomal fraction).
  • the washed precipitate was resuspended in the above Medium A to prepare a microsomal fraction having a protein concentration of 1 mg Zm1.
  • Example 2 Example 2
  • Atsushi solution 24 mM Tris-hydrochloric acid (pH 7.5), 5 Om chloride, 8 mM magnesium sulfate, 1.S SmgZml ⁇ serum albumin, 1 mM DTT) 800 gZm 1 test substance (1% dimers hoki) Cid (hereinafter referred to as DMSO) solution
  • DMSO 1% dimers hoki
  • TLC plate which is sometimes referred to as a TLC plate below
  • a developing solvent of hexane ⁇ ethyl ether / acetic acid 75: 25: 1, ⁇ / ⁇
  • the TLC plate was dried at room temperature, and the dried TLC plate was exposed to an imaging plate (Fuji Film Co., Ltd.) for 16 hours.
  • the activity of monoacyl and lysine lucyltransferase when the test substance is added (measured value A + measured value ⁇ 2, hereinafter referred to as activity value 1)
  • the sections corresponding to the development positions of 1,2-diacylglycerol and triglyceride on the TLC plate were also calculated for the test substance-free group and the background group.
  • the inhibition rate which is an index for evaluating the enzyme inhibitory ability
  • the accumulated rate The relationship with the inhibition rate in Reference Examples 1 and 2 described below, which is based on the direct measurement of fat mass, shows at least dose dependence in a specific concentration range. It was confirmed that the correlation was positive.
  • Reference example 1
  • the following treatment was performed on the mesenteric adipose tissue after washing.
  • the adipose tissue extracted in (1-1) above was treated with collagenase (Type I or Vin, Sigma), penicillin (Gibco), streptomycin (GIB VIIIC) and amphotericin (Gibco), respectively.
  • Dulbecco's modified eigland (4.5 g LD-glucose and 584 mg / l) added to a final concentration of lmgZm1, 100 cut_: 011, 100 / X g / m1 and 250 ngZm1 (LL-glutamine-containing, GIPCO)
  • the pieces were cut into about 5 mm squares using scissors in about 3 O Oml.
  • Dulbecco's modified Eagle's medium (4.5 g / LD-glucose) Medium and 584 mg / LL—containing glutamine, gibconed earth, hereafter sometimes referred to as FBS-containing medium) and suspended in 50 ml of nylon mesh (420 S [mesh size 25 ⁇ ], Sangen) (Industry). The filtrate was collected and centrifuged at room temperature at 1800 rpm for 5 minutes. The liquid layer was gently removed by decantation, and the precipitate was suspended again in 5 Oml of an FBS-containing medium.
  • the operation of centrifuging, removing the liquid layer, and suspending in a FBS-containing medium was further performed twice on the suspension in the same manner as described above to prepare a suspension (120 ml).
  • the suspension was dispensed into cell culture flasks (T150 for adherent cells, Iwaki Glass Co., Ltd.) in an amount of 3 Oml each and cultured at 37 ° C. in the presence of 5% CO 2 .
  • Two to three hours after the start of the culture the medium was removed, and the vessel wall of the flask was washed with 15 ml of the above-mentioned phosphate buffer.
  • phosphate buffer FBS-containing media 30 m 1 flask, and cultured at 3 7 ° C, 5% C 0 2 presence .
  • the medium was removed, the wall of the flask was washed once with 15 ml of a phosphate buffer solution, and then the trypsin-ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) solution (0.05%) was added to the flask.
  • EDTA trypsin-ethylenediaminetetraacetic acid
  • the FBS-containing medium 100 mu 1 containing prostaglandin J 2 were added to each Ueru additional 2 days, 5% C0 2 presence after incubation at 37 ° C, each Ueru the medium was removed, 10 ⁇ g / m 1 insulin, 5 M 15-Dokishi - FBS-containing containing prostaglandin J 2, a test substance 50 M, 0. 5% DMSO (the Wako pure drug Industries, Ltd.) - ⁇ 12 '14 medium was added to 100 1 each Ueru, it was similarly cultured in the test substance untreated silage, 10 ⁇ gZni l incidents Yurin instead of the culture medium, 5 ⁇ 1 5- Dokishi one ⁇ 12 '14 -.
  • the Oiruretsudo ⁇ staining solution The medium containing the solution was removed, and 1 ⁇ l of a 20% aqueous solution of triethyl phosphate was added to each well, after which the 20% aqueous solution of triethyl phosphate was removed from each well, and 100 1 of the same aqueous solution was newly added to each well.
  • a cell lysate (2% SDS, 0.2N NaOH) lO Ojul was added to each well, and the mixture was incubated at 37 ° C for 3 hours or more.
  • Part 2 (2-1) Preparation of adipose tissue piece
  • Dulbecco's Modified Eagle Medium-Low Darco ⁇ "S (1 g containing Og / LD-glucose and 584 mg / LL-glutamine, Gibco) was added to a 48-well plate (for adhesion cell culture, Sumitomo Belite).
  • the test substance dissolved in DMS ⁇ was added so that the final concentration of the test substance was 50 i ⁇ and the final concentration of DMSO was 0.5 ° / 0 . . each Ueru, the (2 1) were placed the prepared fat Ojen 1:50 to 0 Omg with, 5% C0 2 presence 3
  • a developing solvent of hexane: ethyl ether: acetic acid (75: 25: 1) is placed in a closed container, and the TLC plate is developed using the developing solvent. The pieces were dried at room temperature. An imaging plate (Fuji Film Co., Ltd.) was exposed to the dried TLC plate for 4 to 5 hours.
  • the imaging plates Bio Image Analyzer (BAS 2000, Fuji Film Co.) was analyzed, the said measured 4 C] radioactivity of the portion corresponding to the deployed position of Toridariseraido on TLC plates (hereinafter, measurements 5)
  • test compound a known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-1
  • 7-amine ie, compound A
  • the inhibition rate at each test concentration was calculated using the same method as described above. The results are shown in Table 2 together with the results of the former.
  • PCR a 50 ⁇ ⁇ PCR reaction solution containing 5 ⁇ L of buffer attached to ExTaq polymerase and 4 / iL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase, and use it for PCR. did.
  • the PCR was carried out under the conditions that the heat cycle consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute was repeated 50 times, and finally the temperature was kept at 72 ° C for 5 minutes. I was After PCR, a PCR product showing about 1.2 Kbp was recovered by agarose electrophoresis.
  • a plasmid prepared by subcloning the recovered PCR product into ⁇ T7—B1 uevector (Novagen soil) was used to transform E.coli JM10 9 strain competent cells (Toyobo). From the cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / niL ampicillin, the plasmid was Q I AGEN P 1 a sm i d Ma X i Kit (Q
  • the DNA was isolated and purified using AGEN (1AGEN) to obtain a plasmid containing DNA derived from a mouse and having the nucleotide sequence of the present gene (mAT12).
  • AGEN (1AGEN) AGEN (1AGEN) to obtain a plasmid containing DNA derived from a mouse and having the nucleotide sequence of the present gene (mAT12).
  • the plasmid containing the PCR product (approximately 1.2 kbp) obtained in Example 3 was converted into a type III plasmid, and then converted to a Thermo Sequenase II dye kit (Amersham Pharmacia Biotech). ) Using the ABI 373DN A sequence reader (PE Ap 1 ied Biosystems), Sanger's method (F. Sanger, S. Nicklen, AR Cou1sonA, roceedingsoi Natio nal Academyof Science USA (1977), 74, 5463-5467), a mouse-derived book consisting of a DNA having the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of the gene (mAT12) was determined.
  • Example 5 The nucleotide sequence of the gene (mAT12) was determined.
  • PCR reaction solutions containing dNTPmixTre (2.5 mM) 4; L attached to TakaraEx-Taq polymerase were prepared and subjected to PCR.
  • the PCR is performed under the conditions that the heat cycle consisting of 94 ° C for 30 seconds, then 60 ° C for 30 seconds, 72 ° C for 1 minute is repeated 50 times, and finally the temperature is kept at 72 ° C for 5 minutes.
  • a PCR product showing about 1.2 Kbp was recovered by agarose gel electrophoresis. Collected PCR products!
  • E.co1i JM109 strain competent cell Toyobo was transformed with a plasmid prepared by subcloning into T7—B1 uevector (Novagen).
  • QI AGEN Plasmid Maxi Kit QIAGEN
  • a plasmid containing DNA having the nucleotide sequence of the present gene hATl2
  • Plasmid containing the PCR product (about 1.2 kbp) obtained in Example 5 was designated as type III, and then the Thermo Sequenase II gui terminator kit (Amersh Am Pharma cia Biotech) and ABI 373DN Using an A array reader (PEAp lied Biosystems 3 ⁇ 4h), 6
  • the PCR is performed under the conditions that the heat-retention cycle consisting of 94 ° C for 1 minute, then 60 ° C for 30 seconds, and 72 ° C for 1 minute is repeated 30 times, and finally, the temperature is kept at 72 for 5 minutes.
  • the heat-retention cycle consisting of 94 ° C for 1 minute, then 60 ° C for 30 seconds, and 72 ° C for 1 minute is repeated 30 times, and finally, the temperature is kept at 72 for 5 minutes.
  • a PCR product showing 1 Kbp was recovered.
  • the E. coli JM109 strain competent cell (Toyobo) was transformed with a plasmid prepared by subcloning the recovered PCR product into pCMV-Tag2B (Stratagene). Transfer the transformed cells to LB medium containing 50 ⁇ g / mL
  • the plasmid was separated and purified from the culture cells obtained by culturing with OL using QI AGE NQI Ailter Megait (QI AG EN), so that the plasmid was added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 1.
  • a plasmid expressing a fusion protein to which FLAG (amino acid sequence in one-letter code: MDYKDDDDKSPGGSPGLQEF) was added (hereinafter, also referred to as FLAG fusion mAT12 protein) was obtained. Furthermore, the absence of mutation in the plasmid was confirmed by the method described in Example 4.
  • Part 1 Preparation of enucleated cell extract containing FLAG-fused mAT12 protein
  • the FLAG-fused mATl2 protein expression plasmid obtained in Example 8 was transferred to a CHO-K1 cell (Odaimotomoto) using the Lipofec tAMI NE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. Pharmaceutical company).
  • the CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the procedure manual distributed when the cells were purchased. After the culture, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and collected by centrifugation H H.
  • the recovered cells were treated with 50 mM Tris-HCl (H7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2 aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), ImM dithiothreitol (hereinafter referred to as DTT).
  • the suspension was suspended in a buffer solution containing 1/100 volume of the protease inhibitor Kaktenore (Sigma) (hereinafter, referred to as "solution A").
  • solution A the protease inhibitor Kaktenore
  • the suspension was sonicated (x 6 times for 5 seconds under ice-cooling) to crush the cells in the suspension. By centrifugation at, uncrushed cells and nuclear fraction were removed as a precipitate.
  • the protein concentration of the obtained supernatant was measured by the Bradford method using serum albumin as a standard, and the dilution obtained by adding buffer ⁇ to 2 / X g ⁇ L each was removed.
  • Example 11 As control (negative control), as in Example 9 except that CMV-Tag 2B (without insert) was used instead of the plasmid for expressing FLAG fusion mATl2 protein According to the method described above, an enucleated cell extract (control) was obtained.
  • Example 11 As control (negative control), as in Example 9 except that CMV-Tag 2B (without insert) was used instead of the plasmid for expressing FLAG fusion mATl2 protein According to the method described above, an enucleated cell extract (control) was obtained.
  • Example 11 As control (negative control), as in Example 9 except that CMV-Tag 2B (without insert) was used instead of the plasmid for expressing FLAG fusion mATl2 protein According to the method described above, an enucleated cell extract (control) was obtained.
  • the test system in which the FLAG-fused mAT12 protein-enhancing plasmid was introduced had a higher concentration in the enucleated cell extract than the test system in which the control (negative control) plasmid had been introduced. Since the activity of the monoacylglycerol lucinoletransferase was increased, it was confirmed that the protein comprising the amino acid sequence shown in SEQ ID NO: 1 exhibited the activity of monoacylglycerol luciryltransferase.
  • Example 13 Cloung of this gene from mouse (Part 2)
  • a primer consisting of the nucleotide sequence represented by Mouse Normal Adiosec DNA (Biochain) l / xL, SEQ ID NO: 19 19 20 pmo 1, comprising the nucleotide sequence represented by SEQ ID NO: 20 primer 20 20 pmo 1 N T akara Ex -Ta q polymerase (Takara Shuzo Co., Ltd.) 2 U, T akara E xT aq polymerase buffer attached 5 mu L and T Akara A 50 iL PCR reaction solution containing 4 ⁇ L of the dNTP mixture (2.5 mM) attached to ExTaq polymerase was prepared, and subjected to PCR.
  • the PCR was first incubated at 94 ° C for 60 seconds, followed by 40 cycles of incubation at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, and finally at 72 ° C for 4 seconds. The test was performed under the condition of keeping the temperature for one minute. After PCR, a PCR product showing about 1.3 Kbp was recovered by agarose electrophoresis. The E.coli JM109 strain competent cell (Toyobo) was transformed with a plasmid prepared by subcloning the recovered PCR product into pT7-B1uevector (Novagen).
  • the plasmid containing the PCR product (approximately 1.3 kbp) obtained in Example 13 was used as type I, and the ThermoSequenase II dye terminator kit (Amersham Pharmacia Biotech)
  • the Sanga method F. Sanger, S. Nick 1 en, AR Cou 1 son A ⁇ , Proceedingsof was performed using an ABI 373D NA sequence reader (PE Ap 1 ied Biosystems). National A cade Based on my of Science USA (1977), 74, 5463-546 7), the nucleotide sequence of this gene (mAT14) derived from mouse consisting of DNA having the nucleotide sequence shown in SEQ ID NO: 14 was determined.
  • mAT14 nucleotide sequence of this gene
  • a DNA fragment having the human-derived gene (hAT1) is amplified by PCR.
  • Human Adipocyte Marat hon-ready DNA (CLONTECH) 1 / z L represented by SEQ ID NO: 21 Primer consisting of base sequence 21 20 pmo 1, Primer consisting of base sequence shown in SEQ ID NO: 22 20pmol, Takara Ex-TaII polymerase (Takara Shuzo) 2U, buffer attached to Takara ExTaq polymerase
  • Fifty PCR reaction solutions containing 5 ⁇ L and 4 ⁇ L of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase were prepared, and subjected to PCR. PCR was first incubated at 94 ° C for 60 seconds, then at 94 ° C for 30 seconds.
  • the above plasmid was obtained from cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / mL ampicillin. By separation and purification, a plasmid containing a DNA having the nucleotide sequence of the present human-derived gene (hAT14) was obtained.
  • hAT14 human-derived gene
  • the plasmid containing the PCR product (about 1.4 kbp) obtained in Example 15 was designated as type I. Then, using a Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and an ABI 373 DNA sequence reader (PEApplied Biosystems), By the method (F. Sanger, S. Nick 1 en, AR Cou 1 son A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467), the base represented by SEQ ID NO: 16 is obtained. The nucleotide sequence of the human-derived gene (hAT14) consisting of the DNA having the sequence was determined.
  • primer 23 20 pmo 1 that, SEQ ID NO primer 24 having the nucleotide sequence represented by 24 20 pmo 1 N T akara Ex -Ta q polymerase (Takara Shuzo) 2U, Ta kara E xT aq polymerase buffer attached 5 mu L Contact A 50 / L PCR reaction solution containing 4 ⁇ L of dNTP mixture (2.5 mM) attached to the PCR-Takara Ex-Taq polymerase was prepared and subjected to PCR.
  • the PCR was first incubated at 94 ° C for 60 seconds, followed by 40 cycles of incubation at 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally at ⁇ 2 ° C for 5 seconds. The test was performed under the condition of keeping the temperature for minutes. After PCR, a PCR product showing about 1.3 Kbp was recovered by agarose electrophoresis. The E.coli JM109 strain competent cell (Toyobo) was transformed with the plasmid prepared by subcloning the recovered PCR product into pT7-Bluevector (Novagen).
  • the plasmid containing the PCR product (approximately 1.3 kbp) obtained in Example 17 was used as type I, and then converted to a Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech). Using the ABI 373D NA sequence reader (PEAp 1 ied Biosystems), Sanger's method (F. Sanger, S. Nicklen, AR Cou 1 son A, Proceedings of National Ac a The nucleotide sequence of this rat-derived gene (rAT14) consisting of DNA having the nucleotide sequence of SEQ ID NO: 18 was determined by emyof Science USA (1977), 74, 5463-5467).
  • Example 13 0.05 g of the plasmid separated and purified in Example 13; a primer 25 20 pmol consisting of the nucleotide sequence represented by SEQ ID NO: 25; and a primer 26 20 pmo 1 consisting of the nucleotide sequence represented by SEQ ID NO: 26, Takara ExTaq polymerase (Takara Shuzo) 0.5 U, Takara ExTaq polymerase 50 LL PCR reaction containing 5 AZL buffer and 4 d dNTP mixture (2.5 mM) attached to Takara ExTaq polymerase A liquid was prepared and subjected to PCR.
  • Takara ExTaq polymerase Takara ExTaq polymerase 50 LL PCR reaction containing 5 AZL buffer and 4 d dNTP mixture (2.5 mM) attached to Takara ExTaq polymerase A liquid was prepared and subjected to PCR.
  • the PCR was carried out under the conditions of first incubating at 94 ° C for 1 minute, then at 60 ° C for 30 seconds, then at 72 ° C for 1 minute 30 times, and finally at 72 ° C for 5 minutes. I was After PCR, an agarose electrophoresis was used to recover a PCR product exhibiting about 1.1 Kbp.
  • a plasmid prepared by subcloning the recovered CR product into pCMV-.Tag2B (Stratagene) was used to construct E.co1i JM109 strain competent cells (Toyo Was transformed.
  • the plasmid was used using QI AGEN Q IAfi i ter Mega Kit (QI AGEN).
  • QI AGEN an amino acid sequence by letter notation: MDYKDDDDKSPGGS
  • FLAG fusion mAT14 protein an amino acid sequence by letter notation: MDYKDDDDKSPGGS
  • Part 2 Preparation of enucleated cell extract containing FLAG fusion mAT14 protein
  • the Lipofect AMI NE PLUS reagent kit (Invitrogen) was used to prepare CHO-K1 cells (Dainippon, Japan) according to the procedure described in the reagent kit. Pharmaceutical company).
  • the CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the procedure manual distributed when the cells were purchased. After the culture, the cells were washed twice with D-PBS (Gipco), scraped with a cell scraper, and collected by centrifugation. Collect the collected cells in 50 mM Tris-hydrochloric acid (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis
  • Buffer solution containing 2 aminoethyl ether tetraacetic acid hereinafter referred to as EGTA
  • DTT ImM dithiothreitol
  • buffer solution A 1/100 volume of protease inhibitor cocktail
  • pCMV-Tag 2B (without insert) was used in place of the plasmid for expressing FLAG-fused mAT14 protein, except that the method described in Example 20 was used.
  • Example 24 (Preparation of a plasmid for expressing a fusion protein in which the amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 15 is added with FLAG at the amino terminus)
  • Example 8 a PCR method using the hAT-12 plasmid isolated and purified in Example 5, using primers consisting of the nucleotide sequences of SEQ ID NOS: 27 and 28 As a result, a plasmid expressing a fusion protein in which FLAG was added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 3 (hereinafter, also referred to as FLAG fusion hAT-12) was obtained. Furthermore, the absence of mutation in the plasmid was confirmed by the method described in Example 4.
  • Example 15 the hAT-14 plasmid isolated and purified in Example 15 was subjected to PCR using a primer consisting of the nucleotide sequences of SEQ ID NOS: 29 and 30, and was represented by SEQ ID NO: 15.
  • a plasmid expressing a fusion protein in which FLAG was added to the amino acid end of the amino acid sequence (hereinafter sometimes referred to as FLAG fusion hAT-14) was obtained. Further, the absence of mutation in the plasmid was confirmed by the method described in Example 16.
  • Example 25 A plasmid expressing a fusion protein in which FLAG was added to the amino acid end of the amino acid sequence.
  • Part 3 Preparation of enucleated cell extract containing FLAG-fused hATl 2 protein and FLAG-fused hAT14 protein
  • Example 26 The FLAG fusion protein expression plasmid obtained in Example 24 was introduced into CHO-K1 cells, and an enucleated cell extract was prepared in the same manner as in Example 9.
  • Example 26 The FLAG fusion protein expression plasmid obtained in Example 24 was introduced into CHO-K1 cells, and an enucleated cell extract was prepared in the same manner as in Example 9.
  • Example 27 The control (negative control) is described in Example 9 except that pCMV-Tag2B (without insert) is used instead of the plasmid for expressing FLAG-fused hAT-12 protein or the plasmid for expressing FLAG-fused hAT-14 protein.
  • An enucleated cell extract (control) was obtained in the same manner as described above.
  • the test system in which the plasmid for expressing FLAG-fused hAT12 protein and the plasmid for expressing FLAG-fused hAT14 protein were compared with the control system (in comparison with the test system in which the negative control was introduced).
  • the protein comprising the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 15 was converted to monoacyl, 'reseguchi, because of the increased activity of monoacylglycerol lucyltransferase in the enucleated cell extract. It was confirmed that it exhibited the activity of lucasyltransferase.
  • Plasmids obtained in Examples 8, 19 and 24 were treated with restriction enzymes so as to cut at both ends of the gene coding sequence excluding the FLAG sequence.
  • a band of the desired length was cut out from the gel on which the DNA was electrophoresed, and the DNA was purified using a GENECLEANII kit (Takara Shuzo).
  • the reaction was performed using a random prime kit (manufactured by Takara Shuzo) according to the protocol of the kit.
  • the specific activity of the probe was measured by a liquid scintillation counter to confirm that the probe was sufficiently radiolabeled. A portion of the probe was heat-denatured and quenched to form a single strand, and then added to 8 ml of DIGE asy-Hyb solution (Boehringer Mannheim) to obtain a hybrid solution.
  • tissue RNA plot membrane (Mouse Adult No rmal l Tissue mRNA No rthern B lot, Human Adu) previously prehybridized in DIGE as y-Hyb solution lt No rmal l T issue T ota 1 RNA No r tern B lot (all manufactured by BIOCHAIN) was immersed in a hybridization bag to start hybridization.
  • Hybridization conditions are: pre-hybridization: 50 ° C, 3 hours or more, hybridization: 50.
  • mice AT12 and AT14 mRNA are significantly expressed in mouse liver and small intestine, and that human ATI2 and AT14 mRNA are significantly expressed in human small intestine.
  • ATI 2 and AT 14 have monoacylglycerol lucyltransferase activity in cells, have significant expression in the liver and small intestine in mice, and have significant expression in the small intestine in humans.
  • the expression of AT12 and AT14 suggests that AT12 and AT14 are monoacylglycerol acyltransferases involved in triglyceride synthesis in the liver and small intestine.
  • CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) were obtained from the plasmid for expressing the FLAG fusion mAT12 protein obtained in Example 8 using Lipofec tAMINE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. ). After culturing the CHO-K1 cells into which the expression plasmid has been introduced for 72 hours according to the procedure manual distributed at the time of purchase of the cells, Ham, containing 400 ⁇ g Zm1 of G ENET IC IN (Gibco) and 10% FCS, The culture was continued for 7 days while replacing the medium with sF-12 medium (hereinafter referred to as selection medium) and replacing with a new medium as appropriate.
  • selection medium sF-12 medium
  • the stable expression strain was cloned by the ultra-dilution method.
  • a part of each of the obtained cell lines was partially dissolved in 5 OmM Tris-hydrochloric acid (pH 7.5), 0.25 M sucrose, ImM ethylene glycol bis 2 aminoethynol ether tetraacetic acid (hereinafter referred to as EGTA), ImM Dithiothreitol (hereinafter, referred to as DTT) was suspended in a buffer solution (hereinafter, referred to as buffer solution A) containing 1/100 volume of a protease inhibitor cocktail (Sigma).
  • buffer solution A containing 1/100 volume of a protease inhibitor cocktail (Sigma).
  • the cells in the suspension were disrupted by sonicating the suspension (x 6 times under ice-cooling for 5 seconds), and the resulting cells were crushed at 1000 Xg for 30 seconds at 4 ° C. By centrifugation, unbroken cells and the nuclear fraction were removed as a precipitate. The obtained supernatant was subjected to immunoblotting using an anti-FLAG-tagged M2 antibody (Sigma), and cells expressing the most single protein with a molecular weight of about 45 kDa were most expressed. A strain was selected to obtain a stable expression strain of mAT-12. 2004/000976
  • the cell line stably expressing the FLAG fusion mAT12 protein obtained in Example 30 was expanded in a selective medium. After the culture, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and collected by centrifugation.
  • the collected cells were subjected to 5 OmM Tris monohydrochloride (H7.5), 15 OmM sodium chloride, 0.1% ⁇ 3-[(3-Cholam idopropy 1) dime tny la mm on lio] These suspensions were suspended in a buffer solution containing —1—propanesulfonate ⁇ (hereinafter, referred to as CHAPS), 1 mM EGTA, 1 mM DTT, and a 1/1000 volume of protease inhibitor Kakutenore (Sigma). The suspension is sonicated (x 6 times for 6 seconds under ice-cooling) to disrupt the cells in the suspension, and the resulting cell lysate is crushed at 15000Xg for 5 minutes at 4 ° C.
  • CHAPS —1—propanesulfonate ⁇
  • Kakutenore Sigma
  • the purified FLAG fusion mAT12 protein purified fraction 101 obtained in Example 31 was combined with a reaction buffer (24 mM Tris-hydrochloric acid (pH 7.5), 5 OmM salted potassium, 8 mM magnesium sulfate, 1. 25 mg / m 1 ⁇ serum albumin, 1 mM DTT, 0.5 mM 2-monooleoylglycerol) 1 After suspending in 30 ⁇ ⁇ , dissolve in the test substance (dimethylsulfoxide (hereinafter referred to as DMSO)) and add to a final concentration of 1% DMS ⁇ ) Was added and mixed.
  • DMSO dimethylsulfoxide
  • the dried product (fat fraction) was redissolved in 30 ⁇ l of form / methanol (2: 1, v / v), and this solution was then dissolved in a silica gel 150 thin-layer chromatography glass plate ( ⁇ 5 Silica Genome 1 50 o Hexane, dimethyl ether / acetic acid (75: 25: 1, v / v) in a sealed container, and use the developing solvent to prepare the TLC plate. After the development, the TLC plate was dried at room temperature, and the dried TLC plate was exposed with an imaging plate (Fuji Film Co., Ltd.) for 16 hours.
  • an imaging plate Fluji Film Co., Ltd.
  • the activity (measured value I + measured value J 2; hereinafter, referred to as activity value 5) of the monoacylglycerol acyltransferase in the test substance-free group and the background group The activity (measured value + measured value L / 2, hereinafter, referred to as activity value 6) of monoacylglycerol lucyltransferase was calculated. From the obtained activity values, the monoacylglycerol acyltransferase inhibitory ability of the test substance was calculated as an inhibition rate ((activity value 5—activity value 4) ⁇ 100 / (activity value 5—activity value 6)).
  • HepG2 cells Human hepatoma-derived cell line HepG2 cells (Dainippon Pharmaceutical Co., Ltd.) in a 6-well plate until the cells are in a confluent state, the cells are cultured in serum-free D-MEM medium (GIBCO, containing high glucose) for 1 ⁇ . Starved by treatment. Replacing the medium of the cells, the final concentration 12.
  • GEBCO serum-free D-MEM medium
  • the heptane-isopropanol solution containing triglyceride in the cells was collected with a pit, and the collected solution was dried using a centrifugal evaporator.
  • the dried product (fat fraction) thus obtained was redissolved in 30 ⁇ l of form / methanol (2: 1, ⁇ / ⁇ ).
  • this solution was spotted on a glass plate for silica gel 150 thin-layer chromatography ( ⁇ 5 silica gel, 150 Angstrom, Whatman). Hexane ⁇ ethyl ether was placed in a closed container.
  • a developing solvent of acetic acid (75: 25: 1, v / v) was added, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature.
  • the dried TLC plate was exposed to an imaging plate (Fuji Film Co., Ltd.) for 16 hours. After the exposure was completed, the imaging plate was analyzed with a bio-image analyzer (BAS 2500, Fujifinorem Co., Ltd.).
  • the 4 C] radioactivity was measured at the portion corresponding to the development position of triacylglycerol in the TLC plate using the same method as above for the test substance-free area.
  • the 4 C] radioactivity of the portion corresponding to the development position of the above triacylglycerol was measured.
  • the test compound known compound N- [2- (4-benzyl-2-ethylphenyloxy) ethyl] —5,6-dimethyl [1,2,4] triazolo [1,5—a] pyrimidine-1 7-Amine (hereinafter sometimes referred to as Compound A)
  • the amount of triglyceride synthesized at the time of addition 1 is about 31 ° at 10 / ⁇ , assuming that the amount of synthesis in the test substance-free group is 100%. / 0 .
  • the results of calculating the amount of synthesis at each test concentration using the same method as described above are shown in Table 7.
  • the monoasinoleglycerol noreacinoletransferase activity inhibitor can inhibit lipoprotein (VLDL) synthesis by inhibiting hepatic triglyceride synthesis.
  • FIG. 5 shows the time course of triglyceride levels in the blood when Compound A was administered at a dose of 30 mg Zkg, and 3 hours when the dose of Compound A was changed to 0, 10 and 30 mg / kg.
  • FIG. 6 shows the results of measuring the amount of triglyceride in blood after time.
  • the present invention provides a simple method for testing the ability to suppress an increase in fat mass.
  • the present invention by suppressing fat accumulation in the fat system and suppressing an increase in adipose tissue mass, glucose tolerance decreased closely related to fat accumulation in fat tissues, diabetes, hyperlipidemia, hypertension, It is possible to treat or prevent metabolic diseases such as arteriosclerosis and diseases such as coronary artery disease, angina pectoris, and cardiovascular disorders such as myocardial infarction. In addition, it suppresses fat synthesis in the supply of fat to the blood and suppresses the increase in fat mass in the blood. It is possible to treat or prevent arteriosclerotic diseases such as blood heart disease and cerebral infarction.

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Abstract

Méthode simple d’analyse de la capacité à inhiber l’augmentation du niveau de graisse qui est indispensable dans la recherche d’une substance capable d’inhiber l’augmentation du niveau de graisse dans les tissus graisseux. Il s’agit en particulier d’une méthode d’analyse de la capacité à inhiber l’augmentation du niveau de graisse d’une substance test, qui se caractérise par les étapes suivantes : 1) mesurer l’activité du monoacylglycérol acyltransférase dans un système où le monoacylglycérol acyltransférase et la substance test sont mis en contact, et 2) estimer la capacité à inhiber l’augmentation du niveau de graisse de la substance sur la base de différences obtenues en comparant l’activité mesurée à l’étape 1 avec l’activité de référence.
PCT/JP2004/000976 2004-01-30 2004-01-30 Methode d'analyse de la capacite a inhiber l'augmentation du niveau de graisse WO2005073398A1 (fr)

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WO2002000857A2 (fr) * 2000-06-15 2002-01-03 Millennium Pharmaceuticals, Inc. Acyltransférase humaine 53320 nouvellement découverte et ses utilisations
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WO2001090334A2 (fr) * 2000-05-25 2001-11-29 Incyte Genomics, Inc. Enzymes de metabolisation de medicaments
WO2002000857A2 (fr) * 2000-06-15 2002-01-03 Millennium Pharmaceuticals, Inc. Acyltransférase humaine 53320 nouvellement découverte et ses utilisations
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116407636A (zh) * 2023-05-15 2023-07-11 徐州医科大学附属医院 Lnc-CCKAR-5在制备抑制MSCs凋亡、提高治疗效果的药物中的应用
CN116407636B (zh) * 2023-05-15 2023-10-20 徐州医科大学附属医院 Lnc-CCKAR-5在制备促进糖尿病创面修复的药物中的应用

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