WO2016125266A1 - Procédé de criblage d'une substance présentant une activité de prolongation de la durée de vie - Google Patents

Procédé de criblage d'une substance présentant une activité de prolongation de la durée de vie Download PDF

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WO2016125266A1
WO2016125266A1 PCT/JP2015/053092 JP2015053092W WO2016125266A1 WO 2016125266 A1 WO2016125266 A1 WO 2016125266A1 JP 2015053092 W JP2015053092 W JP 2015053092W WO 2016125266 A1 WO2016125266 A1 WO 2016125266A1
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mex
protein
activity
substance
insulin sensitivity
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PCT/JP2015/053092
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English (en)
Japanese (ja)
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秋山　徹
祐介 山角
健昭 小田
祐己 鴨志田
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国立大学法人 東京大学
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a method for screening a substance having one or more activities selected from longevity activity, aging-inhibiting activity, obesity-inhibiting activity, insulin sensitivity-reducing activity, adipose tissue mass-increasing activity, and diabetes-improving activity,
  • the present invention relates to a reagent used for the screening and a screening kit containing the reagent.
  • the present invention relates to a non-human knockout animal in which a part or all of the base sequence encoding Mex-3B protein is destroyed or mutated.
  • the present invention relates to a treatment method comprising the step of knocking out the function of Mex-3B protein in a subject.
  • the mechanism related to life extension control has not been fully elucidated, and the emergence of a more effective life extension agent is awaited.
  • Regarding the elucidation of the mechanism for controlling the longevity for example, the fact that the lifespan is significantly shortened by obesity, but the fact that the lifespan is significantly extended by restricting calorie intake in animal experiments has been elucidated (Non-patent Document 1). ). As a result, it is recognized that elucidation of lipid metabolism control is important for prolonging life.
  • Patent Document 1 describes an apoptosis-inducing gene called D8.
  • D8 described in Patent Document 1 is also referred to as Mex-3B.
  • Mex-3B apoptosis-inducing gene
  • the present invention elucidates the life span control phenomenon from an approach at the gene level, and further extends the life span activity, aging suppression activity, obesity suppression activity, insulin sensitivity reduction improvement activity, fat tissue mass increase suppression activity, and diabetes improvement activity
  • An object of the present invention is to provide a method for screening a substance having one or more activities selected from:
  • the present invention provides a non-human knockout animal in which a part or all of the base sequence encoding Mex-3B protein is destroyed or mutated, and a treatment method including a step of knocking out the function of Mex-3B protein in a subject. The purpose is to do.
  • the present inventors surprisingly show that the Mex-3B protein knockout mouse inhibits fat accumulation; the body weight is reduced and insulin sensitivity is increased. Found that; and extended life.
  • life span activity e.g., life span activity, aging inhibitory activity, obesity inhibitory activity, insulin sensitivity reduction improvement activity, adipose tissue mass increase inhibitory activity, and diabetes improvement activity.
  • the present inventors have found a method of screening a substance having one or more activities selected from
  • the present invention is as follows.
  • a method for screening a substance having one or more activities selected from the activity of suppressing the increase in diabetes and the activity of improving diabetes comprising at least the following steps: (I) contacting a candidate substance or a control with a Mex-3B protein-related factor or a Mex-3B protein-related non-human organism; (Ii) a step of measuring an index value; (Iii) detecting the action of the candidate substance by comparing the result based on (i) and (ii) above with the result of the control; and (iv) selecting a candidate substance whose value serving as the index has changed
  • Mex-3B protein-related factor or a Mex-3B protein-related non-human organism is (1) Mex-3B protein-related factor is (2) a nucleic acid comprising a base sequence encoding Mex-3B protein, (3) Mex-3B protein, (4) an antibody against Mex-3B protein, and (5) a non-human knockout animal in which part or all of the base sequence encoding Mex-3B protein is destroyed or mutated,
  • the method according to [1] which is at least one selected from the group consisting of:
  • Mex-3B protein-related factor is (2) a nucleic acid comprising a base sequence encoding Mex-3B protein, (3) Mex-3B protein, (4) an antibody against Mex-3B protein, and (5) a non-human knockout animal in which part or all of the base sequence encoding Mex-3B protein is destroyed or mutated, Longevity activity, anti-aging activity, obesity-inhibiting activity, activity to reduce insulin sensitivity, suppression of increase in adipose tissue mass, including Mex-3B protein-related factor or Mex-3B protein-related non-human organism selected from the group consisting of A screening reagent for a substance having one or more activities selected from an activity and an activity for improving diabetes.
  • One or more activities selected from the longevity activity, aging inhibition activity, obesity inhibition activity, insulin sensitivity reduction improvement activity, adipose tissue mass increase inhibition activity, and diabetes improvement activity comprising the screening reagent according to [3]
  • Non-human knockout animal in which a part or all of the base sequence encoding Mex-3B protein is destroyed or mutated, extending life span, suppressing aging, suppressing obesity, improving insulin sensitivity reduction, fat
  • a non-human knockout animal having one or more phenotypes selected from the group consisting of suppression of increase in tissue mass and improvement of diabetes.
  • a substance having one or more activities selected from long-lived activity, aging-inhibiting activity, obesity-inhibiting activity, insulin sensitivity-reducing activity, adipose tissue mass-increasing activity, and diabetes-improving activity can be conveniently obtained.
  • the method can be efficiently screened.
  • FIG. 1 is a diagram showing gene targeting used for the production of the Mex-3B knockout mouse of the present invention.
  • FIG. 2 is a fluorescence photograph showing the results of in-situ hybridization of G418-resistant clones used for the production of Mex-3B knockout mice.
  • FIG. 3 is a photograph showing the results of genotyping PCR analysis of Mex-3 + / + , Mex-3 +/ ⁇ and Mex-3B ⁇ / ⁇ mice used for the production of Mex-3B knockout mice.
  • FIG. 4 is a photograph showing an analysis result by Western blotting using an anti-Mex-3B antibody used for the production of a Mex-3B knockout mouse.
  • FIG. 1 is a diagram showing gene targeting used for the production of the Mex-3B knockout mouse of the present invention.
  • FIG. 2 is a fluorescence photograph showing the results of in-situ hybridization of G418-resistant clones used for the production of Mex-3B knockout mice.
  • FIG. 3
  • FIG. 5 is a graph showing the change over time in the body weight of Mex-3 + / + , Mex-3 +/ ⁇ and Mex-3B ⁇ / ⁇ mice.
  • FIG. 6 is a graph showing the lifespan of wild-type mice (WT), Mex-3B heterodeficient mice (HT), and Mex-3B homodeficient (knockout: KO) mice.
  • FIG. 7 is a photograph and graph showing an increase in adipose tissue of Mex-3B knockout mice and wild type mice.
  • FIG. 8 is a graph showing insulin sensitivity of 60-day-old male Mex-3B knockout mice and wild-type mice.
  • FIG. 9 is a graph showing insulin sensitivity of 600-day-old male Mex-3B knockout mice and wild-type mice.
  • FIG. 10 shows the results of an analysis of the mouse aging expression system.
  • FIG. 11 shows the change in body weight in the high fat diet load experiment of mice and the result of the insulin load experiment.
  • FIG. 12 shows the results of measurement of adipose tissue weight in mice loaded with a high fat diet.
  • FIG. 13 shows the measurement results of serum components of mice loaded with a high fat diet.
  • the present invention relates to a method for screening a substance having one or more activities selected from longevity activity, aging-inhibiting activity, obesity-inhibiting activity, insulin sensitivity-reducing activity, adipose tissue mass-increasing activity, and diabetes-improving activity,
  • the present invention relates to a reagent used for the screening and a screening kit containing the reagent.
  • the present invention relates to a non-human knockout animal in which part or all of the base sequence encoding Mex-3B protein is destroyed or mutated.
  • the present invention relates to a treatment method comprising the step of knocking out the function of Mex-3B protein in a subject. The present invention will be described below.
  • the present invention uses a Mex-3B protein-related factor or a Mex-3B protein-related non-human organism to select a long-lived activity, aging-inhibiting activity, obesity-inhibiting activity, or insulin sensitivity reduction from among candidate substances.
  • the present invention relates to a method for screening a substance having one or more activities selected from the activity for improving the above, activity for suppressing the increase in the amount of adipose tissue, and activity for improving diabetes.
  • the outline of the present invention will be described below.
  • Mex-3B protein is known to have post-transcriptional regulation and apoptosis-inducing functions.
  • apoptosis by enhancing the expression of the apoptosis promoting factor Bim by post-transcriptional control.
  • the structure of the Mex-3B protein it is known that there are two KH domains, which are RNA binding domains, on the N-terminal side, and a RING finger domain involved in ubiquitination on the C-terminal side.
  • the present inventors prepared a knockout mouse in which the Mex-3B gene was deleted (FIG. 1), analyzed the characteristics of the knockout mouse, and found the following findings: a) Mex-3B knockout mouse male has a significantly longer lifespan than that of the wild type (FIG. 6) and suppresses aging (FIG. 10); b) Mex-3B knockout mice have small adipose tissue (FIGS. 7 and 12); and c) Mex-3B knockout mice have a significantly reduced body weight compared to wild type (FIG. 5), and males are insulin sensitive. Is increased (FIGS. 8, 9 and 11), and the increase in diabetes marker is suppressed (FIG. 13); Got.
  • Mex-3B knockout mice have a long life span, aging is suppressed, obesity associated with aging is suppressed, an increase in the amount of adipose tissue is also suppressed, insulin sensitivity reduction is improved, and diabetes is improved Was shown to be. From this, it was newly shown that the Mex-3B protein is an RNA-binding protein related to metabolic control and is involved in the lifetime control mechanism.
  • the present inventor considered that a substance that suppresses the expression or function of Mex-3B is a promising substance that is involved in the mechanism for extending the lifetime. Based on this, it is possible to screen for compounds having one or more activities selected from long-lived activity, aging-inhibiting activity, obesity-inhibiting activity, insulin sensitivity-reducing activity, adipose tissue mass-increasing activity, and diabetes-improving activity. It becomes.
  • the screened substance is useful because it can be applied to a long-lived active agent having a completely new mechanism of action.
  • the screening method of the present invention uses the above-mentioned Mex-3B protein-related factor or Mex-3B protein-related non-human organism, from among candidate substances, prolonging life activity, aging-inhibiting activity,
  • An object of the present invention is to search for a substance having one or more activities (hereinafter also referred to as the activity of the present invention) selected from an obesity suppressing activity, an insulin sensitivity reducing activity, an adipose tissue mass increase inhibiting activity, and a diabetes improving activity. To do.
  • the Mex-3B protein-related factor includes any substance that can be contacted with a candidate substance and can search for a substance having long-lived activity. Examples thereof include a nucleic acid containing a base sequence encoding Mex-3B protein, Mex-3B protein and an antibody thereof. Examples of Mex-3B protein-related non-human organisms include knockout animals (preferably non-human mammals) related to Mex-3B protein.
  • sequence data of the Mex-3B protein for example, data registered in a database such as GenBank (NIH genetic sequence database) or DDBJ (DNA Data Bank of Japan) can be used.
  • the cDNA of the human-derived Mex-3B protein has 3534 bases, and NM_032246 (SEQ ID NO: 1) is registered in GenBank, and the amino acid sequence is registered as NP_115622 (SEQ ID NO: 2) of 569 amino acid residues.
  • the Mex-3B protein antibody includes, for example, a method of measuring expression of Mex-3B protein by Western blotting or ELISA using an anti-Mex-3B protein antibody that is specific to Mex-3B protein. can give.
  • the antibody can be prepared using a known method.
  • Examples of knockout animals related to Mex-3B protein include long-lived non-human knockout animals in which Mex-3B protein-related factors disrupt or mutate part or all of the base sequence encoding Mex-3B protein. .
  • a substance that suppresses the function of the Mex-3B protein is a promising substance that participates in a mechanism for extending the lifetime. Therefore, a substance that suppresses the expression of Mex-3B protein is preferable as a candidate for a long-lived active agent in order to promote a long-life mechanism, and further, an aging inhibitor (anti-aging agent), an obesity inhibitor (anti-obesity agent), insulin It is also preferable as a candidate for a sensitivity reduction improving agent (insulin resistance improving agent), an adipose tissue mass increase inhibitor, and a diabetes therapeutic agent.
  • a substance includes any substance as long as it has the above-mentioned properties.
  • These compounds may be novel compounds or known compounds.
  • These substances may form salts, and salts with physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids) are used, especially physiologically acceptable.
  • physiologically acceptable acids eg, inorganic acids
  • bases eg, organic acids
  • the acid addition salts are preferred.
  • salts examples include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • succinic acid tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
  • Examples of the screening method of the present invention include a method in which an expression vector operably linked to the Mex-3B gene is prepared and introduced into cells to express the Mex-3B protein.
  • a method for preparing an expression vector in which a Mex-3B gene is operably linked to a gene encoding a labeling substance and introducing the expression vector into a cell for expression is described below.
  • the labeling substance is used as a detection means for detecting the measurement index of the screening method of the present invention, and any one of a fluorescent labeling substance, a radiolabeling substance, and an antibody labeled with these labeling substances is used.
  • ⁇ GFP green fluorescent protein
  • Examples of the fluorescent labeling substance include GFP, EGFP (Enhanced-humanized GFP) or rsGFP (red-shift GFP), which are mutants modified with GFP (GFP variant). It is also possible to use GFP derived from yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (FPBFP), and Renilla reniformis.
  • the genes encoding them can be used in the present invention.
  • the base sequences of the genes encoding these are known, and commercially available products can be used, or the sequence information can be easily obtained through public databases such as GenBank.
  • the cell into which the expression vector has been introduced is divided into a test cell for contacting the candidate substance and an untreated control cell, and the labeled signal is detected by bringing the candidate substance into contact with the test cell by addition or the like.
  • contact means that the candidate substance is treated so as to interact with the cell into which the expression vector has been introduced.
  • the candidate substance is added to the cell culture system, or the cell is cultured in the presence of the candidate substance. It means to do anything.
  • the “index value” in the screening method of the present invention refers to the expression level of a gene, mRNA or protein that serves as an index for searching for a substance that suppresses the expression of Mex-3B protein.
  • the mRNA expression level or protein expression level of Mex-3B protein can be mentioned.
  • index value in the screening method of the present invention is the degree of interaction between the Mex-3B protein and its target mRNA, and the change in the function of the target mRNA due to the interaction. May be.
  • the expression level of Bim when the expression level of Bim varies due to the variation in the function of the target mRNA, the expression level of Bim can be used as an index.
  • “measurement” in the screening method of the present invention means quantifying the value serving as the index in the cells or animals cultured under appropriate conditions.
  • mRNA or protein of Mex-3B protein When a normal animal is used, the length of life, aging phenotype, body weight, adipose tissue weight, insulin sensitivity, or diabetes marker is measured. Quantification of mRNA or protein can be performed using a known method. mRNA can be quantified by, for example, Northern hybridization or quantitative RT-PCR, and protein can be quantified by, for example, Western blotting using an anti-Mex-3B protein antibody (Current Protocols in Molecular Biology, John Wiley & Sons 1994-2003).
  • the signal intensity (signal intensity) of the labeling substance can be determined by quantifying by a method suitable for each labeling protein such as fluorescence intensity, band density in electrophoresis, or by visual observation. And the candidate substance when signal intensity is reduced rather than a control
  • the expression of the Mex-3B protein is not suppressed, and the recombinant protein in which the expressed labeled protein and the Mex-3B protein are bound emits fluorescence.
  • a candidate substance that can be a substance having the above-mentioned long-lived activity is added, since the expression of Mex-3B protein is inhibited, the label signal emitted from the cell after contacting the candidate substance is It becomes weaker than the intensity of the label signal emitted from the control cells.
  • a candidate substance whose expression level is changed can be selected by measuring and comparing and detecting a specific gene expression level of the cell after contacting the control cell with the candidate substance.
  • the labeling substance can be detected by immunoblot using an anti-labeled protein antibody such as an anti-GFP antibody, so that multiple samples can be processed in a short time, and high-throughput screening is performed. Can do.
  • a candidate substance is administered to an animal or a part thereof, and a value that is a specific index for prolonging life in the present invention described in “Substances with prolonging life activity” is directly measured. And selecting a candidate substance that exhibits a value comparable to or higher than that of the Mex-3B protein knockout animal, which is the control, to screen for a substance having the activity of the present invention. The method of doing is also included. Since this method can evaluate the effect of a substance at an individual level as an in vivo system, it is preferable.
  • the specific value of the “value to be used as an index” is as follows. For the animal administered with the candidate substance, the substance inhibits fat accumulation; decreases body weight; and increases insulin sensitivity. And so on. Candidate substances exhibiting these indices are preferred as substances that can extend the lifetime.
  • the fat accumulation inhibition means that the accumulation of body fat is inhibited by suppressing or delaying the growth of adipose tissue in the body.
  • Adipose tissue refers to a connective tissue composed of adipocytes and having a function of storing energy as fat.
  • the adipose tissue related to the present invention includes any adipose tissue such as those under the skin in the body and those around the internal organs. Examples include epididymal fat tissue and abdominal fat. Fat accumulation can be measured using any known method.
  • the change in body weight refers to the change in body weight over time and can be measured by a known method.
  • Increased insulin sensitivity means a decrease in blood glucose level when insulin is administered, and an increase in insulin sensitivity means that the blood glucose level decreases more quickly due to insulin administration. Any known method for measuring blood glucose level can be used for evaluation of increased insulin sensitivity.
  • mice examples include mice, rats, rabbits, guinea pigs, pigs, sheep and goats other than humans.
  • a mouse that is easy to handle and easy to breed is preferable.
  • a Mex-3B protein knockout animal used as a control can be produced using a known method.
  • the knockout mouse in which a part of the Mex-3B protein shown in FIG. 1 is deleted is preferable for the screening method of the present invention.
  • “Animal or part thereof” includes both the whole body of an animal's body and limited tissues or organs. In the case of limited tissues or organs, it includes those extracted from animals.
  • Examples of the method for administering the candidate substance to the test animal include oral administration, intravenous injection, application, subcutaneous administration, intradermal administration, and intraperitoneal administration.
  • the method is appropriately selected according to the symptoms of the test animal and the nature of the candidate substance. You can choose.
  • the dosage of the candidate substance can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.
  • Candidate substances to be screened according to the present invention include, for example, peptides, proteins, non-peptide compounds, synthetic compounds (polymers or low molecular compounds), fermentation products, cell extracts, cell culture supernatants, and plant extracts. Fluids, tissue extracts of mammals (eg, mice, rats, pigs, cows, sheep, monkeys, humans, etc.), plasma and the like. These compounds may be novel compounds or known compounds. There may be. These candidate substances may form a salt. As described above, the salts of candidate substances include physiologically acceptable acids (for example, inorganic acids and organic acids) and bases (for example, metal acids). ) And the like are used.
  • physiologically acceptable acids for example, inorganic acids and organic acids
  • bases for example, metal acids
  • Mex-3B protein-related factor can be combined with another solvent or solute to form a composition.
  • a composition for example, distilled water, pH buffer reagent, salt, protein, surfactant and the like can be combined.
  • the reaction reagent has a label detectable by an appropriate chemical or physical detection means.
  • a labeling agent used in a measurement method using such a labeling substance for example, a fluorescent substance, an enzyme, a radioisotope, a luminescent substance and the like are used. An ELISA method using an enzyme for labeling is widely used.
  • luminescent substances such as H and 14 C include luciferin, lucigenin, luminol, and luminol derivatives.
  • the reaction medium includes a buffer solution that gives optimum conditions for the reaction or is useful for stabilizing the reaction product, a stabilizer for the reactant, and the like.
  • a candidate substance sample is composed of instructions for interpreting the results obtained by performing screening, reaction reagents, reaction medium in which the reaction is performed, and a substrate that provides a place for the assay.
  • a verification sample for use as a comparison reference or for creating a calibration curve, a detector, and the like may be included.
  • the detection means of the screening method of the present invention include those capable of detecting the label, such as a spectroscope, a radiation detector, and a light scattering detector.
  • Non-human knockout animal of the present invention is a non-human knockout animal in which part or all of the base sequence encoding Mex-3B protein has been disrupted or mutated, and has an extended life span. And having one or more phenotypes selected from the group consisting of suppression of aging, suppression of obesity, improvement of insulin sensitivity reduction, suppression of increase in adipose tissue mass, and improvement of diabetes.
  • a non-human knockout animal in which part or all of the base sequence encoding Mex-3B protein is disrupted or mutated is an animal in which the expression of Mex-3B has been lost or reduced, and should be prepared by methods known to those skilled in the art. Can do.
  • a non-human mammal is preferable, and examples thereof include mice, rats, hamsters, guinea pigs, cows, pigs, goats, etc. Among them, rodents, more preferably mice.
  • a non-human knockout animal can be prepared by arbitrarily converting the chromosomal Mex-3B gene using a genetic engineering technique such as gene targeting.
  • a construct (targeting vector) containing a nonexistent Mex-3B gene is prepared based on a clone isolated from a genomic library, introduced into an ES cell, and homologous recombination is performed.
  • a Mex-3B gene mutant clone in which is generated is obtained.
  • “Mex-3B gene not having the ability to express” refers to the introduction of a mutation that inhibits the expression of Mex-3B, or a mutation that causes loss of the function of the protein encoded by the gene. It is a Mex-3B gene that does not express Mex-3B when transfected into a living body.
  • Mutation can be introduced into the Mex-3B gene by removing a part or all of the base sequence of the Mex-3B gene and inserting or replacing another gene by a known genetic engineering technique.
  • knockout mice in which the expression of Mex-3B is reduced or eliminated can be produced by disrupting the function of the promoter or exon, or shifting the codon reading frame.
  • Examples of the gene to be introduced for destroying the function of the promoter or exon include a drug resistance gene, such as a neomycin resistance gene, a hygromycin resistance gene, preferably a neomycin resistance gene.
  • any gene generally used in the gene targeting method can be used.
  • ES cells can be established from blastocysts of non-human mammals.
  • mouse ES cells include TT2 ES cells established from hybrid primary blastocysts between C57BL / 6 mice and CBA / JNCrj mice.
  • Selection of Mex-3B gene mutant clones in which homologous recombination has occurred from ES cells into which the targeting vector has been introduced can be performed by analyzing the genotype of the clone into which the targeting vector has been introduced. Genotype analysis can be performed by PCR or Southern blotting.
  • Mex- A deletion of the 3B gene can be detected.
  • deletion of the Mex-3B gene can be detected based on the size of DNA detected using the Mex-3B gene or a DNA sequence in the vicinity thereof as a probe.
  • the mutant clone can be easily selected using the drug resistance of the mutant clone.
  • the Mex-3B gene mutant clone thus obtained was injected into a blastocyst or 8-cell stage embryo of a fertilized egg of a non-human mammal, and the uterus of a homologous non-human mammal made into a pseudopregnant state.
  • a chimeric individual composed of cells having a normal Mex-3B locus and cells having a mutated Mex-3B locus.
  • An individual in which a mutation has been introduced into one of the homologous chromosomes (hetero-deficient individual) or an individual in which the mutation has been introduced into both homologous chromosomes (homo-deficient individual) can be obtained by crossing between the chimeric individual and the wild-type individual. In general, heterozygous individuals are obtained by such crossing. Homo deficient individuals can be obtained by crossing heterogene deficient individuals.
  • the acquired knockout animal has one or more phenotypes selected from the group consisting of prolonging lifespan, suppressing aging, suppressing obesity, improving insulin sensitivity, suppressing adipose tissue mass increase, and improving diabetes. Whether or not it has can be evaluated according to the method described in the examples of the present specification.
  • Treatment method including the step of knocking out the function of Mex-3B protein
  • the life of the subject can be prolonged, the aging can be suppressed,
  • One or more selected from suppression of obesity, improvement of insulin sensitivity reduction, suppression of increase in adipose tissue mass, and improvement of diabetes can be achieved. That is, according to the present invention, there is provided a method for treating a subject, comprising a step of knocking out the function of Mex-3B protein in the subject.
  • Mex-3B protein inhibitor that is, a drug that suppresses the expression and / or function of Mex-3B protein
  • Mex-3B protein inhibitor selected by the screening method of the present invention is long-lived activity, aging inhibitory activity, obesity inhibitory activity, insulin sensitivity reduction improving activity, adipose tissue mass increase inhibitory activity, and diabetes improving activity
  • a substance having one or more activities selected from the following can be used. Specifically, siRNA or LNA (Locked Nucleic Acid) targeting Mex-3B can be used, or a low molecular compound having a Mex-3B protein inhibitory action may be used.
  • the administration method of the above Mex-3B protein inhibitor is not particularly limited, and it may be oral administration or parenteral administration.
  • the dose of the Mex-3B protein inhibitor is not particularly limited, and can be appropriately set according to the weight of the subject, the degree of symptoms, the type of active ingredient, and the like.
  • Example 1 The purpose of this example is to create a Mex-3B knockout mouse.
  • the targeting vector was designed as shown in FIG. An approximately 2.5 kb genomic Mex-3B fragment (SEQ ID NO: 1) in BAC clone (GenBank Accession No. AC122736) was replaced with a neomycin resistance cassette (pgkbg2Neo) (SEQ ID NO: 3) to be deleted.
  • This targeting vector was linearized and introduced into half the amount of embryonic stem cells of three 10 cm dishes that became confluent by electroporation. G418 resistant colonies were selected and confirmed to have undergone homologous recombination using primers specific for Mex-3B and the neomycin resistance cassette.
  • the primers used are as follows.
  • the resulting chimeric mouse male was mated with a balb / c mouse female to produce a heterozygote.
  • the genotype of the offspring was identified by Genotyping PCR (FIG. 3). Proteins were collected from Mex-3B + / + or Mex-3B ⁇ / ⁇ thymus and spleen and confirmed by Western blotting for the presence of Mex-3B expression. As a result, it was shown that Mex-3B expression was not observed in the thymus and spleen of Mex-3B ⁇ / ⁇ (FIG. 4). Mex-3B +/ ⁇ mice were backcrossed to balb / c mice more than 10 times. Litters were used as experimental controls. Mex-3B -/- mice were born according to Mendel's law and were healthy for more than 10 months.
  • Example 2 The purpose of this example is to measure the life span of Mex-3B knockout mice and compare it to wild type mice.
  • KO Mex-3B knockout mice prepared in Example 1
  • Example 3 The purpose of this example is to measure the weight of adipose tissue in Mex-3B knockout mice and compare it with wild type mice.
  • the Mex-3B knockout (KO) mouse and wild type (WT) mouse prepared in Example 1 were subjected to statistical processing by measuring the spleen, epididymal fat tissue and kidney by the following method. First, body weights were measured after euthanizing the mice. Thereafter, the mouse was dissected and each organ was removed. The weight of each organ was measured by measuring the weight of each organ and interrupting with the body weight.
  • the vertical axis of the graph represents the relative value when the weight of the organ per WT body weight is 1.
  • Example 4 The purpose of this example is to perform an insulin sensitivity test of Mex-3B knockout mice and compare it with wild-type mice.
  • the Mex-3B knockout (KO) mouse and wild type (WT) mouse prepared in Example 1 were fasted by the following method, an insulin sensitivity test was performed, the body weight was measured, and statistical processing was performed. That is, 60-day-old and 600-day-old male wild-type and Mex-3B knockout mice bred alone were fasted for 17 hours, and then glucose was intraperitoneally administered to each mouse at 2 mg / kg. Thereafter, blood was continuously collected from the tail vein of the mouse, and the blood glucose level was measured to examine insulin sensitivity.
  • KO Mex-3B knockout
  • Example 5 Analysis of mouse aging phenotype 600-day-old BALB line wild-type (WT) and Mex-3B homo-deficient (KO) males and females were sleeped by anesthesia, and the sores (inflammation) around the eyes were observed. Then, the number of individuals with a sore was counted. At the same time, the outer skin was peeled off from each mouse and the spine flexion was observed.
  • WT wild-type
  • KO Mex-3B homo-deficient
  • mice have a reduced aging phenotype
  • mice 13 to 18 weeks old male BALB line wild type mice and 13 to 18 Mex-3B-deficient mice were grouped according to body weight, bred alone, and normal diet (10 kcal % Fat, Normal Diet: ND) and high fat diet (60 kcal% Fat, High Fat Diet: HFD) (Research diets) were fed, and body weight was measured for 13 weeks. After breeding, the animals were fasted for 6 hours and sacrificed, and the weight of epididymal fat tissue and subcutaneous fat tissue in each group were measured. Student's t-test was used for the significant difference test of body weight and tissue weight.
  • the measurement result of body weight is shown in FIG. 11A, and the measurement result of adipose tissue weight is shown in FIG.
  • Mex-3B-deficient male mice are alleviated from worsening obesity associated with high-fat diet loading.
  • the increase in adipose tissue associated with high-fat diet load is moderated.
  • Insulin tolerance test Insulin Tolerance Test (ITT) Human recombinant Insulin (Invitrogen) diluted with PBS to 1 IU (International Unit) / kg was injected into the peritoneal cavity of BALB line wild-type and Mex-3B KO mice fasted for 4 hours under single breeding conditions. It was administered internally. Thereafter, blood glucose levels in the tail vein were measured over time at 0, 15, 30, 60, 90, and 120 minutes using Asensia Breeze 2 (Bayer Pharmaceutical Co., Ltd.). Student's t-test was used for the significant difference test.
  • ITT Insulin Tolerance Test
  • mice were administered a normal diet (ND) or a high fat diet (HFD) for 12 weeks at 8 weeks of age.
  • Blood was analyzed from each group of mice, and serum levels of Glucose, total cholesterol, glucoalbumin, and hemoglobin A1c were measured.
  • Mouse blood was collected from the inferior vena cava using a syringe and a 23G needle during anesthesia. After blood collection, the blood was transferred to a 1.5 mL tube, left to stand at room temperature for 1 hour after inversion stirring. Thereafter, the mixture was centrifuged at 4 ° C.
  • Mex-3B-deficient male mice have an increased insulin sensitivity as well as an increase in life span and resistance to obesity caused by a high fat diet or overeating.
  • aged Mex-3B-deficient male mice have been confirmed to be resistant to aging because the sores around the eyes (inflammation) and the significant flexion of the spine that appear with aging are reduced.
  • the results were consistent with the extended lifespan.
  • Mex-3B-deficient mice with enhanced insulin sensitivity have an anti-obesity / diabetes phenotype. It seems that it is suitable.
  • Mex-3B contributes to metabolic control in animals. Therefore, Mex-3B-deficient mice have a lean form due to the absence of metabolic control by this Mex-3B, and insulin sensitivity is enhanced. As a result, a mechanism similar to caloric restriction works, and Mex-3B It is possible that the deficient male mouse has an extended life span. This indicates that a Mex-3B inhibitor can be a therapeutic agent for obesity and diabetes, and at the same time, is considered to be useful as an anti-aging agent that delays the progression of aging.
  • the longevity activity inhibition by Mex-3B protein is avoided, the longevity activity, the aging inhibition activity, the obesity inhibition activity, the insulin sensitivity reduction improvement activity, the adipose tissue mass increase inhibition activity, and the diabetes improvement
  • a compound having one or more activities selected from the activities can be searched.
  • obesity can be prevented by administering a substance searched by the above screening method to animals such as humans and promoting the lifespan control mechanism.
  • the screening method of the present invention comprises a novel long-life drug having a new mechanism of action, an aging inhibitor (anti-aging agent), an obesity inhibitor (anti-obesity agent), and an insulin sensitivity-reducing agent (insulin resistance). It is expected to contribute to the development of an improving agent), an adipose tissue growth inhibitor, a therapeutic agent for diabetes, and the like.

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Abstract

La présente invention aborde le problème consistant à fournir un procédé de criblage d'une substance qui présente une ou plusieurs activités choisies parmi une activité de prolongation de la durée de vie, une activité anti-vieillissement, une activité d'inhibition de l'obésité, une activité d'amélioration d'une diminution de la sensibilité à l'insuline, une activité de prévention d'une augmentation de la quantité de tissu adipeux et une activité d'amélioration du diabète. Afin de résoudre ce problème, l'invention décrit un procédé qui comporte : (i) une étape consistant à mettre en contact des substances candidates et une substance témoin avec un facteur associé à la protéine Mex-3B ou un organisme non humain associé à la protéine Mex-3B; (ii) une étape consistant à mesurer une valeur servant d'indice; (iii) une étape consistant à comparer les résultats des substances candidates au résultat de la substance témoin sur la base des étapes (i) et (ii), et détecter ainsi les effets des substances candidates; et (iv) une étape consistant à sélectionner une substance candidate qui montre un changement dans la valeur d'indice.
PCT/JP2015/053092 2015-02-04 2015-02-04 Procédé de criblage d'une substance présentant une activité de prolongation de la durée de vie WO2016125266A1 (fr)

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Cited By (1)

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JPWO2019098194A1 (ja) * 2017-11-14 2021-01-14 国立大学法人広島大学 老化状態の評価方法、情報の提示方法、及び老化状態を改善又は予防する物質のスクリーニング方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2019098194A1 (ja) * 2017-11-14 2021-01-14 国立大学法人広島大学 老化状態の評価方法、情報の提示方法、及び老化状態を改善又は予防する物質のスクリーニング方法

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