WO2010140694A1 - Procédé pour le criblage d'inhibiteur utilisant un facteur capable de stimuler la production de peptide amyloïde bêta, et inhibiteur obtenu par celui-ci - Google Patents

Procédé pour le criblage d'inhibiteur utilisant un facteur capable de stimuler la production de peptide amyloïde bêta, et inhibiteur obtenu par celui-ci Download PDF

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WO2010140694A1
WO2010140694A1 PCT/JP2010/059568 JP2010059568W WO2010140694A1 WO 2010140694 A1 WO2010140694 A1 WO 2010140694A1 JP 2010059568 W JP2010059568 W JP 2010059568W WO 2010140694 A1 WO2010140694 A1 WO 2010140694A1
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protein
refseq
substance
gene
secretase
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PCT/JP2010/059568
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English (en)
Japanese (ja)
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オー． ラース シャンバリィ
スザンヌ フリークマン
ジエン フー
康博 寺西
博之 中川
順 左海
孝洋 木原
泰三 石川
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大日本住友製薬株式会社
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to a ⁇ -amyloid production inhibitor that is the cause of Alzheimer's disease, a prophylactic and therapeutic agent for Alzheimer's disease, and a screening method for such a drug.
  • ⁇ -amyloid (A ⁇ ) peptide which accumulates in the brain of Alzheimer's disease (AD), is produced by being excised from the precursor protein amyloid precursor protein (APP) by ⁇ -secretase and ⁇ -secretase, which is polymerized to form senile plaques Is formed. Accumulation of A ⁇ is a lesion that occurs early in AD with high specificity in AD brain. Furthermore, in familial Alzheimer's disease (FAD), A ⁇ is considered to be a causative agent of AD because the mutation of the etiological genes APP and presenilin (PS) increases the production of highly aggregated A ⁇ 42 (amyloid hypothesis). It is regarded as a promising therapeutic target for radical treatment of AD (Non-patent Document 1).
  • a ⁇ peptide production can be suppressed by inhibiting ⁇ -secretase, the responsible enzyme responsible for the final cleavage.
  • ⁇ -secretase inhibitors that have been reported so far also inhibit cleavage of Notch, which is another physiological substrate of the enzyme, and inhibit signals involved in development and differentiation.
  • Peripheral side effects such as immune abnormalities are a problem (Non-patent Document 2).
  • an abnormality occurs in the signal pathway via Notch, not only a serious abnormality occurs in the generation and formation of tissues and organs, but it may cause cancer.
  • the Notch gene has been clarified as an oncogenic gene, and it is also known that regulating the activity of ⁇ -secretase that cleaves Notch has an anticancer effect (Non-patent Document 3).
  • NSAIDs non-steroidal anti-inflammatory drugs
  • ⁇ -secretase modulator activity ⁇ -secretase modulator activity
  • ⁇ -secretase is a transmembrane aspartic protease composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (Non-patent Document 5).
  • PS presenilin
  • NCT nicastrin
  • Aph-1 and Pen-2 Non-patent Document 5
  • TMP21 was identified as a molecule that binds to ⁇ -secretase and controls its activity (Non-patent Document 6).
  • the existence of other ⁇ -secretase activity regulators is not known at all.
  • a gene encoding a membrane-bound protein (ATP2A2, FLOT2) is knocked down by the RNAi method, production of A ⁇ is reported to decrease (Non-patent Documents 7 and 8).
  • An object of the present invention is to identify a novel factor that binds to ⁇ -secretase and promotes / enhances its activity, and to suppress the expression and function of the factor, a ⁇ -secretase inhibitor or modulator, A ⁇ It is to provide a production inhibitor and an AD preventive / therapeutic agent, and to provide a method for screening such a drug using the factor.
  • the present inventors have conceived that there are molecules other than TMP21 that bind to ⁇ -secretase and control A ⁇ production in brain tissue, and that bind to ⁇ -secretase using an inhibitor pull-down method.
  • HEK-APP human embryonic kidney cell line
  • APP human embryonic kidney cell line
  • the present invention is as follows.
  • the following proteins (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) synaptophysin; (P16) solute carrier family 2 (
  • [6] In mammals, the following proteins: (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-ne
  • the method comprises selecting a substance that decreases the expression level of at least one gene selected from (G1) to (G25) or the activity of at least one protein selected from (P1) to (P25).
  • a method for screening an A ⁇ production inhibitor comprises selecting a substance that decreases the expression level of at least one gene selected from (G1) to (G25) or the activity of at least one protein selected from (P1) to (P25).
  • a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c): A reporter gene under the control of the transcriptional regulatory region of (a) (a1) gene Gn (n is any integer from 1 to 25) or (a2) gene Gn (n is any integer from 1 to 25) A step of contacting a test substance with a cell to be expressed (b) a step of measuring the expression level of (b1) gene Gn or (b2) reporter gene in the cell (c) a measurement in the absence of the test substance [14] A method for screening an A ⁇ production inhibitory substance, comprising the following steps (a) to (c): selecting a substance that reduces the expression level as a candidate for an A ⁇ production inhibitory substance compared to (A) contacting a cell expressing amyloid precursor protein (APP) with at least one protein selected from the above (P1) to (P25) and a test substance (b) measuring the activity of the protein ( c) The step of selecting a substance that reduces the activity of the protein compared to the
  • a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c): (A) contacting at least one protein selected from (P1) to (P25) and a test substance with a cell expressing ⁇ -secretase or a cell membrane fraction thereof; and (b) measuring ⁇ -secretase activity. Step (c) selecting a substance that reduces or modulates ⁇ -secretase activity compared to that measured in the absence of the test substance [18]
  • the protein is provided by the cell itself, The method according to [17].
  • a screening method for an A ⁇ production inhibitor which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer of 1 to 25) and ⁇ -secretase.
  • a screening method for an A ⁇ production inhibitor comprising the following steps (a) to (c): (A) contacting protein Pn (n is an integer of 1 to 25) and ⁇ -secretase with a test substance (b) measuring the binding activity between protein Pn and ⁇ -secretase (c ) A step of selecting a substance that reduces the binding activity as compared with the case where it is measured in the absence of the test substance [21] non-introducing at least one gene selected from (G1) to (G25) A method for screening or evaluating an efficacy of a therapeutic or prophylactic agent for Alzheimer's disease, comprising selecting a substance that improves the phenotype reflecting the pathological condition of Alzheimer's disease in a human animal.
  • a screening or drug efficacy evaluation method for a therapeutic or prophylactic agent for Alzheimer's disease comprising the following steps (a) to (c): (A) a step of administering a test substance to a non-human animal into which at least one gene selected from the above (G1) to (G25) has been introduced; (b) at least one expression reflecting the pathological condition of Alzheimer's disease in the animal A step of evaluating the type (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is not administered, [23] the phenotype is brain tissue, cerebrospinal fluid, Selected from the group consisting of A ⁇ amount in tissues such as blood, neuronal cell death, central nervous system inflammatory response, cognitive ability, amyloid plaque accumulation, cerebral blood flow and cerebral glucose metabolism 22].
  • a method for determining the onset or risk of developing Alzheimer's disease or cancer comprising the following steps (a) to (c): (A) Providing a sample derived from a test animal (b) Expression level of at least one gene selected from (G1) to (G25) in the sample or selected from (P1) to (P25) A step of measuring the activity of at least one protein (c) is the subject animal having an increased expression level or activity compared to the case of measuring in a sample derived from a normal animal developing Alzheimer's disease or cancer? The process of determining that the risk of developing in the future is high
  • the present invention by using a novel protein that regulates the activity of ⁇ -secretase, it becomes possible to screen for compounds that inhibit the production of A ⁇ and to develop medical treatments using antibodies and nucleic acids. Thus, it is useful for the prevention and treatment of diseases such as AD and cancer.
  • FIG. 2 is a schematic diagram (A) of the principle of an inhibitor pull-down method and a diagram showing that a known component of ⁇ -secretase is pulled down by the method. It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to a HEK-APP cell (transduction siRNA amount: 0.1pmol / well).
  • the present invention includes the following proteins: (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) synaptophysin; (P16) solute carrier family 2 (facilitated glucose transporter), member 3;
  • Protein Pn [(P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) ) synaptophysin; (P16) solute carrier family 2 (facilitated glucose transporter), member 3; (P
  • Protein Pn is a cell of humans and other warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, dogs, pigs, sheep, cows, monkeys, etc.) [eg, hepatocytes, spleen cells, neurons, glia.
  • Cells pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, lung cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts , Mammary cells or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, cerebrum) Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n (a) an amino acid sequence having about 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2n; (b) in the amino acid sequence represented by SEQ ID NO: 2n, an amino acid sequence in which 1 to 50 amino acids are substituted and / or deleted and / or inserted and / or added; (c) the amino acid sequence of an ortholog in another mammal of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n; or (d) The amino acid sequence of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n or the splice variant, allelic variant or polymorphism of the ortholog of (c) above.
  • homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both).
  • similar amino acids means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn).
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms include NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 1997 (1997))], Needleman et al., J. Mol.
  • the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n” is about 80% or more of the amino acid sequence represented by SEQ ID NO: 2n,
  • the amino acid sequence preferably has about 90% or more, more preferably about 95% or more, still more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more.
  • a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2n includes the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2n, and the sequence No .: A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by 2n.
  • activity refers to an activity that promotes the production of A ⁇ .
  • substantially the same quality means that the properties are qualitatively the same, for example, physiologically or pharmacologically. Therefore, it is preferable that the A ⁇ production promoting activities are equivalent, but quantitative factors such as the degree of these activities (eg, about 0.1 to about 10 times) and the molecular weight of the protein may be different.
  • a ⁇ production promoting activity can be measured by measuring and comparing the amount of A ⁇ produced (preferably the amount of A ⁇ 40 or A ⁇ 42 produced) in the presence and absence of a protein according to a method known per se. .
  • the protein Pn in the present invention as shown in (b) above, for example, (i) 1 to 50, preferably 1 to 30, more preferably 1 in the amino acid sequence represented by SEQ ID NO: 2n ⁇ 10, more preferably 1 to several (5, 4, 3 or 2) amino acid sequences deleted, (ii) 1 to 50, preferably 1 to the amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence to which 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are added; (iii) an amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence into which 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3 or 2) amino acids are inserted, (iv) a sequence 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably in the amino acid sequence represented by the number: 2n It is also included so-called muteins such as proteins containing 1 to several (5, 4, 3 or 2) amino acid sequence number
  • Preferred examples of the protein P1 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (RefSeq No. NP_006036), or their orthologs in other mammals (for example, , Mice (RefSeq No. NP_056546), chimpanzees (RefSeq No. XP_001168714), dogs (RefSeq No. XP_534457), and their splice variants (eg UniProtKB Nos. O75110-1, O75110-2 etc.), alleles Examples include mutants and polymorphisms.
  • Preferred examples of the protein P2 include, for example, a human protein (RefSeq No. NP_006171) consisting of the amino acid sequence represented by SEQ ID NO: 4, or those in other mammals.
  • Orthologs eg, mice (RefSeq No. NP_001020245), rats (RefSeq No. NP_036863), cows (RefSeq No. NP_001068693), chimpanzees (RefSeq No. XP_001135401), dogs (RefSeq No. XP_856422) etc.
  • those Examples include splice variants (for example, RefSeq No.
  • NP_001007098, NP_001018074, NP_001018075, NP_001018076, etc. allelic variants, polymorphisms, and the like.
  • Preferred examples of the protein P3 include, for example, a human protein (RefSeq No. NP_068771) consisting of the amino acid sequence represented by SEQ ID NO: 6, or an ortholog thereof (for example, Mice (RefSeq No. NP_034618), rats (RefSeq No.NP_001071119), cows (RefSeq No. NP_001075075), chimpanzees (RefSeq No.
  • protein P4 oiled-coil domain-containing protein 136) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (RefSeq No. NP_073579), or an ortholog thereof in other mammals (For example, mouse (RefSeq No.
  • NP_663549 rat (RefSeq No. XP_342653), cattle (RefSeq No. XP_872956), chimpanzee (RefSeq No. XP_519361), etc.) and their splice variants (for example, UniProtKB Nos. Q96JN2 -1, Q96JN2-2, Q96JN2-3, Q96JN2-4, etc.), allelic variants, polymorphisms (eg refSNP Nos. Rs3816887 (Asp / His), rs4728137 (Glu / Gln), rs8180870 (Thr / Ala), etc.) ) Etc.
  • Preferred examples of the protein P5 include, for example, a human protein (RefSeq No. NP_001185) consisting of the amino acid sequence represented by SEQ ID NO: 10, or other mammals Orthologs (eg mice (RefSeq No. NP_032252), rats (RefSeq No. NP_446136), dogs (RefSeq No. XP_855233), etc.), and their splice variants, allelic variants, polymorphisms, etc. .
  • Preferred examples of the protein P6 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 12 (RefSeq No. NP_005871), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_062768), rats (RefSeq No. NP_114468), cattle (RefSeq No. NP_001035581), chimpanzees (RefSeq No. XP_528644), dogs (RefSeq No.
  • protein P7 Vehicle-associated membrane protein-associated protein A
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 14, or those in other mammals.
  • Orthologs for example, mice (RefSeq No. NP_038961), rats (RefSeq No. NP_113819), cattle (RefSeq No. NP_001069201), chimpanzees (RefSeq No.
  • XP_512022) dogs
  • dogs dogs
  • RefSeq No. XP_537332 dogs
  • Examples include splice variants (for example, RefSeq No. NP_919415), allelic variants, and polymorphisms.
  • Preferred examples of the protein P8 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 16 (RefSeq No. NP_443117), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_001028805), rats (RefSeq No. NP_598295), cattle (RefSeq No.
  • Preferred examples of the protein P9 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 18 (RefSeq No. NP_001768), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_034711), rat (RefSeq No. NP_062068), cattle (RefSeq No.
  • NP_777133 chimpanzee
  • chimpanzee RefSeq No. XP_516636
  • dog RefSeq No. NP_001074190
  • splice variants for example, UniProtKB Nos. Q08722-1, Q08722-2, Q08722-3, Q08722-4, etc.
  • allelic variants for example, refSNP No. rs11546646 (Pro / Ala), etc.
  • Preferred examples of the protein P10 include, for example, a human protein (RefSeq No.
  • NP_005794 consisting of the amino acid sequence represented by SEQ ID NO: 20, or an ortholog thereof (for example, mouse ( RefSeq No. NP_032053), rat (RefSeq No. NP_073192), cattle (RefSeq No. NP_001070355), chimpanzee (RefSeq No. NP_001035840), dog (RefSeq No. XP_848778) etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs1802470 (Asp / Tyr) etc.) and the like.
  • Preferred examples of the protein P11 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_818932), or an ortholog thereof (eg, rat) in other mammals.
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_818932), or an ortholog thereof (eg, rat) in other mammals.
  • RefSeq No. NP_067713 cattle
  • RefSeq No. XP_872829 chimpanzee
  • RefSeq No. XP_525314 dogs
  • their splice variants for example, UniProtKB Nos.
  • Preferred examples of the protein P12 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 24 (RefSeq No. NP_002915), or their orthologs in other mammals (Ref. For example, mice (RefSeq No. NP_036010), rats (RefSeq No. NP_062216), cattle (RefSeq No.
  • NP_776594 chimpanzees
  • chimpanzees RefSeq No. XP_001159739
  • their splice variants for example, UniProtKB Nos. P49802- 1, P49802-2, P49802-3, P49802-4, P49802-5, etc.
  • allelic variants polymorphisms
  • polymorphisms eg refSNP No. rs12746550 (Met / Leu), rs17851953 (Gln / His), etc.
  • Preferred examples of the protein P13 include, for example, a human protein (RefSeq No.
  • NP_001026866 consisting of the amino acid sequence represented by SEQ ID NO: 26, or an ortholog (eg, mouse (RefSeq) in other mammals. No. NP_035246), rat (RefSeq No. NP_001012167), chimpanzee (RefSeq No. XP_524268), dog (RefSeq No. XP_541616) and the like, and their splice variants, allelic variants, and polymorphisms.
  • Preferred examples of the protein P14 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 28 (RefSeq No.
  • NP_003624 or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_031956), rats (RefSeq No. NP_001003401), chimpanzees (RefSeq No. XP_526897), dogs (RefSeq No. XP_544373), etc.) and their splice variants, allelic variants, polymorphisms (for example, RefSNP No. rs11557041 (Phe / Leu), rs11747630 (Thr / Asn), rs16872126 (Ile / Ser), etc.).
  • the protein P15 includes, for example, a human protein (RefSeq No. NP_003170) consisting of the amino acid sequence represented by SEQ ID NO: 30, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals) NP_033331), rats (RefSeq No. NP_036796), cattle (RefSeq No. NP_776388), chimpanzees (RefSeq No. XP_521056), dogs (RefSeq No. XP_855381), etc.) and their splice variants, allelic variants, many Examples include molds.
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 30, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals) NP_033331)
  • rats RefSeq No. NP_036796
  • cattle RefSeq No. NP_776388
  • Preferred examples of the protein P16 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 32 (RefSeq No. NP_008862), or other mammals Orthologs of them (eg, mice (RefSeq No. NP_035531), rats (RefSeq No. NP_058798), cattle (RefSeq No. NP_777028), chimpanzees (RefSeq No. XP_508989), dogs (RefSeq No.
  • NP_0010033008 examples include splice variants, allelic variants, and polymorphisms (eg, refSNP No. rs17728193 (Val / Leu)).
  • Preferred examples of the protein P17 (syntaxin binding protein 1) include, for example, a human protein (RefSeq No. NP_001027392) consisting of the amino acid sequence represented by SEQ ID NO: 34, or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_033321), rat (RefSeq No. NP_037170), bovine (RefSeq No.
  • NP_777044 splice variants
  • P61764-1 e.g UniProtKB Nos. P61764-1, P61764-2 etc.
  • allelic variants And polymorphism e.g UniProtKB Nos. P61764-1, P61764-2 etc.
  • Preferred examples of the protein P18 (growth associated protein 43) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 36 (RefSeq No. NP_002036), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_032109), rat (RefSeq No. NP_058891), cow (RefSeq No. NP_976234), chimpanzee (RefSeq No.
  • NP_001009816 dog (RefSeq No. XP_535747 etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs6291 (Val / Ile), rs11557762 (Lys / Glu), rs11557763 (Glu / Asp), etc.) and the like.
  • Preferred examples of protein P19 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 38 (RefSeq No. NP_001668), or those in other mammals Orthologs (eg, mice (RefSeq No.
  • NP_033851 rats (RefSeq No. NP_037245), cattle (RefSeq No. NP_001030411), chimpanzees (RefSeq No. NP_001092025), dogs (RefSeq No. NP_001003283), etc.)
  • Splice variants for example, refSNP No. rs11545848 (Lys / Gln), rs1545849 (Met / Val), etc.
  • Preferred examples of the protein P20 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 40 (RefSeq No. NP_000889), or an ortholog thereof in other mammals (For example, mice (RefSeq No. NP_766366), rats (RefSeq No. NP_037330), cattle (RefSeq No. NP_808813), dogs (RefSeq No. XP_855549), etc.), and their splice variants, allelic variants, and polymorphisms (For example, refSNP No. rs12845783 (Ser / Arg)).
  • protein P21 (Muscarinic acetylcholine receptor M1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 42 (RefSeq No. NP_000729), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_031724), rat (RefSeq No. NP_542951), cattle (RefSeq No XP_001255658), chimpanzee (RefSeq No. XP_001159938), dog (RefSeq No. XP_540897), etc.) and their splice variants and allelic variants And polymorphism.
  • Preferred examples of the protein P22 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 44 (RefSeq No. NP_066000), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_766449), rats (RefSeq No. NP_001128087), chimpanzees (RefSeq No. XP_526378), dogs (RefSeq No. XP_545285), etc., as well as their splice variants, allelic variants, polymorphisms (for example, refSNP No. rs2276717 (Gly / Arg), etc.).
  • Preferred examples of the protein P23 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 46 (RefSeq No. NP_077718), or an ortholog thereof in other mammals ( For example, mice (RefSeq No. NP_083548), rats (RefSeq No. NP_001008525), cattle (RefSeq No. NP_001033111), dogs (RefSeq No. XP_533960), etc.), as well as their splice variants, allelic variants, polymorphisms ( For example, refSNP No. rs1142530 (Pro / Leu) etc.).
  • Preferred examples of the protein P24 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 48 (RefSeq No. NP_443131), or those in other mammals Orthologs (eg, mouse (RefSeq No. NP_036144), rat (RefSeq No. XP_344572), etc.) and their splice variants (eg, UniProtKB Nos. Q7Z2K8-1, Q7Z2K8-2 etc.), allelic variants, polymorphisms (For example, refSNP No.
  • Preferred examples of the protein P25 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 50 (RefSeq No. NP_001098999), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_001034088), cattle (RefSeq No. NP_777263), chimpanzees (RefSeq No.
  • XP_001150768 dogs (RefSeq No. XP_849357), and their splice variants (eg UniProtKB Nos. Q9Y2Q0-1) , Q9Y2Q0-2, etc.), allelic variants, polymorphisms (for example, refSNP No. rs3792687 (Thr / Met) etc.) and the like.
  • the “substance that suppresses the expression of protein Pn” refers to the transcription level of the gene Gn encoding protein Pn, the level of post-transcriptional regulation, the level of translation into protein Pn, the level of post-translational modification, etc. It may act. Therefore, substances that suppress the expression of protein Pn include, for example, substances that inhibit the transcription of gene Gn (eg, antigene), substances that inhibit the processing of early transcription products into mRNA, and transport of mRNA to the cytoplasm.
  • substances that suppress the expression of protein Pn include, for example, substances that inhibit the transcription of gene Gn (eg, antigene), substances that inhibit the processing of early transcription products into mRNA, and transport of mRNA to the cytoplasm.
  • Inhibiting substances inhibiting translation of mRNA to protein Pn (eg, antisense nucleic acid, miRNA) or degrading mRNA (eg, siRNA, ribozyme, miRNA), inhibiting post-translational modification of the initial translation product Substances are included. Any substance that acts at any stage can be preferably used, but a substance that complementarily binds to mRNA and inhibits translation into protein Pn or decomposes mRNA is preferable.
  • a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or one of them A nucleic acid containing a portion.
  • the base sequence substantially complementary to the base sequence of mRNA of gene Gn can bind to the target sequence of mRNA and inhibit its translation under physiological conditions in mammals (or cleave the target sequence).
  • a base sequence having a degree of complementarity specifically, for example, a region that overlaps with a base sequence that is completely complementary to the base sequence of the mRNA (that is, a base sequence of the complementary strand of the mRNA).
  • the base sequence complementary or substantially complementary to the base sequence of the gene Gn mRNA is (a) SEQ ID NO: 2n-1 (n is any integer from 1 to 25) A complementary or substantially complementary nucleotide sequence to the nucleotide sequence represented, or (b) a complementary strand of the nucleotide sequence represented by "SEQ ID NO: 2n-1 (n is any integer from 1 to 25)" Is a base sequence that hybridizes under stringent conditions, and has substantially the same quality of activity as a protein consisting of the amino acid sequence represented by SEQ ID NO: 2n (n is an integer from 1 to 25) Examples of the sequence that encodes a protein include complementary or substantially complementary base sequences.
  • the stringent conditions are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washes at 0.2 ⁇ SSC / 0.1% SDS / 50 to 65 ° C., and those skilled in the art will know the conditions for hybridization that give the same stringency. It can be selected appropriately.
  • mRNA of gene G1 include human ATP9A (RefSeq Accession No. NM_006045) containing the nucleotide sequence represented by SEQ ID NO: 1, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_015731), chimpanzee (RefSeq No. XM_001168714), dog (RefSeq No. XM_534457), etc.), and their splice variants, allelic variants, and polymorphisms.
  • Preferred examples of mRNA of the gene G2 include, for example, human NTRK2 (RefSeq No.
  • NM_006180 consisting of the base acid sequence represented by SEQ ID NO: 3, or their orthologs in other mammals (eg, mouse) (RefSeq No. NM_001025074), rat (RefSeq No. NM_012731), cattle (RefSeq No. NM_001075225), chimpanzee (RefSeq No. XM_001135401), dogs (RefSeq No. XM_851329), etc.) and their splice variants (for example, RefSeq No.
  • mRNA of the gene G3 include, for example, human ELAVL4 (RefSeq No. NM_021952) consisting of the base sequence represented by SEQ ID NO: 5, or their orthologs (eg, mouse ( RefSeq No. NM_010488), rat (RefSeq No. NM_001077651), cattle (RefSeq No. NM_001081606), chimpanzee (RefSeq No.
  • mRNA of the gene G4 include, for example, human NAG6 (RefSeq No. NM_022742) consisting of the base sequence represented by SEQ ID NO: 7, or their orthologs (for example, mouse ( RefSeq No. NM_145574), rat (RefSeq No. XM_342652), cattle (RefSeq No.
  • mRNA of gene G5 include, for example, human HCN2 (RefSeq No. NM_001194) consisting of the base sequence represented by SEQ ID NO: 9, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_008226), rat (RefSeq No.
  • mRNA of the gene G6 includes, for example, human DNAJA2 (RefSeq No. NM_005880) consisting of the base sequence represented by SEQ ID NO: 11, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_019794), rat (RefSeq No. NM_032079), cattle (RefSeq No. NM_001040491), chimpanzee (RefSeq No.
  • VAPA VAPA
  • human VAPA RefSeq No. NM_003574
  • SEQ ID NO: 13 the base sequence represented by SEQ ID NO: 13
  • rat RefSeq No. NM_031631
  • cattle RefSeq No.
  • mRNA of the gene G8 include, for example, human SLC2A13 (RefSeq No. NM_052885) consisting of the nucleotide sequence represented by SEQ ID NO: 15, or their orthologs (eg, mouse ( RefSeq No. NM_001033633), rat (RefSeq No.
  • mRNA of the gene G9 include, for example, human CD47 (RefSeq No. NM_001777) consisting of the base sequence represented by SEQ ID NO: 17, or their orthologs in other mammals (eg, mouse ( RefSeq No. NM_010581), rat (RefSeq No.
  • mRNA of the gene G10 include, for example, human FLOT1 (RefSeq No. NM_005803) consisting of the base sequence represented by SEQ ID NO: 19, or their orthologs (for example, mouse ( RefSeq No.
  • NM_008027 rat (RefSeq No. NM_022701), cattle (RefSeq No. NM_001076887), chimpanzee (RefSeq No. NM_001042381), dog (RefSeq No. XM_843685) etc.), and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs1802470 (G / T) etc.).
  • G11 EPB41L1
  • human EPB41L1 RefSeq Accession No.
  • NM_177996 containing the base sequence represented by SEQ ID NO: 21 or an ortholog (eg, rat (RefSeq) in other mammals) No. NM_021681), cattle (RefSeq No. XM_867736), chimpanzee (RefSeq No. XM_525314), dogs (RefSeq No. XM_542979), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
  • Preferred examples of mRNA of the gene G12 (RGS7) include human RGS7 (RefSeq Accession No.
  • NM_002924 containing the nucleotide sequence represented by SEQ ID NO: 23, or their orthologs in other mammals (eg, mouse (RefSeq No. NM_011880), rat (RefSeq No. NM_019343), cattle (RefSeq No. NM_174169), chimpanzee (RefSeq No. XM_001159739)), and their splice variants, allelic variants, and polymorphisms. .
  • mRNA of gene G13 PLD3
  • human PLD3 RefSeq Accession No.
  • NM_001031696 containing the base sequence represented by SEQ ID NO: 25, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_011116), rat (RefSeq No. NM_001012167), chimpanzee (RefSeq No. XM_524268), dog (RefSeq No. XM_541616), etc., and their splice variants, allelic variants, and polymorphisms. .
  • Preferred examples of mRNA of the gene G14 (ENC1) include human ENC1 (RefSeq Accession No.
  • NM_003633 containing the nucleotide sequence represented by SEQ ID NO: 27, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007930), rat (RefSeq No. NM_001003401), chimpanzee (RefSeq No. XM_526897), dog (RefSeq No. XM_544373), etc., and their splice variants, allelic variants, and polymorphisms .
  • Preferred examples of mRNA of the gene G15 include human SYP (RefSeq Accession No.
  • NM_003179 containing the nucleotide sequence represented by SEQ ID NO: 29, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009305), rat (RefSeq No. NM_012664), cattle (RefSeq No. NM_173963), chimpanzee (RefSeq No. XM_521056), dog (RefSeq No. XM_850288), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA. As a preferable example of mRNA of gene G16 (SLC2A3), human SLC2A3 (RefSeq Accession No.
  • NM_006931 containing the base sequence represented by SEQ ID NO: 31 or their orthologs (for example, mouse (RefSeq) in other mammals) No. NM_011401), rat (RefSeq No. NM_017102), cattle (RefSeq No. NM_174603), chimpanzee (RefSeq No. XM_508989), dog (RefSeq No. NM_001003308) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA. As a preferable example of mRNA of gene G17 (STXBP1), human STXBP1 (RefSeq Accession No.
  • NM_001032221 containing the base sequence represented by SEQ ID NO: 33, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009295), rat (RefSeq No. NM_013038), bovine (RefSeq No. NM_174619), etc.), and their splice variants, allelic variants, and polymorphisms.
  • mouse RefSeq No. NM_009295
  • rat RefSeq No. NM_013038
  • bovine RefSeq No. NM_174619
  • NM_008083 rat (RefSeq No. NM_017195), cattle (RefSeq No. NM_203358), chimpanzee (RefSeq No. NM_001009816), dog (RefSeq No. XM_535747), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G19 ATP1B1
  • human ATP1B1 RefSeq Accession No. NM_001677 containing the base sequence represented by SEQ ID NO: 37, or their orthologs in other mammals (for example, mouse (RefSeq No.
  • NM_009721 rat (RefSeq No. NM_013113), cattle (RefSeq No. NM_001035334), chimpanzee (RefSeq No. NM_001098555), dog (RefSeq No. NM_001003283) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G20 MAOB
  • human MAOB RefSeq Accession No. NM_000898 containing the base sequence represented by SEQ ID NO: 39, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No.
  • mRNA of the gene G21 include human CHRM1 (RefSeq Accession No. NM_000738) containing the nucleotide sequence represented by SEQ ID NO: 41, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007698), rat (RefSeq No.
  • NM_080773 cattle (RefSeq No. XM_001255657), chimpanzee (RefSeq No. XM_001159938), dog (RefSeq No. XM_540897), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G22 SLC7A14
  • human SLC7A14 RefSeq Accession No. NM_020949
  • containing the base sequence represented by SEQ ID NO: 43 or their orthologs in other mammals (for example, mouse (RefSeq No. NM_172861), rat (RefSeq No.
  • NM_001134615 chimpanzee
  • chimpanzee RefSeq No. XM_526378
  • dog RefSeq No. XM_545285
  • mRNA of the gene G23 include human NDUFS7 (RefSeq Accession No. NM_024407) containing the nucleotide sequence represented by SEQ ID NO: 45, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_029272), rat (RefSeq No. NM_001008525), cattle (RefSeq No.
  • NM_001038022 dog (RefSeq No. XM_533960), etc.) and their splice variants, allelic variants, and polymorphisms .
  • GPRIN1 human GPRIN1 (RefSeq Accession No. NM_052899) containing the base sequence represented by SEQ ID NO: 47, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_012014), rat (RefSeq No. XM_344571), etc., and their splice variants, allelic variants, and polymorphisms.
  • mRNA of gene G25 ATP8A1
  • human ATP8A1 (RefSeq Accession No. NM_001105529) containing the base sequence represented by SEQ ID NO: 49, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_001038999), cattle (RefSeq No. NM_174838), chimpanzee (RefSeq No. XM_001150768), dogs (RefSeq No. XM_844264), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
  • Part of the base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn means that it can specifically bind to the mRNA of gene Gn and translates the protein from the mRNA.
  • the length and the position are not particularly limited as long as they can inhibit (or degrade the mRNA), but at least 10 bases that are complementary or substantially complementary to the target sequence from the viewpoint of sequence specificity. As mentioned above, it contains about 15 bases or more, more preferably about 20 bases or more.
  • the nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof is preferably exemplified by any of the following (a) to (c): Is done.
  • (a) Antisense nucleic acid for gene Gn mRNA (b) Ribozyme nucleic acid for gene Gn mRNA (c) a nucleic acid having RNAi activity against the mRNA of gene Gn or a precursor thereof
  • the “antisense nucleic acid against mRNA of gene Gn” includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. It is a nucleic acid and has a function of suppressing protein synthesis by forming a specific and stable duplex with a target mRNA.
  • Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base such as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base).
  • RNA double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-rigid Having a side chain group such as sugar (eg, monosaccharide), having an intercurrent compound (eg, acridine, psoralen
  • nucleoside may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
  • the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of gene Gn.
  • the intron sequence can be determined by comparing the genome sequence and the cDNA base sequence of the gene Gn using a homology search program such as BLAST or FASTA.
  • the target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, translation into protein Pn is inhibited.
  • the entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases, and a long sequence of mRNA or the initial transcript.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto.
  • 5 'end hairpin loop of gene Gn may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the gene Gn and the initial transcription product to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA to form triple strands ( A triplex) that can inhibit transcription to RNA (antigene).
  • the nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.) may be substituted.
  • the base moiety pyrimidine, purine
  • BNA LNA
  • ENA ENA
  • the antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of gene Gn, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman) Etc.) can be prepared by synthesizing a complementary sequence thereto.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
  • Ribozyme nucleic acid for gene Gn mRNA As another preferred example of a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof, the mRNA is used in the coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme”, in a narrow sense, refers to RNA having an enzyme activity for cleaving nucleic acids, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends (about 10 bases in total) adjacent to the part that takes the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate.
  • the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)].
  • ribozymes are used in the form of expression vectors containing the DNA that encodes them, in order to promote the transfer of transcripts to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked to a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • siRNA siRNA or miRNA for gene Gn mRNA
  • a double-stranded RNA consisting of an oligo RNA complementary to the mRNA of gene Gn and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of the gene Gn mRNA. Defined as encompassed by nucleic acid containing base sequence or part thereof.
  • RNAi RNA interference
  • So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.
  • siRNA can be designed according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence information of the target gene.
  • Examples of the target sequence of siRNA include, but are not limited to, AA + (N) 19 , AA + (N) 21 or NA + (N) 21 (N is an arbitrary base).
  • the position of the target sequence is not particularly limited.
  • the selected candidate target sequence group whether or not there is homology in the 16-17 base sequence in the mRNA other than the target is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed.
  • AA + (N) 19 AA + (N) 21 or NA + (N) 21 (N is an arbitrary base) is used as the target sequence
  • the target sequence whose specificity has been confirmed is AA (or NA) or later.
  • Two strands consisting of a sense strand having a TT or UU 3 'end overhang at 19-21 bases and an antisense strand having a sequence complementary to the 19-21 base and a TT or UU 3' end overhang Strand RNA may be designed as siRNA.
  • siRNA short hairpin RNA
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are combined with the short strand. It can be designed by linking via a linker sequence.
  • siRNA Target Finder http://www.ambion.com/jp/techlib/misc/siRNA_finder.html
  • insert design tool for pSilencer TM Expression Vector http: / /www.ambion.com/techlib/misc/psilencer_converter.html
  • GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) .
  • the ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a single hairpin RNA (shRNA) serving as a siRNA precursor and cleaving it with a dicer.
  • shRNA single hairpin RNA
  • a nucleic acid designed to generate siRNA for the gene Gn mRNA in vivo also refers to a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the gene Gn mRNA or one of the nucleotide sequences. Defined as encompassed by a nucleic acid containing a moiety. Examples of such nucleic acids include expression vectors constructed so as to express the above-mentioned shRNA and siRNA.
  • shRNA is an oligo containing a base sequence in which the sense strand and the antisense strand of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 5 to 25 bases) long enough to form an appropriate loop structure.
  • Vectors expressing shRNA include tandem type and stem loop (hairpin) type.
  • the former is a tandem-linked siRNA sense cassette and antisense strand expression cassette. Each strand is expressed in a cell and annealed to form a double-stranded siRNA (dsRNA). It is.
  • dsRNA double-stranded siRNA
  • the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA.
  • a pol II promoter for example, a CMV immediate early promoter
  • a pol III promoter is generally used.
  • the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like.
  • a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
  • the siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector.
  • vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids, and the like.
  • Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn or a part thereof is provided in a special form such as a liposome or a microsphere, applied to gene therapy, It can be given in an added form.
  • the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol.
  • Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.).
  • nucleic acids can be attached via bases, sugars, intramolecular nucleoside linkages.
  • examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the protein Pn expression inhibitory activity of these nucleic acids can be examined using a transformant into which the gene Gn has been introduced, a gene Gn expression system in vivo or in vitro, or a protein Pn translation system in vivo or in vitro.
  • the substance that suppresses the expression of protein Pn in the present invention is not limited to a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of gene Gn as described above or a part thereof, but includes protein Pn Other substances such as low molecular weight compounds may be used as long as they directly or indirectly inhibit the production of.
  • the substance may be one that suppresses the expression of any one protein selected from proteins P1 to P25, or may be one that can suppress the expression of two or more proteins. Such a substance can be obtained, for example, by the screening method of the present invention described later.
  • the “substance that suppresses the function of protein Pn” may be any substance as long as the protein Pn once functionally produced suppresses the promotion of A ⁇ production. For example, it binds to protein Pn.
  • a substance that suppresses A ⁇ production a substance that inhibits the binding activity between protein Pn and ⁇ -secretase (for example, a substance that inhibits or modulates the promotion of ⁇ -secretase activity due to the binding between protein Pn and ⁇ -secretase) And substances that inhibit the binding or complex formation of protein Pn and ⁇ -secretase), substances that inhibit the transfer of protein Pn to the cell membrane, and the like.
  • examples of the substance that suppresses the function of protein Pn include an antibody against protein Pn.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known methods for producing antibodies or antisera.
  • the isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG.
  • the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen.
  • CDR complementarity determining region
  • Fab, Fab ′, F (ab ') 2 such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having a protein stabilizing action such as polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • the antibody preferably a monoclonal antibody
  • the antibody has a reduced risk of being antigenic when administered to a human.
  • Antibodies specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., particularly preferably fully human antibodies.
  • Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to a conventional method.
  • fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using.
  • a more preferable substance that suppresses the function of protein Pn is a low-molecular compound suitable for Lipinski's Rule.
  • the low molecular weight compound may be one that suppresses the function of any one protein selected from the proteins P1 to P25, and may be one that can suppress the function of two or more proteins. Such a compound can be obtained, for example, using the screening method of the present invention described later.
  • a substance that suppresses the expression or function of protein Pn suppresses A ⁇ production, and thus is useful for improving the pathology of Alzheimer's disease (AD) or cancer.
  • the substance since the substance may not inhibit the cleavage of Notch protein, the substance has a further advantageous effect that it has a low risk of causing peripheral side effects such as intestinal epithelialization disorder and immune abnormality. Therefore, drugs containing substances that suppress the expression or function of protein Pn are ⁇ -secretase inhibitors or modulators, A ⁇ production inhibitors, diseases involving A ⁇ (eg, AD), and diseases involving Notch protein cleavage. It can be used as a preventive and / or therapeutic agent for (eg, cancer).
  • Only one type of substance that suppresses the expression or function of protein Pn may be used, or two or more types may be used in combination.
  • these substances may suppress the expression or function of the same protein, or two or more kinds of substances may be suppressed. It may be one that suppresses the expression or function of different proteins.
  • two or more substances that suppress the expression or function of protein Pn may be formulated as separate medicaments, or may be blended in the same pharmaceutical composition.
  • the respective preparations may be administered simultaneously or at intervals.
  • the administration route may be the same or different.
  • the dose described below indicates the dose of a single substance that suppresses the expression or function of protein Pn. However, even when two or more substances are used in combination, the dose does not adversely affect the administration subject. Similar dosages can be used for each substance.
  • An antisense nucleic acid of the present invention capable of binding to a transcription product of a gene Gn complementary to an antisense nucleic acid, a ribozyme nucleic acid, siRNA and a precursor thereof, and inhibiting protein translation from the transcription product SiRNAs (or ribozymes) that can cleave the transcripts of nucleic acids and gene Gn transcripts that are homologous (or complementary), and shRNA that is the precursor of the siRNA (hereinafter, comprehensive) May be referred to as “the nucleic acid of the present invention”, an agent for inhibiting and / or treating an A ⁇ production inhibitor, a disease involving A ⁇ (eg, AD) and a disease involving cleavage of Notch protein (eg, cancer) Can be used as The pharmaceutical containing the nucleic acid of the present invention is used as it is, or as a pharmaceutical composition of an appropriate dosage form, as a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat,
  • the nucleic acid of the present invention When used as an A ⁇ production inhibitor or a preventive and / or therapeutic agent for a disease involving A ⁇ , it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention may be used alone or inserted in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector. You can also.
  • the nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
  • the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .
  • the nucleic acid of the present invention may be administered per se or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • composition for parenteral administration for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent.
  • Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solub
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the nucleic acid of the present invention is preferably contained, for example, usually in an amount of about 0.01 to 500 mg per dosage unit dosage form.
  • the dosage of the above-mentioned medicament containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for treatment / prevention of AD, the nucleic acid of the present invention It is convenient to administer about 0.0001 to 20 mg / kg body weight by intravenous injection once a day to about 6 months. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention.
  • active ingredients include AD therapeutic agents typified by acetylcholinesterase inhibitors, ⁇ -secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), ⁇ -secretase inhibitors, ⁇ -secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 ⁇ ⁇ ⁇ ⁇ (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • ⁇ -secretase inhibitors eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure
  • An antibody against protein Pn, or a low molecular compound that suppresses the expression or function of protein Pn The activity can be inhibited, or the interaction (complex formation) between protein Pn and ⁇ -secretase can be inhibited. Therefore, these substances suppress the expression or function of protein Pn in vivo and inhibit ⁇ -secretase activity. Therefore, A ⁇ production inhibitors, diseases involving A ⁇ (eg, AD) and Notch protein cleavage It can be used as a preventive and / or therapeutic agent for a disease involved (eg, cancer).
  • the medicament containing the above antibody or low molecular weight compound can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey). Etc.) orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
  • a human or mammal eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey.
  • Etc. orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
  • the above-described antibodies and low-molecular compounds may be administered per se, or may be administered as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the above antibody or low molecular compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • composition for parenteral administration for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent.
  • Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared by, for example, dissolving, suspending or emulsifying the antibody or low molecular compound of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solub
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody or low molecular weight compound is preferably contained in an amount of usually 0.1 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms per dosage unit form.
  • the dose of the above-mentioned medicament containing the above-mentioned antibody or low-molecular compound or a salt thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of AD Is usually about 0.0001 to 20 mg / kg body weight for a single dose of antibody or low molecular weight compound, about 1 to 5 times a day for low molecular weight compound, orally or parenterally, 1 to several days for antibody Conveniently administered once a month by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the above-described antibody or low-molecular compound.
  • active ingredients include, for example, AD therapeutic agents typified by acetylcholinesterase inhibitors, ⁇ -secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), ⁇ -secretase inhibitors, ⁇ -secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 ⁇ (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • ⁇ -secretase inhibitors eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbony
  • a compound or a salt thereof that suppresses the expression and / or function of protein Pn is an A ⁇ production inhibitor, a disease involving A ⁇ (eg, AD, etc.) or a disease involving cleavage of Notch protein (eg, cancer). It can be used as a prophylactic and / or therapeutic agent. Therefore, protein Pn or a partial peptide thereof, or a cell producing the same can be used as a tool for screening a substance having an inhibitory effect on A ⁇ production by using the expression level and / or activity of the protein (gene) as an index. Can be used.
  • Screening method (I) When screening for a compound or a salt thereof that suppresses the function of protein Pn, the screening method is used in the presence and absence of a test substance for protein Pn or a partial peptide thereof or a cell capable of producing the same. Incubating (culturing) and comparing the activity of protein Pn under both conditions.
  • Cells having the ability to produce the protein Pn used in the above screening methods include human or other mammalian cells that naturally express them or biological samples containing them (eg, blood, tissues, organs, etc.) If there is no particular limitation. In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance is administered to the living body itself, and these biological samples are isolated after a certain period of time. May be. Examples of cells having the ability to produce protein Pn or a partial peptide thereof include various transformants prepared by known and commonly used genetic engineering techniques. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells, CHO cells are preferably used.
  • a base sequence represented by DNA encoding protein Pn or a partial peptide thereof that is, SEQ ID NO: 2n-1 (n is any integer of 1 to 25) or a complementary strand of the base sequence
  • a DNA containing a base sequence encoding a polypeptide that hybridizes under stringent conditions and has the same quality of activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2n) in a promoter in an appropriate expression vector And can be prepared by introducing it into a host animal cell.
  • a DNA encoding the protein Pn or a partial peptide thereof is synthesized, for example, based on the base sequence represented by SEQ ID NO: 2n-1, using a suitable oligonucleotide as a probe or primer, and a cell that produces the protein Pn described above -It can be cloned from tissue-derived cDNA or cDNA library using hybridization or PCR.
  • Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
  • the cloned DNA is obtained using a known kit such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc.
  • the nucleotide sequence can be converted according to a method known per se such as the Kunkel method or a method analogous thereto.
  • the cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired.
  • the DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • Expression vectors include animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as ⁇ phage; animal viruses such as retrovirus, vaccinia virus, adenovirus A vector or the like is used.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR ⁇ promoter For example, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter It is done. Of these, CMV promoter, SR ⁇ promoter and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do.
  • Selectable markers include, for example, dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter abbreviated as amp r ), neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like.
  • dhfr dihydrofolate reductase gene
  • amp r ampicillin resistance gene
  • neomycin resistance gene hereinafter sometimes abbreviated as neo r, include G418 resistance
  • the target gene can also be selected using a medium that does not contain thymidine.
  • Protein Pn-expressing cells can be produced by transforming a host with an expression vector containing the DNA encoding the protein Pn described above.
  • Hosts include mammalian cells such as HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO Cells (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells and the like can be used.
  • mammalian cells such as HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO Cells (
  • Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like.
  • PEG method New Cell Engineering Experiment Protocol
  • electroporation method microinjection method
  • lipofection method lipofection method and the like.
  • the methods described in Cell Engineering Supplement 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) can be used.
  • mammalian cells having the ability to produce the native protein Pn, or tissues / organs containing the cells are, for example, a minimum essential medium containing about 5 to 20% fetal calf serum ( MEM) [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199,519 ( 1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)].
  • MEM fetal calf serum
  • DMEM Dulbecco's Modified Eagle Medium
  • RPMI 1640 Medium RPMI 1640 Medium
  • the pH of the medium is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C, with aeration and agitation as necessary.
  • Protein Pn or a partial peptide thereof may be the aforementioned transformed cells (or non-mammalian cells obtained by introducing the gene Gn into bacteria such as Escherichia coli and Bacillus subtilis, yeast and insect cells), or the native protein Pn. It can be separated and purified from a culture obtained by culturing mammalian cells having the ability to produce according to a method known per se.
  • the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or
  • a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by freeze-thawing or the like is appropriately used.
  • extracting protein Pn or its partial peptide from the membrane fraction after disrupting the cells or cells in the same manner as described above, cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to obtain a fraction containing cell membrane.
  • the cell membrane fraction is purified by density gradient centrifugation or the like.
  • methods such as fractionating a culture supernatant from a culture by centrifugation or filtration, are used. Isolation and purification of the protein Pn or its partial peptide contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. Examples of such methods include the use of solubilities such as salting out and solvent precipitation; mainly the differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point such as point electrophoresis is used. These methods can be combined as appropriate.
  • the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
  • the protein Pn produced by the transformed cell or its partial peptide can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
  • protein Pn or a partial peptide thereof can be synthesized in vitro using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using the RNA encoding it as a template.
  • a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding the protein Pn or a partial peptide thereof as a template.
  • any one protein selected from (P1) to (P25) may be used, or two or more proteins may be combined. It may be used.
  • two or more types of proteins are provided in the form of cells, it is preferable to use one type of cell that produces all of the two or more types of proteins.
  • test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. Alternatively, a known one may be used.
  • an A ⁇ production system Preferably coexist.
  • the A ⁇ production system cells expressing APP, which is a precursor of A ⁇ , are preferably used.
  • protein Pn or its partial peptide is provided as a cell which has the capability to produce it, it is preferable that the cell itself expresses APP.
  • the contact of the test substance with the cells may be performed by, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid).
  • the test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time.
  • concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 nM, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
  • the contact between the protein or peptide and the test substance is performed by adding both to the culture medium of the cell expressing APP and incubating the cell for a certain period of time.
  • the concentration of the protein Pn or a partial peptide to be added is appropriately selected for each protein or peptide, for example, in the range of about 0.1 to about 100 ⁇ g / ml.
  • cells that produce protein Pn are provided in the form of a non-human mammal individual, an animal that naturally expresses protein Pn may be used, or a gene that incorporates gene Gn so that Pn is overexpressed Recombinant animals (transgenic (Tg) animals) may be used.
  • Tg transgenic
  • a knock-in animal in which the endogenous gene Gn of the animal is replaced with the human gene Gn can also be used.
  • cells expressing APP are inherent in the animal individual.
  • the state of the individual animal is not particularly limited, and may be an AD model animal such as Tg2576 mouse or APP23 Tg mouse.
  • the administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, rectal administration, intranasal administration, intraventricular administration and the like.
  • a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day for about 1 to 6 months.
  • the protein Pn activity in the above screening method can be measured by measuring the production amount of A ⁇ by a method known per se.
  • Test samples for measuring activity include cultured cells that express APP (including tissue culture and organ culture), protein Pn or its partial peptide, or cells that have the ability to produce it (cells that express APP).
  • APP tissue culture and organ culture
  • cells that have the ability to produce it cells that express APP.
  • the test substance is administered to a non-human mammal individual in which the culture supernatant of the cultured cells produces the proteins Pn and APP
  • Examples thereof include bodily fluids collected from the individual, such as blood, cerebrospinal fluid, and the like, or cell, tissue, and organ extracts, such as the brain or homogenates of tissue sections thereof.
  • the production amount of A ⁇ is the amount of A ⁇ (eg, A ⁇ 38, A ⁇ 40, A ⁇ 42, etc., preferably A ⁇ 40 and / or A ⁇ 42) in the test sample, for example, various immunological methods such as ELISA using anti-A ⁇ antibody and Western blotting. It can be performed by measuring using a technique, mass spectrometry or the like.
  • the activity of protein Pn in the presence of the test substance is, for example, about 10% or more compared to the activity in the absence of the test substance
  • the substance is inhibited, preferably about 20% or more, more preferably 30% or more, and even more preferably about 50% or more
  • the test substance is treated as a protein Pn function inhibitory substance, and thus a substance having an A ⁇ production inhibitory action. Can be selected as a candidate.
  • Screening method (Ia) in a non-human animal into which at least one gene selected from (G1) to (G25) has been introduced, screening for a therapeutic or prophylactic agent for AD using an improvement in phenotype reflecting the pathological condition of AD as an index, The efficacy of the drug obtained by the above screening method or other screening methods against AD can be evaluated. Specifically, the method includes the following steps (a) to (c).
  • the method can be carried out in the same manner as in the case of administering the test substance to a non-human mammal individual producing the proteins Pn and APP.
  • phenotypes reflecting the pathology of AD include, for example, (1) A ⁇ amount in tissues such as brain tissue, cerebrospinal fluid, blood, (2) neuronal cell death, (3) central nervous system Inflammatory response of the system, (4) reduced cognitive ability, (5) accumulation of amyloid plaques, (6) deterioration of cerebral blood flow, (7) reduction of cerebral glucose metabolism, but not limited thereto .
  • tissues such as brain tissue, cerebrospinal fluid, blood
  • neuronal cell death e.g., central nervous system Inflammatory response of the system
  • reduced cognitive ability e.g., those known per se can be used (for example, as a method for evaluating cognitive ability, a Y-shaped maze test or the like can be mentioned).
  • the activity of protein Pn can be measured using the increased activity of ⁇ -secretase as an indicator.
  • the screening method includes: (A) contacting the protein Pn and ⁇ -secretase with a test substance; (B) measuring ⁇ -secretase activity, and (c) selecting a substance that reduces or modulates ⁇ -secretase activity compared to when measured in the absence of the test substance. .
  • ⁇ -secretase for example, a cell expressing it or a cell membrane fraction thereof can be used.
  • the protein Pn may be provided by a cell that expresses ⁇ -secretase itself.
  • ⁇ -secretase activity is determined by adding APP or Notch as a substrate, APP metabolite fragments (A ⁇ 38, A ⁇ 40, A ⁇ 42, AICD, etc.) or Notch metabolites (NICD), ELISA, Western blotting, reporter gene assay It can be measured by detecting using mass spectrometry or the like.
  • the ⁇ -secretase activity in the presence of the test substance is about 10% or more, preferably about 20% or more, more preferably 30%, compared to the activity in the absence of the test substance. %, More preferably about 50% or more, the test substance can be selected as a protein Pn function-inhibiting substance, and thus a candidate for an A ⁇ production-inhibiting substance.
  • a control a cell or an animal in which the gene Gn is knocked out or knocked down is used as a control, and in the presence of a test substance in the test cell or animal and the control.
  • An A ⁇ production inhibitor can also be selected by measuring and comparing the activity of the protein Pn.
  • the present invention also relates to a method for screening an A ⁇ production inhibitor, comprising comparing the expression of the protein (gene) in a cell having the ability to produce protein Pn in the presence and absence of the test substance.
  • the cells used in this method, the type of test substance, the mode of contact between the test substance and the cells, and the like are the same as in the method using the activity of the protein Pn as an index.
  • the expression level of protein Pn is a nucleic acid that can hybridize with the above-described DNA encoding protein Pn under highly stringent conditions, that is, the nucleotide sequence represented by SEQ ID NO: n or a complementary nucleotide sequence and string It can be measured at the RNA level by detecting the mRNA of the gene Gn using a nucleic acid that can hybridize under gentle conditions (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”). Alternatively, the expression level can be measured at the protein level by detecting these proteins using an antibody against the above-described protein Pn (hereinafter sometimes referred to as “the detection antibody of the present invention”). .
  • the present invention (A) Cells having the ability to produce protein Pn are cultured in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention.
  • a method for screening an A ⁇ production inhibitor characterized by measuring and comparing, and (b) culturing cells capable of producing protein Pn in the presence and absence of a test substance, under both conditions
  • Provided is a method for screening an A ⁇ production inhibitor, characterized in that the amount of the protein is measured and compared using the detection antibody of the present invention.
  • the measurement of the amount of mRNA or protein of protein Pn can be specifically performed as follows.
  • blood, a specific organ (for example, brain), or a tissue or cell isolated from the organ is obtained.
  • the mRNA of the protein Pn contained in the obtained cell can be quantified by, for example, extracting mRNA from the cell etc. by a normal method and using, for example, a technique such as RT-PCR, or a known Northern Northern It can also be quantified by blot analysis.
  • the amount of protein Pn can be quantified using Western blot analysis or various immunoassay methods described in detail below.
  • a transformant introduced with the gene Gn is prepared according to the above method, and the protein Pn contained in the transformant or mRNA encoding the same can be quantified and analyzed in the same manner as in (i) above. it can.
  • the measurement of the amount of protein Pn in the screening method of (b) above is, for example, (I) The detection antibody of the present invention, the sample solution and the labeled protein Pn are reacted competitively, and the protein Pn in the sample solution is detected by detecting the labeled protein bound to the antibody. How to quantify, (Ii) The sample solution is reacted with the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously, and then the labeling agent on the insolubilized carrier. For example, a method of quantifying C protein Pn in a sample solution by measuring the amount (activity) of.
  • the two types of antibodies recognize different portions of the protein Pn.
  • the other antibody can react with the C-terminal part of the protein.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent material for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
  • the method for quantifying protein Pn using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • a sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and another labeled detection antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier.
  • the protein Pn in the sample solution can be quantified by measuring the amount or activity of the labeling agent.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the insolubilization method can be the same as those described above.
  • the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
  • the detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • a competitive method the protein Pn in the sample solution and the labeled protein Pn are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody. Separation (B / F separation) and the amount of labeling of either B or F is measured to quantify the protein Pn in the sample solution.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody)
  • a solid phase is used as the primary antibody.
  • Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
  • the protein Pn in the sample solution and the immobilized protein Pn are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the sample solution in the sample solution is separated. After reacting protein Pn with an excess amount of labeled antibody, and then adding immobilized protein Pn to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
  • nephrometry the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured.
  • Laser nephrometry using laser scattering is preferably used even when the amount of protein Pn in the sample solution is very small and only a small amount of precipitate can be obtained.
  • the expression level of protein Pn (mRNA amount or protein amount) in the presence of the test substance is about 10% less than that in the absence of the test substance.
  • the test substance is inhibited by 10% or more, preferably about 20% or more, more preferably about 30% or more, and even more preferably about 50% or more, the test substance is expressed as a protein Pn expression-suppressing substance, and thus has an effect of inhibiting A ⁇ production.
  • a cell containing a reporter gene under the control of the transcriptional regulatory region in the gene Gn can be used instead of the cell expressing the gene Gn.
  • a cell may be a cell, tissue, organ or individual of a transgenic animal into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of gene Gn is introduced.
  • a reporter gene eg, luciferase, GFP, etc.
  • the expression level of protein Pn can be evaluated by measuring the expression level of the reporter gene using a conventional method.
  • a protein or gene in which the gene Gn is knocked out or knocked down is used as a control, and the protein in the presence of the test substance in the test cell or animal and the control
  • An A ⁇ production inhibitor can also be selected by measuring and comparing the expression level of Pn.
  • the present invention also provides a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c).
  • a step of bringing protein Pn and ⁇ -secretase into contact with a test substance (b) A step of measuring the binding activity between protein Pn and ⁇ -secretase (c) When measured in the absence of the test substance
  • the step of selecting the substance that decreases the binding activity compared to the above may be exemplified by measuring the amount of binding as shown in the following, as an indicator of binding activity, or by measuring the increased activity of ⁇ -secretase (the former Can be performed according to the above screening method (Ib)).
  • the screening method includes (1) Measuring and comparing the amount of labeled protein Pn bound to ⁇ -secretase when the labeled protein Pn and ⁇ -secretase are contacted in the presence and absence of the test substance. Or (2) the labeled protein Pn and the cell or membrane of the labeled protein Pn when the ⁇ -secretase producing cell or its membrane fraction is contacted in the presence and absence of the test substance The amount of binding to the fraction is measured and compared.
  • Protein Pn can be labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like according to a conventional method.
  • radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • enzyme those which are stable and have high specific activity are preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent material for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • the amount of label (protein Pn) that binds to ⁇ -secretase in the presence of the test substance is larger than that in the absence of the test substance.
  • the test substance suppresses the binding activity between protein Pn and ⁇ -secretase.
  • the substance can be selected as a candidate of a substance having an A ⁇ production inhibitory action.
  • a substance that suppresses the expression or function of protein Pn obtained by using any one of the screening methods of the present invention is a disease involving A ⁇ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer). ) Is useful as a preventive and / or therapeutic agent.
  • the substance obtained using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as a low molecular weight compound that suppresses the expression or function of the above protein Pn, and the same administration route And administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Can do.
  • mammals eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.
  • the present invention relates to A ⁇ -related diseases (eg, AD) or Notch protein cleavage characterized by measuring the expression level of protein Pn in a sample collected from a test animal
  • test animals include humans and other mammals, preferably humans, mice, rats, rabbits, dogs, monkeys and the like that are widely used as experimental animals.
  • the measurement target sample include blood, plasma, serum, cerebrospinal fluid, lymph fluid, saliva, mucous membrane, urine, tears, semen, joint fluid, biopsy sample, and the like.
  • the expression level of gene Gn and the amount of protein Pn in the sample can be measured by the same method as described in the above screening method using the expression level of the gene or the protein as an index.
  • the expression level of the gene Gn or the protein Pn in the sample collected from the test animal is significantly higher than the expression level of the gene Gn or the protein Pn in the sample collected from the normal animal. If the test animal has a disease associated with A ⁇ (eg, AD) or a disease associated with Notch protein cleavage (eg, cancer), or can be determined to have a high risk of developing in the future. .
  • a ⁇ eg, AD
  • Notch protein cleavage eg, cancer
  • the expression level in a normal animal is identified in advance, for example, the average value + 2SD is defined as a cutoff value, and the expression level of gene Gn or the amount of protein Pn in a sample collected from the test animal When the off-value is exceeded, the test animal has developed a disease involving A ⁇ (eg, AD), a disease involving cleavage of Notch protein (eg, cancer), or has a high risk of developing in the future It can also be determined.
  • a ⁇ eg, AD
  • Notch protein eg, cancer
  • an animal having the gene Gn knocked out or knocked down may be used as a control.
  • An animal in which the gene Gn is knocked out or knocked down is also useful as a tool for analyzing the function of the protein Pn.
  • Example 1 Identification of brain ⁇ -secretase binding protein (1)
  • ⁇ -secretase activity is strongly detected in the microsomal fraction, surfactant-insoluble membrane fraction (DRM), synaptic membranes, and synaptic vesicles. Therefore, each of these fractions was prepared, and the ⁇ -secretase complex was purified by an inhibitor pull-down method.
  • DRM surfactant-insoluble membrane fraction
  • synaptic membranes synaptic membranes
  • synaptic vesicles synaptic vesicles. Therefore, each of these fractions was prepared, and the ⁇ -secretase complex was purified by an inhibitor pull-down method.
  • Preparation of fraction containing brain ⁇ -secretase SD rats were obtained from B & K Universal (Sollentuna, Sweden), and human brain tissue was obtained from Huddinge Brain Bank (Huddinge, Sweden).
  • Rat or human brain tissue was homogenized in buffer A (20 mM Hepes, 50 mM KCl, 2 mM EGTA, pH 7.5 plus complete protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA)) Thereafter, cell debris and nuclear components were removed by centrifugation at 1,000 xg and 10,000 xg, and then a microsomal fraction was prepared from the precipitate obtained by centrifugation at 100,000 xg. The prepared microsomal fraction was solubilized with buffer A containing 1% (w / v) CHAPSO, and then the solubilized ⁇ -secretase complex was prepared by centrifugation at 100,000 ⁇ g.
  • the DRM fraction was prepared according to the method of Hur et al. (Hur JY et al., FEBS J. (2008) 275: 1174-87). That is, brain tissue was homogenized in Buffer B (20 mM Hepes (pH 7.5), 50 mM KCl, 2 mM EGTA added with complete protease inhibitor mixture), and the microsomal fraction was obtained by centrifugation as described above. Prepared. Resuspend the obtained microsomal fraction in Buffer C (20 mM Tris / HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2.0% CHAPSO), and prepare the DRM fraction by the sucrose density gradient method. .
  • Synaptic membranes and synaptic vesicles were prepared according to the method of Cohen et al. (Cohen J. et al., Org Chem (2004), 69, 7344-7347). That is, brain tissue was homogenized in Buffer D (0.32 M sucrose, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.5 mM CaCl 2 ) and centrifuged at 17,300 ⁇ g. The obtained precipitate is resuspended in Buffer E (1 mM NaHCO 3 , 0.32 M sucrose), and a synaptosome fraction is prepared by a sucrose density gradient method.
  • the synaptosome fraction is solubilized with 6 mM Tris-HCl pH 8.1, centrifuged at 48,250 ⁇ g, and separated into a LP1 membrane fraction and a supernatant fraction containing synaptic vesicles.
  • the synaptic membrane is prepared by further separating the LP1 membrane fraction by the sucrose density gradient method.
  • Synaptic vesicles are prepared by centrifuging the supernatant fraction at 100,000 x g.
  • the prepared synaptic membrane and the synaptic vesicle are solubilized with buffer A containing 1% (w / v) CHAPSO to prepare a solubilized ⁇ -secretase complex.
  • Example 2 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (1) About the thing identified by the said method, the influence with respect to A (beta) production was evaluated by RNAi method.
  • HEK-APP cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum.
  • the siRNA of the novel factor can be purchased from Ambion etc., or the method reported by Teramoto R. et al. (Teramoto, R. et al., FEBS Lett. (2005) 579, 2878-2882) and It was designed by the method described in Patent Publication 2006-236153 and synthesized from Invitrogen.
  • siRNA was introduced into HEK-APP cells using the siRNA introduction reagent Lipofectamine TM RNAiMAX Transfection Reagent (Invitrogen). 48 hours after introduction, the culture medium was converted to Opti-MEM I (Invitrogen), and further cultured for 24 hours, and the culture supernatant was collected. The number of viable cells was measured by the WST8 method, and siRNAs showing cytotoxicity of 30% or more were excluded from the test. The knockdown efficiency of the target gene was confirmed by real-time PCR. As a target gene primer, Applied Biosystems Pre-Developed TaqMan (registered trademark) Assay Reagents was used, and the GAPDH gene was used as an internal standard.
  • Applied Biosystems Pre-Developed TaqMan registered trademark
  • the amounts of A ⁇ 40 and A ⁇ 42 in the culture supernatant were measured by ELISA (Wako Chemicals, Osaka, Japan). The inhibition rate was calculated with the value in siRNA-untreated cells (MOCK) as 100%. As a result, it was confirmed that the amount of A ⁇ 40 and A ⁇ 42 was decreased by decreasing the expression level of presenilin 1 (PS1), which is a constituent factor of ⁇ -secretase, used as a test system control (FIGS. 3 and 4). Medium “PSEN1”).
  • PS1 presenilin 1
  • ATP2A2 and FLOT2 have been reported to reduce A ⁇ production by siRNA knockdown, confirming that this method is effective as a novel method for identifying ⁇ -secretase-binding A ⁇ production regulators. ing.
  • the knowledge which reduces the amount of A (beta) 40 and A (beta) 42 by reducing the expression level of a novel factor was acquired (FIGS. 3, 4).
  • Example 3 Identification of binding protein of brain ⁇ -secretase (2) Using the microsomal fraction obtained in Example 1, a plurality of novel ⁇ -secretase constituents that specifically bind to novel ⁇ -secretase and are expressed in the brain were found (Table 3).
  • Example 4 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (2)
  • siRNA knockdown of a novel binding factor (2) For the novel ⁇ -secretase component identified in Example 3 above, the effect on A ⁇ production was evaluated by the RNAi method in the same manner as described in Example 2.
  • Example 5 Identification of binding protein of brain ⁇ -secretase (3) Using the DRM fraction obtained in Example 1, a plurality of novel ⁇ -secretase components that specifically bind to the novel ⁇ -secretase and are expressed in the brain were found (Tables 5, 17 to 21). . Further, from the synaptic membrane and the synaptic vesicle fraction obtained in Example 1, a plurality of novel ⁇ -secretase constituents that were newly specifically bound to ⁇ -secretase and expressed in the brain were found (Tables 5 and 22). To 27).
  • Example 6 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (3)
  • the effect on A ⁇ production was evaluated by the RNAi method in the same manner as in Example 2.
  • the screening method provided by the present invention can select a substance having an action of suppressing the expression and function of a novel protein that up-regulates the activity of ⁇ -secretase
  • the substance obtained by this screening method is As a ⁇ production and / or Notch cleavage inhibitor or modulator, it is expected as a prophylactic and therapeutic agent for diseases such as AD and cancer.
  • these novel proteins may not affect the Notch cleavage activity of ⁇ -secretase
  • the substance obtained by this screening method does not inhibit the cleavage of other physiological substrates such as Notch, It may be able to inhibit only ⁇ -secretase activity, and is expected as a safe preventive and therapeutic drug for diseases such as AD with few side effects.
  • the present invention is based on a Japanese patent application filed on June 4, 2009, Japanese Patent Application No. 2009-135589, the entire contents of which are included in this specification.

Abstract

La présente invention concerne un agent thérapeutique nouveau et efficace pour la maladie d'Alzheimer. La présente invention concerne spécifiquement : un inhibiteur de production de β-amyloïde (Aβ), qui comprend une substance capable d'inhiber l'expression ou la fonction d'une protéine choisie parmi l'ATPase IIA transporteur de phospholipide probable ; un précurseur de récepteur de facteurs de croissance BDNF/NT-3 ; la protéine 4 de type ELAV ; la protéine 136 contenant un domaine hélicoïdal ; le canal 2 déclenché par nucléotide cyclique activé par hyperpolarisation potassium/sodium ; le membre 2 de la sous-famille A d'homologues de DnaJ ; la protéine A associée à la protéine membranaire associée aux vésicules ; un cotransporteur de myo-inositol protonique ; l'antigène de surface de leucocytes CD47 ; la flotilline-1 ; band-4.1-like 1; un régulateur de la signalisation de protéine G 7 ; la phospholipase D3 ; la protéine 1 d'ectoderme-cortex neural ; la synaptophysine ; la famille de véhicule de soluté 2 (un transporteur de glucose facilité), membre 3 ; la protéine de liaison de syntaxine 1 ; la protéine associée à la croissance 43 ; le polypeptide bêta 1, ATPase, transporteur de Na+/K+ ; l'amine oxydase [contenant de la flavine] B ; le récepteur M1 d'acétylcholine muscarinique ; un transporteur d'acide aminé cationique probable ; la protéine 7 NADH déshydrogénase à fer-soufre ; un inducteur régulé par la protéine G de croissance de neurite ; et l'ATPase transporteur de phospholipide probable 1A ; et un procédé pour cribler une substance inhibitrice de production de Aβ ou un agent prophylactique/thérapeutique pour la maladie d'Alzheimer, qui utilise la protéine.
PCT/JP2010/059568 2009-06-04 2010-06-04 Procédé pour le criblage d'inhibiteur utilisant un facteur capable de stimuler la production de peptide amyloïde bêta, et inhibiteur obtenu par celui-ci WO2010140694A1 (fr)

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CN102539758A (zh) * 2011-12-26 2012-07-04 中南大学 一种人肺腺癌蛋白质标志物Flotillin-1的应用方法
WO2012091138A1 (fr) * 2010-12-28 2012-07-05 大日本住友製薬株式会社 Médicament de diagnostic et procédé de diagnostic pour la maladie d'alzheimer
WO2018196782A1 (fr) * 2017-04-27 2018-11-01 The University Of Hong Kong Utilisation d'inhibiteurs de hcn pour le traitement du cancer
WO2021177448A1 (fr) * 2020-03-06 2021-09-10 公益財団法人神戸医療産業都市推進機構 Procédé de criblage
US20210309726A1 (en) * 2018-05-21 2021-10-07 New York University Treatment of melanoma brain metastasis by inhibition of amyloid precursor protein cleavage
WO2023039395A1 (fr) * 2021-09-07 2023-03-16 Yale University Méthodes de traitement d'affections neurodégénératives et compositions pour celles-ci

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Cited By (12)

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Publication number Priority date Publication date Assignee Title
WO2012091138A1 (fr) * 2010-12-28 2012-07-05 大日本住友製薬株式会社 Médicament de diagnostic et procédé de diagnostic pour la maladie d'alzheimer
CN103370620A (zh) * 2010-12-28 2013-10-23 大日本住友制药株式会社 阿尔茨海默病的诊断药和诊断方法
KR20140004701A (ko) * 2010-12-28 2014-01-13 다이닛본 스미토모 세이야꾸 가부시끼가이샤 알츠하이머 병의 진단약 및 진단 방법
JP5894085B2 (ja) * 2010-12-28 2016-03-23 大日本住友製薬株式会社 アルツハイマー病の診断薬及び診断方法
CN105548561A (zh) * 2010-12-28 2016-05-04 大日本住友制药株式会社 阿尔茨海默病的诊断药和诊断方法
KR101883515B1 (ko) 2010-12-28 2018-07-30 다이닛본 스미토모 세이야꾸 가부시끼가이샤 알츠하이머 병의 진단약 및 진단 방법
US10393757B2 (en) 2010-12-28 2019-08-27 Dainippon Sumitomo Pharma Co., Ltd. Diagnostic drug and diagnostic method for Alzheimer's disease
CN102539758A (zh) * 2011-12-26 2012-07-04 中南大学 一种人肺腺癌蛋白质标志物Flotillin-1的应用方法
WO2018196782A1 (fr) * 2017-04-27 2018-11-01 The University Of Hong Kong Utilisation d'inhibiteurs de hcn pour le traitement du cancer
US20210309726A1 (en) * 2018-05-21 2021-10-07 New York University Treatment of melanoma brain metastasis by inhibition of amyloid precursor protein cleavage
WO2021177448A1 (fr) * 2020-03-06 2021-09-10 公益財団法人神戸医療産業都市推進機構 Procédé de criblage
WO2023039395A1 (fr) * 2021-09-07 2023-03-16 Yale University Méthodes de traitement d'affections neurodégénératives et compositions pour celles-ci

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