JP2012135304A - Method for screening inhibitor by using factor for controlling production of amyloid beta - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective drug for treating Alzheimer's disease.SOLUTION: The β-amyloid (Aβ) production inhibitor includes a substance inhibiting the expression or functions of twelve kinds of specific proteins of which the relation with the γ-secretase activity has not been known. The method for screening the Aβ production inhibitor, and the drug for preventing and treating the Alzheimer's disease using the proteins are disclosed.

Description

  The present invention relates to a production inhibitor of β-amyloid that is a cause of Alzheimer's disease, a preventive and therapeutic agent for Alzheimer's disease, and a screening method for such a drug.

  Β-amyloid (Aβ) peptide that accumulates in the brain of Alzheimer's disease (AD) is generated by being excised from the precursor protein amyloid precursor protein (APP) by β-secretase and γ-secretase, and this is polymerized to form senile plaques. Is formed. Accumulation of Aβ is a lesion that is highly specific to AD brain and occurs early in AD. Furthermore, in familial Alzheimer's disease (FAD), Aβ is considered to be a causative agent of AD (amyloid hypothesis) because the production of highly-aggregated Aβ42 is increased by mutations in the pathogenic genes APP and presenilin (PS). It is regarded as a promising therapeutic target for radical treatment of AD (Non-patent Document 1).

Aβ peptide production can be suppressed by inhibiting γ-secretase, the responsible enzyme responsible for the final cleavage. However, γ-secretase inhibitors that have been reported so far also inhibit cleavage of Notch, 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). On the other hand, when an abnormality occurs in a 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 oncogene, and it is also known that regulating the activity of γ-secretase that cleaves Notch has an anticancer effect (Non-patent Document 3).
In recent years, it has been reported that non-steroidal anti-inflammatory drugs (NSAIDs) exhibit specific inhibitory ability (γ-secretase modulator activity) for the production of Aβ42 and are being developed as AD therapeutics (Non-patent Document 4). . In this way, it is predicted that a compound or γ-secretase modulator that inhibits the activity of γ-secretase specifically in the brain can be an ideal therapeutic drug that avoids peripheral side effects in the future.

γ-secretase is a transmembrane aspartic protease composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (Non-patent Document 5). γ-secretase produces Aβ40 and Aβ42, but the detailed control mechanism of Aβ peptide production is not clear. Recently, TMP21 was identified as a molecule that binds to γ-secretase and controls its activity (Non-patent Document 6).
On the other hand, it has been reported that when a gene encoding a membrane-bound protein (ATP2A2, FLOT2) is knocked down by the RNAi method, the production of Aβ decreases (Non-patent Documents 7 and 8).
Furthermore, the present inventors have identified a plurality of factors that bind to γ-secretase and control the production of Aβ (Patent Document 1).

Mattson, M.P., Nature (2004) 430, 631-639 Wong, G.T. et al., J. Biol. Chem. (2004) 279, 12876 Purow B., Curr. Pharm. Biotechnol. (2009) 10 (2): 154-60 Kukar, T., Nat. Med. (2005) 11, 545-50 Haass, C., EMBO J. (2004) 23, 483-488 Chen, F. et al., Nature (2006) 440, 1208-12 Green, K.N. et al., J. Cell Biol. (2008) 181, 1107-1116 Schneider, A. et al., J. Neurosci. (2008) 28, 2874-2882

International Publication No. 2010/140694

  There is a strong need for the development of effective AD therapeutics. An object of the present invention is to identify a novel factor that binds to γ-secretase and promotes / enhances its activity, and to suppress the expression and function of the factor, a γ-secretase inhibitor or modulator, Aβ 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 is a molecule that binds to γ-secretase and controls Aβ production in brain tissue, and used an inhibitor pull-down method (TOrAC method) (International Publication No. 2010/053115). Thus, a protein that binds to γ-secretase was searched from human and rat brain, and a plurality of novel γ-secretase binding factors expressed in the brain were found. Furthermore, in the human embryonic kidney cell line (HEK-APP) overexpressing APP, which is a precursor protein of Aβ, expression of these binding factors was knocked down by RNAi method, and the effect on Aβ production ability was determined by ELISA method. evaluated. As a result, with respect to 12 factors whose relationship with γ-secretase activity was not known, knowledge was obtained that the production of Aβ can be specifically suppressed by reducing their expression level, and the present invention is completed. It came to.

That is, the present invention is as follows.
[1] The following proteins:
(P1) F-box only protein 2;
(P2) 14-3-3 protein eta;
(P3) 60S ribosomal protein L22;
(P4) Endoplasmin;
(P5) Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2;
(P6) Prostaglandin E synthase 3;
(P7) Heat shock protein HSP 90-alpha;
(P8) Protein disulfide-isomerase;
(P9) Cytochrome c;
(P10) Phospholipid scramblase 3;
(P11) Vesicle-associated membrane protein 1; and
(P12) Tetraspanin-2;
An Aβ production inhibitor comprising one or more substances that suppress the expression or function of a protein Pn selected from (n is an integer of 1 to 12).
[2] The agent according to [1] above, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) FBXO2;
(G2) YWHAH;
(G3) RPL22;
(G4) HSP90B1;
(G5) GNG2;
(G6) PTGES3;
(G7) HSP90AA1;
(G8) P4HB;
(G9) CYCS;
(G10) PLSCR3;
(G11) VAMP1; or
(G12) TSPAN2;
(B) a ribozyme nucleic acid against the transcription product of gene Gn (c) a nucleic acid having RNAi activity against the transcription product of gene Gn or a precursor thereof [3] a substance that suppresses the function of protein Pn The agent according to [1] above, which is a substance selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase [4] Aβ is Aβ40 or Aβ42 [1] The agent according to any one of [3].
[5] The agent according to any one of [1] to [4] above, for treating or preventing Alzheimer's disease or cancer.
[6] In mammals, the following proteins:
(P1) F-box only protein 2;
(P2) 14-3-3 protein eta;
(P3) 60S ribosomal protein L22;
(P4) Endoplasmin;
(P5) Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2;
(P6) Prostaglandin E synthase 3;
(P7) Heat shock protein HSP 90-alpha;
(P8) Protein disulfide-isomerase;
(P9) Cytochrome c;
(P10) Phospholipid scramblase 3;
(P11) Vesicle-associated membrane protein 1; and
(P12) Tetraspanin-2;
A method for inhibiting Aβ production in the mammal, comprising inhibiting the expression or function of a protein Pn selected from (n is an integer of 1 to 12).
[7] The method of [6] above, comprising administering to the mammal an effective amount of one or more substances that suppress the expression or function of protein Pn.
[8] The method according to [7] above, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) FBXO2;
(G2) YWHAH;
(G3) RPL22;
(G4) HSP90B1;
(G5) GNG2;
(G6) PTGES3;
(G7) HSP90AA1;
(G8) P4HB;
(G9) CYCS;
(G10) PLSCR3;
(G11) VAMP1; or
(G12) TSPAN2;
An antisense nucleic acid against the transcription product of (b) a ribozyme nucleic acid against the transcription product of gene Gn (c) a nucleic acid having RNAi activity against the transcription product of gene Gn or a precursor thereof [9] a substance that suppresses the function of protein Pn The method according to [7] above, which is a substance selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase [10] Aβ is Aβ40 or Aβ42 [6] The method according to any one of [9].
[11] The method according to any one of [6] to [10] above, for treating or preventing Alzheimer's disease or cancer.
[12] The method comprises selecting a substance that reduces the expression level of at least one gene selected from (G1) to (G12) or the activity of at least one protein selected from (P1) to (P12). A method for screening an Aβ production inhibitor.
[13] A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) a reporter gene under the control of the transcriptional regulatory region of (a1) gene Gn (n is any integer from 1 to 12) or (a2) gene Gn (n is any integer from 1 to 12) 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 And [14] A screening method for an Aβ production inhibitory substance, which includes the following steps (a) to (c): selecting a substance that reduces the expression level as a candidate for an Aβ production inhibitory substance.
(A) contacting the cell expressing amyloid precursor protein (APP) with at least one protein selected from (P1) to (P12) 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 case where it is measured in the absence of a test substance [15] The protein is provided by the cell itself, [14] the method of.
[16] The method according to [14] or [15] above, wherein the activity of the protein is measured using the production amount of Aβ as an index.
[17] A method for screening an Aβ production inhibitor comprising the following steps (a) to (c):
(A) a step of contacting at least one protein selected from (P1) to (P12) and a test substance with a cell expressing γ-secretase or a cell membrane fraction thereof; and (b) measuring γ-secretase activity. Step (c) selecting a substance that reduces or modulates γ-secretase activity compared to when measured in the absence of a test substance [18] The protein is provided by the cell itself, The method according to [17].
[19] A method for screening an Aβ production inhibitor, which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer of 1 to 12) and γ-secretase.
[20] A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) a step of bringing protein Pn (n is an integer from 1 to 12) and γ-secretase into contact with a test substance (b) a step of measuring the binding activity of protein Pn and γ-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 (G12) 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.
[22] 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 (G12) has been introduced; (b) at least one expression reflecting the pathology 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 the amount of Aβ 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].
[24] 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 test animal-derived sample (b) Expression level of at least one gene selected from (G1) to (G12) in the sample or selected from (P1) to (P12) 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

  According to the present invention, by using a novel protein that regulates the activity of γ-secretase, it becomes possible to screen for compounds that inhibit the production of Aβ 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.

It is a figure which shows the structure of Affigel-DAPT + Cholesterol resin. It is a figure which shows that the known component of (gamma) -secretase was pulled down using Affigel-DAPT + Cholesterol resin. It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to HEK-APP cell (introduction siRNA amount: 0.1pmol / well).

The present invention includes the following proteins:
(P1) F-box only protein 2;
(P2) 14-3-3 protein eta;
(P3) 60S ribosomal protein L22;
(P4) Endoplasmin;
(P5) Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2;
(P6) Prostaglandin E synthase 3;
(P7) Heat shock protein HSP 90-alpha;
(P8) Protein disulfide-isomerase;
(P9) Cytochrome c;
(P10) Phospholipid scramblase 3;
(P11) Vesicle-associated membrane protein 1; and
(P12) Tetraspanin-2;
An Aβ production inhibitor comprising a substance that suppresses the expression of a protein Pn selected from (n is an integer of 1 to 12) or a substance that suppresses a function is provided.
Protein Pn [(P1) F-box only protein 2; (P2) 14-3-3 protein eta; (P3) 60S ribosomal protein L22; (P4) Endoplasmin; (P5) Guanine nucleotide-binding protein G ( I) / G (S) / G (O) subunit gamma-2; (P6) Prostaglandin E synthase 3; (P7) Heat shock protein HSP 90-alpha; (P8) Protein disulfide-isomerase; (P9) Cytochrome c; (P10) Phospholipid scramblase 3; (P11) Vesicle-associated membrane protein 1; and (P12) Tetraspanin-2;] is an amino acid sequence represented by SEQ ID NO: 2n (n is an integer from 1 to 12) Is a protein comprising the same or substantially the same amino acid sequence. In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
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 β 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 or 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, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (eg, smooth muscle, Skeletal muscle), lungs, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, White adipose tissue, brown adipose tissue), etc.], etc.] may be isolated and purified by protein separation and purification techniques known per se.

“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 orthologue splice variant, allelic variant or polymorphism of (c) above.

  As used herein, “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 percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "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) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  The homology of amino acid sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). 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 are NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program It includes built-in 'or the like, may they likewise preferably used.

In the above (a), more preferably, 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.

Here, “activity” refers to an activity that promotes the production of Aβ. Further, “substantially the same quality” means that the properties are qualitatively the same, for example, physiologically or pharmacologically. Therefore, it is preferable that the Aβ production promoting activity is 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.
Aβ production promoting activity can be measured by measuring and comparing the amount of Aβ produced (preferably the amount of Aβ40 or Aβ42 produced) in the presence and absence of a protein according to a method known per se. .

Further, as 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 in the amino acid sequence represented by SEQ ID NO: 2n, preferably 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 in 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 Number: 1 to 50 in the amino acid sequence represented by 2n, preferably 1 to 30, more preferably 1 to 10, more preferably It is also included so-called muteins such as proteins containing 1 to several (5, 4, 3, or 2) the amino acid sequence number of amino acids are substituted with other amino acids, or (v) amino acid sequence comprising a combination thereof.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the Aβ production promoting activity is retained.

  Preferred examples of the protein P1 (F-box only protein 2) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (RefSeq No. NP_036300), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_789818), chimpanzees (RefSeq No. XP_514389), dogs (RefSeq No. XP_535406), rats (RefSeq No. NP_445963), cattle (RefSeq No. NP_001069037), etc.) and their splice variants, Examples include allelic variants and polymorphisms (eg, refSNP No. rs75074481 (Val / Leu), rs9614 (Lys / Thr)).

  As a preferable example of protein P2 (14-3-3 protein eta), for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 4 (RefSeq No. NP_003396), or an ortholog thereof in other mammals ( For example, mouse (RefSeq No. NP_035868), rat (RefSeq No. NP_037184), cow (RefSeq No. NP_776917), chimpanzee (RefSeq No. XP_001151357), etc., and their splice variants (for example, RefSeq Nos. XP_001151423, XP_001150958, XP_001151164, XP_001151028, XP_001151226, XP_515092, XP_001151095, etc.), allelic variants, polymorphisms and the like.

  Preferred examples of the protein P3 (60S ribosomal protein L22) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 6 (RefSeq No. NP_000974), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_033105), rats (RefSeq No. NP_112366), dogs (RefSeq No. XP_536725) etc., and their splice variants (eg RefSeq No. XP_849312 etc.), allelic variants, polymorphisms, etc. It is done.

  Preferred examples of protein P4 (Endoplasmin) include, for example, a human protein (RefSeq No. NP_003290) consisting of the amino acid sequence represented by SEQ ID NO: 8, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals. NP_035761), rats (RefSeq No. NP_001012197), cattle (RefSeq No. NP_777125), chimpanzees (RefSeq No. XP_001158681), dogs (RefSeq No. NP_001003327), and their splice variants (eg, RefSeq Nos. XP_001158799, XP_001158841, XP_001158740, XP_001157342, XP_001158377, etc.), allelic variants, polymorphisms (for example, refSNP No. rs34482425 (Lys / Asn), etc.).

  Preferred examples of protein P5 (Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 10 ( RefSeq No. NP_444292), or their orthologs in other mammals (eg, mouse (RefSeq No. NP_034445), rat (RefSeq No. XP_002725075), dog (RefSeq No. XP_853464), chimpanzee (RefSeq No. XP_522854), Bovine (RefSeq No. NP_776497), and their splice variants (for example, RefSeq Nos. XP_001158267, XP_001158222, XP_001158380, XP_001158320, NP_001033726, XP_002725074, etc.), allelic variants, polymorphisms and the like.

  Preferred examples of the protein P6 (Prostaglandin E synthase 3) include, for example, a human protein (RefSeq No. NP_006592) consisting of the amino acid sequence represented by SEQ ID NO: 12, or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_062740), rat (RefSeq No. NP_001014294), cattle (RefSeq No. NP_001007807), chimpanzee (RefSeq No. XP_001159085), dog (RefSeq No. XP_848910), etc.) and their splice variants (for example, refSNP No. XP_001159134 etc.), allelic variants, polymorphisms and the like.

  As a preferable example of the protein P7 (Heat shock protein HSP 90-alpha), for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 14 (RefSeq No. NP_001017963), or their orthologs in other mammals ( For example, mice (RefSeq No. NP_034610), rats (RefSeq No. NP_786937), cattle (RefSeq No. NP_001012688), chimpanzees (RefSeq No. NP_001092042), dogs (RefSeq No. XP_537557), and their splice variants (For example, RefSeq Nos. NP_005339, XP_868585, XP_868588, etc.), allelic variants, polymorphisms (for example, refSNP No. rs8005905 (Met / Leu), rs35446359 (Gln / Arg, etc.)) and the like.

  Preferred examples of protein P8 (Protein disulfide-isomerase) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 16 (RefSeq No. NP_000909), or an ortholog thereof in other mammals (eg, mouse) (RefSeq No. NP_035162), rat (RefSeq No. NP_037130), cattle (RefSeq No. NP_776560), chimpanzee (RefSeq No. XP_001164396), dog (RefSeq No. XP_540488), etc.) and their splice variants (for example, RefSeq Nos. XP_001164327, XP_001164362, XP_511745, XP_001164216, XP_001164286, XP_001164146, XP_001164258, etc.), allelic variants, polymorphisms (for example, refSNP No. rs62077233 (His / Arg, etc.)).

  Preferred examples of the protein P9 (Cytochrome c) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 18 (RefSeq No. NP_061820), or an ortholog thereof (eg, mouse (RefSeq) in other mammals). No. NP_031834), rat (RefSeq No. NP_036971), cattle (RefSeq No. NP_001039526), chimpanzee (RefSeq No. NP_001065289), dog (RefSeq No. NP_001183974), etc.), and their splice variants, allelic variants, Polymorphism (for example, refSNP No. rs11548795 (Lys / Arg) etc.) and the like.

  Preferred examples of the protein P10 (Phospholipid scramblase 3) include, for example, a human protein (RefSeq No. NP_065093) consisting of the amino acid sequence represented by SEQ ID NO: 20, or an ortholog (eg, mouse ( RefSeq No. NP_076053), rat (RefSeq No. NP_001012139), cattle (RefSeq No. NP_001039518), chimpanzee (RefSeq No. XP_001174792), dogs (RefSeq No. XP_546589) etc.) and their splice variants (eg refSNP) No.NP_001161969), allelic variants, polymorphisms (eg, refSNP Nos. Rs3744549 (Val / lle), rs34961966 (Met / lle), etc.).

  Preferred examples of the protein P11 (Vesicle-associated membrane protein 1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_055046), or an ortholog thereof in other mammals (for example, , Rat (RefSeq No. NP_037222), cattle (RefSeq No. NP_001069098), chimpanzee (RefSeq No. XP_001169040), dog (RefSeq No. XP_543853), mouse (RefSeq No. NP_033522), etc., and their splice variants ( For example, RefSeq No. NP_058439, NP_954740, NP_001074026, etc.), allelic variants, polymorphisms and the like can be mentioned.

  Preferred examples of the protein P12 (Tetraspanin-2) include, for example, a human protein (RefSeq No. NP_005716) consisting of the amino acid sequence represented by SEQ ID NO: 24, or an ortholog thereof (for example, mouse ( RefSeq No. NP_081809), rat (RefSeq No. NP_072111), cattle (RefSeq No. NP_001029829), chimpanzee (RefSeq No. XP_513675), dog (RefSeq No. XP_848759 etc.), and their splice variants, allelic variants, Examples include polymorphisms (for example, refSNP No. rs34749181 (Val / Ala), rs9659602 (Arg / Leu, etc.)).

  In the present invention, 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, as a substance that suppresses the expression of protein Pn, for example, a substance that inhibits the transcription of gene Gn (eg, antigene), a substance that inhibits the processing of early transcription products into mRNA, and the transport of mRNA to the cytoplasm. Inhibiting substances, inhibiting translation from 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. However, a substance that binds complementarily to mRNA and inhibits translation into protein Pn or decomposes mRNA is preferable.

As a substance that specifically inhibits the translation of mRNA of gene Gn from protein Pn (or degrades mRNA), preferably 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 nucleotide sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 97% or more.

  The “base sequence homology” in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gaps; filtering = ON; It can be calculated by match score = 1; mismatch score = -3).

  More specifically, 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 an integer of 1 to 12) Complementary or substantially complementary nucleotide sequence to the nucleotide sequence represented, or (b) complementary strand of the nucleotide sequence represented by "SEQ ID NO: 2n-1 (n is any integer from 1 to 12)" Is a nucleotide 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 12) Examples of the sequence that encodes a protein include complementary or substantially complementary base sequences. Here, “substantially the same quality of activity” has the same meaning as described above.

  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, such as 6 × SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washings at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C., etc., those skilled in the art will be able to determine the hybridization conditions that give the same stringency. It can be selected appropriately.

  As a preferable example of mRNA of gene G1 (FBXO2), human FBXO2 (RefSeq Accession No. NM_012168) containing the nucleotide sequence represented by SEQ ID NO: 1, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_176848), chimpanzee (RefSeq No. XM_514389), dog (RefSeq No. XM_535406), rat (RefSeq No. NM_053511), bovine (RefSeq No. NM_001075569), etc.), and their splice variants, allelic variants, Examples thereof include mRNAs such as polymorphisms (for example, refSNP No. rs75074481 (G / C), rs9614 (A / C), etc.).

  Preferred examples of mRNA of gene G2 (YWHAH) include, for example, human YWHAH (RefSeq Accession No. NM_003405) consisting of a base acid sequence represented by SEQ ID NO: 3, or their orthologs in other mammals (for example, Mice (RefSeq No. NM_011738), rats (RefSeq No. NM_013052), cattle (RefSeq No. NM_174492), chimpanzees (RefSeq No. XM_001151357), and their splice variants (eg, RefSeq No. XM_001151423, XM_001150958, XM — 001151164, XM — 001151028, XM — 001151226, XM — 515092, XM — 001151095, etc.), allelic variants, and polymorphisms.

  Preferred examples of mRNA of gene G3 (RPL22) include, for example, human RPL22 (RefSeq Accession No. NM_000983) consisting of the base sequence represented by SEQ ID NO: 5, or their orthologs (for example, mice) in other mammals (RefSeq No. NM_009079), rat (RefSeq No. NM_031104), dog (RefSeq No. XM_536725), etc., as well as their splice variants (eg, RefSeq No. XM_844219 etc.), allelic variants, polymorphic mRNAs Can be given.

  Preferred examples of mRNA of gene G4 (HSP90B1) include, for example, human HSP90B1 (RefSeq Accession No. NM_003299) consisting of the base sequence represented by SEQ ID NO: 7, or their orthologs (for example, mouse) in other mammals (RefSeq No. NM_011631), rat (RefSeq No. NM_001012197), cattle (RefSeq No. NM_174700), chimpanzee (RefSeq No. XM_001158681), dogs (RefSeq No. NM_001003327), etc.) and their splice variants (for example, RefSeq No. XM_001158799, XM_001158841, XM_001158740, XM_001157342, XM_001158377, etc.), allelic variants, and polymorphisms (for example, refSNP No. rs34482425 (Lys / Asn) etc.).

  Preferred examples of mRNA of gene G5 (GNG2) include, for example, human GNG2 (RefSeq Accession No. NM_053064) consisting of the base sequence represented by SEQ ID NO: 9, or an ortholog thereof (eg, mouse) in other mammals (RefSeq No. NM_010315), rat (RefSeq No. XM_002725029), dog (RefSeq No. XM_848371), cow (RefSeq No. NM_174072), chimpanzee (RefSeq No. XM_522854), etc., and their splice variants (for example, RefSeq No. XM — 001158267, XM — 001158222, XM — 001158380, XM — 001158320, NM — 001038637, XM — 002725028, etc.), allelic variants, and polymorphisms.

  Preferred examples of mRNA of the gene G6 (PTGES3) include, for example, human PTGES3 (RefSeq Accession No. NM_006601) consisting of the base sequence represented by SEQ ID NO: 11, or their orthologs in other mammals (eg, mouse) (RefSeq No. NM_019766), rat (RefSeq No. NM_001014272), cow (RefSeq No. NM_001007806), chimpanzee (RefSeq No. XM_001159085), dog (RefSeq No. XM_843817), etc.) and their splice variants (for example, RefSeq No. XM — 001159134, etc.), allelic mutants, polymorphic mRNAs.

  Preferred examples of mRNA of the gene G7 (HSP90AA1) include, for example, human HSP90AA1 (RefSeq Accession No. NM_001017963) consisting of the base sequence represented by SEQ ID NO: 13, or those orthologs (for example, mice) in other mammals (RefSeq No. NM_010480), rat (RefSeq No. NM_175761), cow (RefSeq No. NM_001012670), chimpanzee (RefSeq No. NM_001098572), dog (RefSeq No. XM_537557), etc., and their splice variants (for example, RefSeq No. NM — 005348, XM — 863492, XM — 863495, etc.), allelic variants, polymorphisms (eg, refSNP No. rs8005905 (Met / Leu), rs35446359 (Gln / Arg, etc.)) and the like.

  Preferred examples of mRNA of the gene G8 (P4HB) include, for example, human P4HB (RefSeq Accession No. NM_000918) consisting of the base sequence represented by SEQ ID NO: 15, or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NM_011032), rat (RefSeq No. NM_012998), cattle (RefSeq No. NM_174135), chimpanzee (RefSeq No. XM_001164396), dog (RefSeq No. XM_540488), etc.) and their splice variants (for example, RefSeq No. XM — 001164327, XM — 001164362, XM — 511745, XM — 001164216, XM — 001164286, XM — 001164146, XM — 001164258, etc.), allelic variants, and polymorphisms (eg, refSNP No. rs62077233 (His / Arg), etc.).

  Preferred examples of mRNA of the gene G9 (CYCS) include, for example, human CYCS (RefSeq Accession No. NM_018947) consisting of the base sequence represented by SEQ ID NO: 17, or their orthologs in other mammals (eg, mouse) (RefSeq No. NM_007808), rat (RefSeq No. NM_012839), cattle (RefSeq No. NM_001046061), chimpanzee (RefSeq No. NM_001071821), dog (RefSeq No. NM_001197045), etc.) and their splice variants and allelic variants Body and polymorphism (for example, refSNP No. rs11548795 (Lys / Arg) etc.).

  Preferred examples of mRNA of gene G10 (PLSCR3) include, for example, human PLSCR3 (RefSeq Accession No. NM_020360) consisting of the base sequence represented by SEQ ID NO: 19, or their orthologs (eg, mouse) in other mammals (RefSeq No. NM_023564), rat (RefSeq No. NM_001012139), bovine (RefSeq No. NM_001046053), chimpanzee (RefSeq No. XM_001174792), dog (RefSeq No. XM_546589), etc.) and their splice variants (for example, RefSeq No. NM_001168497 etc.), allelic variants, and polymorphisms (for example, refSNP No. rs3744549 (Val / lle), rs34961966 (Met / lle), etc.) and the like.

  Preferred examples of mRNA of the gene G11 (VAMP1) include human VAMP1 (RefSeq Accession No. NM_014231) containing the base sequence represented by SEQ ID NO: 21, or their orthologs in other mammals (eg, rat (RefSeq No. NM_013090), cattle (RefSeq No. NM_001075630), chimpanzee (RefSeq No. XM_001169040), dogs (RefSeq No. XM_543853), mice (RefSeq No. NM_009496) etc., and their splice variants (eg RefSeq No NM_016830, NM_199245, NM_001080557, etc.), allelic variants, and polymorphisms.

  As a preferable example of mRNA of gene G12 (TSPAN2), human TSPAN2 (RefSeq Accession No. NM_005725) containing the nucleotide sequence represented by SEQ ID NO: 23, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_027533), rat (RefSeq No. NM_022589), cattle (RefSeq No. NM_001034657), chimpanzee (RefSeq No. XM_513675), dog (RefSeq No. XM_843666 etc.), and their splice variants, allelic variants, many Examples thereof include mRNA such as refSNP Nors34749181 (Val / Ala), rs9659602 (Arg / Leu) and the like.

  “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.

Specifically, a nucleic acid containing 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 mRNA of gene Gn
(c) a nucleic acid having RNAi activity against the mRNA of gene Gn or a precursor thereof

(a) Antisense nucleic acid against mRNA of gene Gn In the present invention, 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). They may be 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), chelate compound (eg, metal, having radioactivity) It may be one containing a metal, boron, oxidizing metal, etc., one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid etc.). Here, the “nucleoside”, “nucleotide” and “nucleic acid” 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, for example, 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.

  As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by the 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. In view of easiness of synthesis, antigenicity, intracellular migration, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. Specifically, 5 'end hairpin loop of gene Gn, 5' end 6-base pair repeat, 5 'end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region , 3 ′ end palindromic region or 3 ′ end hairpin loop, etc. may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.

  Furthermore, 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. For example, in order to prevent degradation by a hydrolase such as nuclease, 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. In addition, 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. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

  The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by cross-linking 2 'oxygen and 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleic Acids , 22, 1619-21, 2003) can also be preferably used.

  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. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.

(b) 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 gene Gn mRNA, or a part thereof, the mRNA is used as a coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage 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 several bases at both ends (about 10 bases in total) adjacent to the part having 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. When the mRNA of gene Gn has a double-stranded structure by itself, 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. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

(c) siRNA or miRNA for gene Gn mRNA
In the present specification, 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 nucleotide sequence of the gene Gn mRNA. Defined as encompassed by nucleic acid containing base sequence or part thereof. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to the RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon was confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.

The siRNA can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). 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. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed. For example, when AA + (N) ₁₉ , AA + (N) ₂₁ or NA + (N) ₂₁ (N is an arbitrary base) is used as a 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. In addition, for short hairpin RNA (shRNA) which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.

The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and insert design tool for pSilencer ^™ 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 the above-described antisense nucleic acid in order to improve stability, specific activity and the like.

  siRNA is synthesized by each of the sense strand and antisense strand of the target sequence on the 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.

  In the present specification, a nucleic acid designed to generate an siRNA against the mRNA of the gene Gn in vivo is also a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the gene Gn mRNA. 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 a sense sequence and an antisense strand of a 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. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. Vectors expressing shRNA include tandem type and stem loop (hairpin) type. In the former, siRNA sense strand expression cassette and antisense strand expression cassette are linked in tandem, and each strand is expressed and annealed in the cell to form double stranded siRNA (dsRNA). It is. On the other hand, 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. As the promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but in order to accurately transcribe a short RNA, a pol III promoter is generally used. Examples of polIII promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoter, and human valine-tRNA promoter. Further, 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. As such vectors, virus vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, animal cell expression plasmids and the like are used.

  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. In this way, 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.). These can be attached to the 3 'or 5' end of nucleic acids and 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. Such 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 the proteins P1 to P12, 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.

  In the present invention, 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 Aβ production. For example, it binds to protein Pn. A substance that suppresses Aβ production, a substance that inhibits the binding activity between protein Pn and γ-secretase (for example, a substance that inhibits or modulates the promotion of γ-secretase activity due to the binding between protein Pn and γ-secretase) And substances that inhibit the binding or complex formation of protein Pn and γ-secretase), substances that inhibit the transfer of protein Pn to the cell membrane, and the like. Examples of the “substance that modulates γ-secretase activity” include substances that change the substrate selectivity of APP or Notch serving as a substrate for γ-secretase activity, or change the production ratio of Aβ species.

Specifically, 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 antibody or antiserum production methods. 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. In addition to a complete antibody molecule, for example, 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) They may be modified derivatives thereof.

  In a preferred embodiment, since an antibody against the protein Pn is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal antibody) has a reduced risk of being antigenic when administered to a human. An antibody, specifically a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. In addition, a fully human antibody can be produced from a human-human (or mouse) hybridoma, but in order to provide a large amount of antibody stably and at low cost, a human antibody-producing mouse or a phage display method is used. It is desirable to manufacture using.

  Since protein Pn plays an important role in the production of Aβ peptide via γ-secretase, the substance that suppresses the function of protein Pn is excellent in brain migration (blood-brain barrier permeability) and cell membrane permeability. Desirable material. Therefore, a more preferable substance that suppresses the function of protein Pn is a low molecular weight compound that meets 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 P12, or 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 Aβ production, and thus is useful for improving the pathology of Alzheimer's disease (AD) or cancer. In addition, since the substance may not inhibit Notch protein cleavage, it has an additional advantageous effect that it has a low risk of causing peripheral side effects such as intestinal epithelialization disorders and immune abnormalities.

Therefore, a drug containing a substance that suppresses the expression or function of protein Pn is a γ-secretase inhibitor or modulator, an Aβ production inhibitor, a disease involving Aβ (eg, AD), or a disease involving Notch protein cleavage. It can be used as a prophylactic 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. When two or more kinds of substances that suppress the expression or function of protein Pn are 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.
In addition, 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. When two or more kinds of substances that suppress the expression or function of protein Pn are formulated as separate medicaments, the respective preparations may be administered simultaneously or at intervals. Moreover, the administration route may be the same or different. The dosage described below indicates the dosage of one substance that suppresses the expression or function of protein Pn, but even in the case of using a combination of two or more substances, it does not adversely affect the administration subject. Similar dosages can be used for each substance.
In the case of using a combination of two or more substances that suppress the expression or function of protein Pn, the combination of the two or more different proteins may be any two or more proteins selected from proteins P1 to P12. Is mentioned. Alternatively, any one or more proteins selected from the proteins P1 to P12 and the following proteins disclosed in WO 2010/140694:
Probable phospholipid-transporting ATPase IIA (gene name: ATP9A);
BDNF / NT-3 growth factors receptor precursor (gene name: NTRK2);
ELAV-like protein 4 (gene name: ELAVL4);
Coiled-coil domain-containing protein 136 (gene name: NAG6);
Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (gene name: HCN2);
DnaJ homolog subfamily A member 2 (gene name: DNAJA2);
Vesicle-associated membrane protein-associated protein A (gene name: VAPA);
Proton myo-inositol cotransporter (gene name: SLC2A13);
Leukocyte surface antigen CD47 (gene: CD47);
Flotillin-1 (gene name: FLOT1);
Band 4.1 like 1 (gene name: EPB41L1);
Regulator of G-protein signaling 7 (gene name: RGS7);
Phospholipase D3 (gene name: PLD3);
Ectoderm-neural cortex protein 1 (gene name: ENC1);
synaptophysin (gene name: SYP);
solute carrier family 2 (facilitated glucose transporter), member 3 (gene name: SLC2A3);
syntaxin binding protein 1 (gene name: STXBP1);
growth associated protein 43 (gene name: GAP43);
ATPase, Na + / K + transporting, beta 1 polypeptide (gene name: ATP1B1);
Amine oxidase [flavin-containing] B (gene name: MAOB);
Muscarinic acetylcholine receptor M1 (gene name: CHRM1);
Probable compress amino acid transporter (gene name: SLC7A14);
NADH dehydrogenase iron-sulfur protein 7 (gene name: NDUFS7);
G-protein-regulated inducer of neurite outgrowth (gene name: GPRIN1); and
Probable phospholipid-transporting ATPase 1A (gene name: ATP8A1);
A combination of any one or more proteins selected from may be used. Alternatively, a pharmaceutical composition containing two or more substances that suppress the expression or function of any two or more proteins selected from the 25 types of proteins disclosed in WO 2010/140694. It is also included in the present invention.
For example, examples of the combination of the two proteins include the following. Hereinafter, for convenience, each protein is represented by the corresponding gene name, and the two combinations are represented by “first protein-second protein”.
YWHAH-FBXO2, RPL22-FBXO2, RPL22-YWHAH, HSP90B1-FBXO2, HSP90B1-YWHAH, HSP90B1-RPL22, GNG2-FBXO2, GNG2-YWHAH, GNG2-RPL22, GNG2-HSP90B1, PTGES3-FBXO2, PTGES3-Y RPL22, PTGES3-HSP90B1, PTGES3-GNG2, HSP90AA1-FBXO2, HSP90AA1-YWHAH, HSP90AA1-RPL22, HSP90AA1-HSP90B1, HSP90AA1-GNG2, HSP90AA1-PTGES3, P4HB-FBXO2, P4HB, P4HB, P4HB, H4B P4HB-GNG2, P4HB-PTGES3, P4HB-HSP90AA1, CYCS-FBXO2, CYCS-YWHAH, CYCS-RPL22, CYCS-HSP90B1, CYCS-GNG2, CYCS-PTGES3, CYCS-HSP90AA1, CYCS-P4FBX2, PL YWHAH, PLSCR3-RPL22, PLSCR3-HSP90B1, PLSCR3-GNG2, PLSCR3-PTGES3, PLSCR3-HSP90AA1, PLSCR3-P4HB, PLSCR3-CYCS, VAMP1-FBXO2, VAMP1-YWHAH, VAMP1-RPL22, VAMP1-HSP90B1, VAMP1-GNG2, VAMP1-PTGES3, VAMP1-HSP90AA1, VAMP1-P4HB, VAMP1-CYCS, VAMP1-PLSCR3, TSPAN2-FBXO2, TSPAN2-YWHAH, TSPAN2-RPL22, TSPAN2-HSP90B1, TSPAN2-GNG2, TSPAN2-PTGES3, TSPAN2-HSP90AA1, TSPAN2- P4HB, TSPAN2-CYCS, TSPAN2-PLSCR3, TSPAN2-VAMP1, ATP9A-FBXO2, ATP9A-YWHAH, ATP9A-RPL22, ATP9A-HSP90B1, ATP9A-GNG2, ATP9A-PTGES3, ATP9A-H SP90AA1, ATP9A-P4HB, ATP9A-CYCS, ATP9A-PLSCR3, ATP9A-VAMP1, ATP9A-TSPAN2, NTRK2-FBXO2, NTRK2-YWHAH, NTRK2-RPL22, NTRK2-HSP90B1, NTRK2-PTGES3, NTRK2-HSP903, NTRK2-HSPAA NTRK2-P4HB, NTRK2-CYCS, NTRK2-PLSCR3, NTRK2-VAMP1, NTRK2-TSPAN2, NTRK2-ATP9A, Elavl4-FBXO2, Elavl4-YWHAH, Elavl4-RPL22, Elavl4-HSP90B1, Elavl4-GNG2, Elavl4-PTGES3, Elavl4- HSP90AA1, Elavl4-P4HB, Elavl4-CYCS, Elavl4-PLSCR3, Elavl4-VAMP1, Elavl4-TSPAN2, Elavl4-ATP9A, Elavl4-NTRK2, NAG6-FBXO2, NAG6-YWHAH, NAG6-RPL22, NAG6-HSP90B1, NAG6-GNG2, NAG6-PTGES3, NAG6-HSP90AA1, NAG6-P4HB, NAG6-CYCS, NAG6-PLSCR3, NAG6-VAMP1, NAG6-TSPAN2, NAG6-ATP9A, NAG6-NTRK2, NAG6-Elavl4, Hcn2-FBXO2, Hcn2-YWHAH, Hcn2- RPL22, Hcn2-HSP90B1, Hcn2-GNG2, Hcn2-PTGES3, Hcn2-HSP90AA1, Hcn2-P4HB, Hcn2-CYCS, Hcn2-PLSCR3, Hcn2-VAMP1, Hcn2-TSPAN2, Hcn2-ATP9A, Hcn2-NTRK2, Hcn2-Elavl4, Hcn2-NAG6, Dnaja2-FBXO2, Dnaja2-YWHAH, Dnaja2-RPL22, Dnaja2-HSP90B1, Dnaja2-GNG2, Dnaja2-PTGES3, Dnaja2-HSP90AA1, Dnaja2-P4HB, Dnaja2-CYCS, Dnaja2-PLSCR3, Dnaja2-VAMP1, Dnaja2-VAMP1 TSP AN2, Dnaja2-ATP9A, Dnaja2-NTRK2, Dnaja2-Elavl4, Dnaja2-NAG6, Dnaja2-Hcn2, VAPA-FBXO2, VAPA-YWHAH, VAPA-RPL22, VAPA-HSP90B1, VAPA-GNG2, VAPA-PTGES3, VAPA-SP VAPA-P4HB, VAPA-CYCS, VAPA-PLSCR3, VAPA-VAMP1, VAPA-TSPAN2, VAPA-ATP9A, VAPA-NTRK2, VAPA-Elavl4, VAPA-NAG6, VAPA-Hcn2, VAPA-Dnaja2, SLC2A13-FBXO2-LC YWHAH, SLC2A13-RPL22, SLC2A13-HSP90B1, SLC2A13-GNG2, SLC2A13-PTGES3, SLC2A13-HSP90AA1, SLC2A13-P4HB, SLC2A13-CYCS, SLC2A13-PLSCR3, SLC2A13-VTPALC3T SLC2A13-Elavl4, SLC2A13-NAG6, SLC2A13-Hcn2, SLC2A13-Dnaja2, SLC2A13-VAPA, CD47-FBXO2, CD47-YWHAH, CD47-RPL22, CD47-HSP90B1, CD47-GNG2, CD47-PTGES3, CD47-SPGE903, CD47-SPGE90 P4HB, CD47-CYCS, CD47-PLSCR3, CD47-VAMP1, CD47-TSPAN2, CD47-ATP9A, CD47-NTRK2, CD47-Elavl4, CD47-NAG6, CD47-Hcn2, CD47-Dnaja2, CD47-VAPA, CD47-SLC2A13, FLOT1-FBXO2, FLOT1-YWHAH, FLOT1-RPL22, FLOT1-HSP90B1, FLOT1-GNG2, FLOT1-PTGES3, FLOT1-HSP90AA1, FLOT1-P4HB, FLOT1-CYCS, FLOT1-PLSCR3, FLOT1-VAMP1, FLOT1-TSPAN2, FLOT1- ATP 9A, FLOT1-NTRK2, FLOT1-Elavl4, FLOT1-NAG6, FLOT1-Hcn2, FLOT1-Dnaja2, FLOT1-VAPA, FLOT1-SLC2A13, FLOT1-CD47, ATP2A2-FBXO2, ATP2A2-YWHAH, ATP2A2-RPL22, ATP2A2-HSP90B1, ATP2A2-GNG2, ATP2A2-PTGES3, ATP2A2-HSP90AA1, ATP2A2-P4HB, ATP2A2-CYCS, ATP2A2-PLSCR3, ATP2A2-VAMP1, ATP2A2-TSPAN2, ATP2A2-ATP9A, ATP2A2-NTRK2, ATP2A2-GTP2 Hcn2, ATP2A2-Dnaja2, ATP2A2-VAPA, ATP2A2-SLC2A13, ATP2A2-CD47, ATP2A2-FLOT1, FLOT2-FBXO2, FLOT2-YWHAH, FLOT2-RPL22, FLOT2-HSP90B1, FLOT2-PTGES3, FLOT2-HSP90AA FLOT2-P4HB, FLOT2-CYCS, FLOT2-PLSCR3, FLOT2-VAMP1, FLOT2-TSPAN2, FLOT2-ATP9A, FLOT2-NTRK2, FLOT2-Elavl4, FLOT2-NAG6, FLOT2-Hcn2, FLOT2-Dnaja2, FLOT2-VAPA, FLOT2- SLC2A13, FLOT2-CD47, FLOT2-FLOT1, FLOT2-ATP2A2, EPB41L1-FBXO2, EPB41L1-YWHAH, EPB41L1-RPL22, EPB41L1-HSP90B1, EPB41L1-GNG2, EPB41L1-PTGES3, EPB41L1-HSP90AA1, EPB41L1 EPB41L1-PLSCR3, EPB41L1-VAMP1, EPB41L1-TSPAN2, EPB41L1-ATP9A, EPB41L1-NTRK2, EPB41L1-Elavl4, EPB41L1-NAG6, EPB41L1-Hcn2, EPB41L1-Dnaja2, EPB41L1-VAPA, EPB41L1-SLC2A13, EPB41L1-CD47, EPB41L1-FLOT1, EPB41L1-ATP2A2, EPB41L1-FLOT2, RGS7-FBXO2, RGS7-YWHAH, RGS7-RPL22, RGS7-HSP90B1, RGS7-GGE2, RGS7-PT3 HSP90AA1, RGS7-P4HB, RGS7-CYCS, RGS7-PLSCR3, RGS7-VAMP1, RGS7-TSPAN2, RGS7-ATP9A, RGS7-NTRK2, RGS7-Elavl4, RGS7-NAG6, RGS7-Hcn2, RGS7-Dnaja2, RGS7-VAPA RGS7-SLC2A13, RGS7-CD47, RGS7-FLOT1, RGS7-ATP2A2, RGS7-FLOT2, RGS7-EPB41L1, PLD3-FBXO2, PLD3-YWHAH, PLD3-RPL22, PLD3-HSP90B1, PLD3-GNG2, PLD3-PTGES3, PLD3- HSP90AA1, PLD3-P4HB, PLD3-CYCS, PLD3-PLSCR3, PLD3-VAMP1, PLD3-TSPAN2, PLD3-ATP9A, PLD3-NTRK2, PLD3-Elavl4, PLD3-NAG6, PLD3-Hcn2, PLD3-Dnaja2, PLD3-VAPA, PLD3-SLC2A13, PLD3-CD47, PLD3-FLOT1, PLD3-ATP2A2, PLD3-FLOT2, PLD3-EPB41L1, PLD3-RGS7, ENC1-FBXO2, ENC1-YWHAH, ENC1-RPL22, ENC1-HSP90B1, ENC1-GNG2, ENC1- PTGES3, ENC1-HSP90AA1, ENC1-P4HB, ENC1-CYCS, ENC1-PLSCR3, ENC1-VAMP1, ENC1-TSPAN2, ENC1-ATP9A, ENC1-NTRK2, ENC1-Elavl4, ENC1-NAG6, ENC1-Hcn2, ENC1-Dnaja2, ENC1-VAPA, ENC1-SLC2A13, ENC1-CD47, ENC1-FLOT1, ENC1-ATP2A2, ENC1-FLOT2, ENC1-EPB41L1, ENC1-RGS7, ENC1-PLD3, SYP-FBXO2, SYP-YWHAH, SYP-RPL22, SYP-HSP90B1, SYP-GNG2, SYP-PTGES3, SYP-HSP90AA1, SYP-P4HB, SYP- CYCS, SYP-PLSCR3, SYP-VAMP1, SYP-TSPAN2, SYP-ATP9A, SYP-NTRK2, SYP-Elavl4, SYP-NAG6, SYP-Hcn2, SYP-Dnaja2, SYP-VAPA, SYP-SLC2A13, SYP-CD47, SYP-FLOT1, SYP-ATP2A2, SYP-FLOT2, SYP-EPB41L1, SYP-RGS7, SYP-PLD3, SYP-ENC1, SLC2A3-FBXO2, SLC2A3-YWHAH, SLC2A3-RPL22, SLC2A3-HSP90B1, SLC2A3-GNG2, SLC2A3- PTGES3, SLC2A3-HSP90AA1, SLC2A3-P4HB, SLC2A3-CYCS, SLC2A3-PLSCR3, SLC2A3-VAMP1, SLC2A3-TSPAN2, SLC2A3-ATP9A, SLC2A3-NTRK2, SLC2A3-Elavl4, SLC2A3-NAG3A2, SLC2A3-NAG3A2, SLC2A3-VAPA, SLC2A3-SLC2A13, SLC2A3-CD47, SLC2A3-FLOT1, SLC2A3-ATP2A2, SLC2A3-FLOT2, SLC2A3-EPB41L1, SLC2A3-RGS7, SLC2A3-PLD3, SLC2A3-ENP1, SLC2A3-BPF, STX2A3-BP YWHAH, STXBP1-RPL22, STXBP1-HSP90B1, STXBP1-GNG2, STXBP1-PTGES3, STXBP1-HSP90AA1, STXBP1-P4HB, STXBP1-CYCS, STXBP1-PLSCR3, STXBP1-VAMP1, STXBP1-TSPAN2, STXBP1-ATP9RK, STXBP1-ATP9RK STXBP1-Elavl4, STXBP1-NAG6, STXBP1-Hcn2, STXBP1-Dnaja2, STXBP1-VAPA, STXBP1-SLC2A13, STXBP1-CD47, STXBP1-FLOT1, STXBP1-ATP2A2, STXBP1-FLOT2, STXBP1-EPB41L1, STXBP1-RGS7, STXBP1-PLD3, STXBP1-ENC1, STXBP1-ENC1, SYP, STXBP1-SLC2A3, GAP43-FBXO2, GAP43-YWHAH, GAP43-RPL22, GAP43-HSP90B1, GAP43-GNG2, GAP43-PTGES3, GAP43-HSP90AA1, GAP43-P4HB, GAP43-CYCS, GAP43-PLSCRV1, GAP43-PLSCRVAMP GAP43-TSPAN2, GAP43-ATP9A, GAP43-NTRK2, GAP43-Elavl4, GAP43-NAG6, GAP43-Hcn2, GAP43-Dnaja2, GAP43-VAPA, GAP43-SLC2A13, GAP43-CD47, GAP43-FLOT1, GAP43-ATP2A2, GAP43-ATP2A2, GAP43 FLOT2, GAP43-EPB41L1, GAP43-RGS7, GAP43-PLD3, GAP43-ENC1, GAP43-SYP, GAP43-SLC2A3, GAP43-STXBP1, ATP1B1-FBXO2, ATP1B1-YWHAH, ATP1B1-RPL22, ATP1B1-TP1G1, ATP1B1-PTGES3, ATP1B1-HSP90AA1, ATP1B1-P4HB, ATP1B1-CYCS, ATP1B1-PLSCR3, ATP1B1-VAMP1, ATP1B1-TSPAN2, ATP1B1-ATP9A, ATP1B1-NTRK2, ATP1B1-Elavl4, ATP1B1-B6 Dnaja2, ATP1B1-VAPA, ATP1B1-SLC2A13, ATP1B1-CD47, ATP1B1-FLOT1, ATP1B1-ATP2A2, ATP1B1-FLOT2, ATP1B1-EPB41L1, ATP1B1-RGS7, ATP1B1-PLD3, ATP1B1-ENC1, ATP1B1-SYP, ATP1B1-SLC2A3, ATP1B1-STXBP1, ATP1B1-GAP43, MAOB-FBXO2, MAOB-YWHAH, MAOB-RPL22, MAOB-HSP90B1, MAOB-GNG2, MAOB-PTGES3, MAOB-HSP90AA1, MAOB-P4HB CYCS, MAOB-PLSCR3, MAOB-VAMP1, MAOB-TSPAN2, MAOB-ATP9A, MAOB-NTRK2, MAOB-Elavl4, MAOB-NAG6, MAOB-Hcn2, MAOB-Dnaja2, MAOB-VAPA, MAOB-SLC2A13, MAOB-CD47, MAOB-FLOT1, MAOB-ATP2A2, MAOB-FLOT2, MAOB-EPB41L1, MAOB-RGS7, MAOB-PLD3, MAOB-ENC1, MAOB-SYP, MAOB-SLC2A3, MAOB-STXBP1, MAOB-GAP43, MAOB-ATP1B1, CHRM1- FBXO2, CHRM1-YWHAH, CHRM1-RPL22, CHRM1-HSP90B1, CHRM1-GNG2, CHRM1-PTGES3, CHRM1-HSP90AA1, CHRM1-P4HB, CHRM1-CYCS, CHRM1-PLSCR3, CHRM1-VAMP1, CHRM1-TSPAN2, CHRM1-ATP9A, CHRM1-NTRK2, CHRM1-Elavl4, CHRM1-NAG6, CHRM1-Hcn2, CHRM1-Dnaja2, CHRM1-VAPA, CHRM1-SLC2A13, CHRM1-CD47, CHRM1-FLOT1, CHRM1-ATP2A2, CHRM1-FLOT2, CHRM1-EPB41L1, CHRM1- RGS7, CHRM1-PLD3, CHRM1-ENC1, CHRM1-SYP, CHRM1-SLC2A3, CHRM1-STXBP1, CHRM1-GAP43, CHRM1-ATP1B1, CHRM1-MAOB, SLC7A14-FBXO2, SLC7A14-YWHAH, SLC7A14-RPL22, SLC90A14H SLC7A14-GNG2, SLC7A14-PTGES3, SLC 7A14-HSP90AA1, SLC7A14-P4HB, SLC7A14-CYCS, SLC7A14-PLSCR3, SLC7A14-VAMP1, SLC7A14-TSPAN2, SLC7A14-ATP9A, SLC7A14-NTRK2, SLC7A14-Elavl4, SLC7A14-LC7ALC7ALC7A14 VAPA, SLC7A14-SLC2A13, SLC7A14-CD47, SLC7A14-FLOT1, SLC7A14-ATP2A2, SLC7A14-FLOT2, SLC7A14-EPB41L1, SLC7A14-RGS7, SLC7A14-PLD3, SLC7A14-SLC7A14-ENC1, SLC7A14-ENC1, ALC7 SLC7A14-GAP43, SLC7A14-ATP1B1, SLC7A14-MAOB, SLC7A14-CHRM1, NDUFS7-FBXO2, NDUFS7-YWHAH, NDUFS7-RPL22, NDUFS7-HSP90B1, NDUFS7-GNG2, NDUFS7NPT7HPT7H CYCS, NDUFS7-PLSCR3, NDUFS7-VAMP1, NDUFS7-TSPAN2, NDUFS7-ATP9A, NDUFS7-NTRK2, NDUFS7-Elavl4, NDUFS7-NAG6, NDUFS7-Hcn2, NDUFS7-Dnaja2, NDUFS7-VAPA, NDUFS7-LCA, NDUFS7-LC NDUFS7-FLOT1, NDUFS7-ATP2A2, NDUFS7-FLOT2, NDUFS7-EPB41L1, NDUFS7-RGS7, NDUFS7-PLD3, NDUFS7-ENC1, NDUFS7-SYP, NDUFS7-SLC2A3, NDUFS7-STXBP1, NDUFS7-B4343 MAOB, NDUFS7-CHRM1, NDUFS7-SLC7A14, GPRIN1-FBXO2, GPRIN1-YWHAH, GPRIN1-RPL22, GPRIN1-HSP90 B1, GPRIN1-GNG2, GPRIN1-PTGES3, GPRIN1-HSP90AA1, GPRIN1-P4HB, GPRIN1-CYCS, GPRIN1-PLSCR3, GPRIN1-VAMP1, GPRIN1-TSPAN2, GPRIN1-ATP9A, GPRIN1-NTRK2, GPRIN1-Elavl4, GPRIN1-NAG6 GPRIN1-Hcn2, GPRIN1-Dnaja2, GPRIN1-VAPA, GPRIN1-SLC2A13, GPRIN1-CD47, GPRIN1-FLOT1, GPRIN1-ATP2A2, GPRIN1-FLOT2, GPRIN1-EPB41L1, GPRIN1-RGS7, GPRIN1-PLD3, GPRIN1-ENC1, GPRIN1-ENC1, SYP, GPRIN1-SLC2A3, GPRIN1-STXBP1, GPRIN1-GAP43, GPRIN1-ATP1B1, GPRIN1-MAOB, GPRIN1-CHRM1, GPRIN1-SLC7A14, GPRIN1-NDUFS7, ATP8A1-FBXO2, ATP8A1-YWHAH, ATP8A90B1H ATP8A1-GNG2, ATP8A1-PTGES3, ATP8A1-HSP90AA1, ATP8A1-P4HB, ATP8A1-CYCS, ATP8A1-PLSCR3, ATP8A1-VAMP1, ATP8A1-TSPAN2, ATP8A1-ATP9A, ATP8A1-NTRK8, ATP8A1-TP6ATP4 Hcn2, ATP8A1-Dnaja2, ATP8A1-VAPA, ATP8A1-SLC2A13, ATP8A1-CD47, ATP8A1-FLOT1, ATP8A1-ATP2A2, ATP8A1-FLOT2, ATP8A1-EPB41L1, ATP8A1-RGS7, ATP8A1, PLD3EN, CTP8A1-PLD3EN ATP8A1-SLC2A3, ATP8A1-STXBP1, ATP8A1-GAP43, ATP8A1-ATP1B1, ATP8A1-MAOB, ATP8A1-CHRM1, ATP8A1-SLC7A14, ATP8A1-NDUFS7, AT P8A1-GPRIN1.
Furthermore, one or more different proteins may be combined with the above two combinations to use three or more combinations.

(1) 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) Are sometimes referred to as “the nucleic acid of the present invention”, and are Aβ production inhibitors, preventive and / or therapeutic agents for diseases involving Aβ (eg, AD) and diseases 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, dog, monkey, etc.). Can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).

When the nucleic acid of the present invention is used as the above-mentioned Aβ production inhibitor, a preventive and / or therapeutic agent for diseases involving Aβ, etc., 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.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .

  The nucleic acids of the invention may be administered per se or as a suitable 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.

  As a 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. As 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. As the 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 solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.

  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. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the 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 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.

  In addition, each said composition may contain another active ingredient, as long as it does not produce an unfavorable interaction by mix | blending with the nucleic acid of this invention. Examples of other active ingredients include AD therapeutic agents typified by acetylcholinesterase inhibitors, γ-secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), γ-secretase inhibitors, β-secretase inhibitors (eg, Statin, hydroxyethylene, and peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 (2003)) that can function as transition state mimics, non-peptidic small molecules Type inhibitors (International Publication No. 2001/87293, International Publication No. 2002/88101, International Publication No. 2002/96897)) and the like.

(2) A drug containing an antibody against protein Pn, a low molecular compound that suppresses the expression or function of protein Pn, etc. 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 γ-secretase can be inhibited. Therefore, these substances suppress the expression or function of protein Pn in vivo and inhibit γ-secretase activity. Therefore, Aβ production inhibitors, diseases involving Aβ (eg, AD) and Notch protein cleavage It can be used as a preventive and / or therapeutic agent for a disease involved (eg, cancer).
The medicine 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.).

  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.

  As a 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. As 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. As the 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 solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing the above-described antibody or a salt thereof with an ordinary suppository base.

  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. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the 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 per dosage unit form, especially 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

  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 per antibody or low molecular weight compound, 1 to 5 times daily for low molecular weight compounds, orally or parenterally, 1 day to several times for antibodies. 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.

  In addition, each said composition may contain another active ingredient, as long as the interaction with the said antibody and low molecular weight compound does not produce an unfavorable interaction. Examples of other active ingredients include AD therapeutic agents typified by acetylcholinesterase inhibitors, γ-secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), γ-secretase inhibitors, β-secretase inhibitors (eg, Statin, hydroxyethylene, and peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 (2003)) that can function as transition state mimics, non-peptidic small molecules Type inhibitors (International Publication No. 2001/87293, International Publication No. 2002/88101, International Publication No. 2002/96897)) and the like.

(3) Screening of drug candidate compounds for diseases As described above, when the expression and / or function of protein Pn is suppressed, Aβ production is suppressed. Therefore, a compound or a salt thereof that suppresses the expression and / or function of protein Pn is an Aβ production inhibitor, a disease involving Aβ (eg, AD, etc.) or a disease (eg, cancer) involving Notch protein cleavage. 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 Aβ production inhibitory effect 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 in the presence of a test substance. 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.

  Specifically, 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 12) 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, for example, a cell that produces the protein Pn by synthesizing an appropriate oligonucleotide as a probe or primer based on the base sequence represented by SEQ ID NO: 2n-1 -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 can be obtained by using a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -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, if 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 λ 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. For example, SRα 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, SRα promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (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. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, 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 DNA encoding the protein Pn.
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.

  Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like. For example, the method described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1973) can be used.

  The transformed cells obtained as described above, mammalian cells having the ability to produce 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)]. The pH of the medium is preferably about 6-8. Incubation is usually performed at about 30 to 40 ° C., and aeration and agitation are added 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.

  For example, when extracting protein Pn or a partial peptide thereof from cultured cells or cytoplasm of cells, 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 For example, 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. On the other hand, when 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. Is used (if necessary, the cell membrane fraction is purified by density gradient centrifugation or the like). Moreover, when protein Pn or its partial peptide is secreted outside a microbial cell (cell), 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. As such methods, methods utilizing the solubility such as salting out and solvent precipitation method; mainly using differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. 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.

  When the protein or peptide thus obtained is a free form, 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. In addition, 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. . Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.

  Furthermore, 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, Escherichia coli lysate, etc., using the RNA encoding it as a template. Alternatively, 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.

As a protein Pn or a partial peptide thereof, or a cell having the ability to produce it, any one protein selected from (P1) to (P12) may be used, or two or more proteins may be combined. It may be used. That is, by using two or more kinds of proteins Pn selected from P1 to P12 or a partial peptide thereof, or a cell having the ability to produce them, a compound or a salt thereof that suppresses the function of two or more kinds of proteins Pn is screened. can do. Alternatively, any one or more proteins selected from the proteins P1 to P12 and one or more proteins selected from the 25 types of proteins disclosed in International Publication No. 2010/140694 are used in combination. Also good. Alternatively, a method for screening a compound or a salt thereof that suppresses the function of any two or more proteins selected from the above-mentioned 25 types of proteins disclosed in WO 2010/140694 is also included in the present invention.
For example, examples of combinations of two proteins include the following. Hereinafter, for convenience, each protein is represented by the corresponding gene name, and the two combinations are represented by “first protein-second protein”.
YWHAH-FBXO2, RPL22-FBXO2, RPL22-YWHAH, HSP90B1-FBXO2, HSP90B1-YWHAH, HSP90B1-RPL22, GNG2-FBXO2, GNG2-YWHAH, GNG2-RPL22, GNG2-HSP90B1, PTGES3-FBXO2, PTGES3-Y RPL22, PTGES3-HSP90B1, PTGES3-GNG2, HSP90AA1-FBXO2, HSP90AA1-YWHAH, HSP90AA1-RPL22, HSP90AA1-HSP90B1, HSP90AA1-GNG2, HSP90AA1-PTGES3, P4HB-FBXO2, P4HB, P4HB, P4HB, H4B P4HB-GNG2, P4HB-PTGES3, P4HB-HSP90AA1, CYCS-FBXO2, CYCS-YWHAH, CYCS-RPL22, CYCS-HSP90B1, CYCS-GNG2, CYCS-PTGES3, CYCS-HSP90AA1, CYCS-P4FBX2, PL YWHAH, PLSCR3-RPL22, PLSCR3-HSP90B1, PLSCR3-GNG2, PLSCR3-PTGES3, PLSCR3-HSP90AA1, PLSCR3-P4HB, PLSCR3-CYCS, VAMP1-FBXO2, VAMP1-YWHAH, VAMP1-RPL22, VAMP1-HSP90B1, VAMP1-GNG2, VAMP1-PTGES3, VAMP1-HSP90AA1, VAMP1-P4HB, VAMP1-CYCS, VAMP1-PLSCR3, TSPAN2-FBXO2, TSPAN2-YWHAH, TSPAN2-RPL22, TSPAN2-HSP90B1, TSPAN2-GNG2, TSPAN2-PTGES3, TSPAN2-HSP90AA1, TSPAN2- P4HB, TSPAN2-CYCS, TSPAN2-PLSCR3, TSPAN2-VAMP1, ATP9A-FBXO2, ATP9A-YWHAH, ATP9A-RPL22, ATP9A-HSP90B1, ATP9A-GNG2, ATP9A-PTGES3, ATP9A-H SP90AA1, ATP9A-P4HB, ATP9A-CYCS, ATP9A-PLSCR3, ATP9A-VAMP1, ATP9A-TSPAN2, NTRK2-FBXO2, NTRK2-YWHAH, NTRK2-RPL22, NTRK2-HSP90B1, NTRK2-PTGES3, NTRK2-HSP903, NTRK2-HSPAA NTRK2-P4HB, NTRK2-CYCS, NTRK2-PLSCR3, NTRK2-VAMP1, NTRK2-TSPAN2, NTRK2-ATP9A, Elavl4-FBXO2, Elavl4-YWHAH, Elavl4-RPL22, Elavl4-HSP90B1, Elavl4-GNG2, Elavl4-PTGES3, Elavl4- HSP90AA1, Elavl4-P4HB, Elavl4-CYCS, Elavl4-PLSCR3, Elavl4-VAMP1, Elavl4-TSPAN2, Elavl4-ATP9A, Elavl4-NTRK2, NAG6-FBXO2, NAG6-YWHAH, NAG6-RPL22, NAG6-HSP90B1, NAG6-GNG2, NAG6-PTGES3, NAG6-HSP90AA1, NAG6-P4HB, NAG6-CYCS, NAG6-PLSCR3, NAG6-VAMP1, NAG6-TSPAN2, NAG6-ATP9A, NAG6-NTRK2, NAG6-Elavl4, Hcn2-FBXO2, Hcn2-YWHAH, Hcn2- RPL22, Hcn2-HSP90B1, Hcn2-GNG2, Hcn2-PTGES3, Hcn2-HSP90AA1, Hcn2-P4HB, Hcn2-CYCS, Hcn2-PLSCR3, Hcn2-VAMP1, Hcn2-TSPAN2, Hcn2-ATP9A, Hcn2-NTRK2, Hcn2-Elavl4, Hcn2-NAG6, Dnaja2-FBXO2, Dnaja2-YWHAH, Dnaja2-RPL22, Dnaja2-HSP90B1, Dnaja2-GNG2, Dnaja2-PTGES3, Dnaja2-HSP90AA1, Dnaja2-P4HB, Dnaja2-CYCS, Dnaja2-PLSCR3, Dnaja2-VAMP1, Dnaja2-VAMP1 TSP AN2, Dnaja2-ATP9A, Dnaja2-NTRK2, Dnaja2-Elavl4, Dnaja2-NAG6, Dnaja2-Hcn2, VAPA-FBXO2, VAPA-YWHAH, VAPA-RPL22, VAPA-HSP90B1, VAPA-GNG2, VAPA-PTGES3, VAPA-SP VAPA-P4HB, VAPA-CYCS, VAPA-PLSCR3, VAPA-VAMP1, VAPA-TSPAN2, VAPA-ATP9A, VAPA-NTRK2, VAPA-Elavl4, VAPA-NAG6, VAPA-Hcn2, VAPA-Dnaja2, SLC2A13-FBXO2-LC YWHAH, SLC2A13-RPL22, SLC2A13-HSP90B1, SLC2A13-GNG2, SLC2A13-PTGES3, SLC2A13-HSP90AA1, SLC2A13-P4HB, SLC2A13-CYCS, SLC2A13-PLSCR3, SLC2A13-VTPALC3T SLC2A13-Elavl4, SLC2A13-NAG6, SLC2A13-Hcn2, SLC2A13-Dnaja2, SLC2A13-VAPA, CD47-FBXO2, CD47-YWHAH, CD47-RPL22, CD47-HSP90B1, CD47-GNG2, CD47-PTGES3, CD47-SPGE903, CD47-SPGE90 P4HB, CD47-CYCS, CD47-PLSCR3, CD47-VAMP1, CD47-TSPAN2, CD47-ATP9A, CD47-NTRK2, CD47-Elavl4, CD47-NAG6, CD47-Hcn2, CD47-Dnaja2, CD47-VAPA, CD47-SLC2A13, FLOT1-FBXO2, FLOT1-YWHAH, FLOT1-RPL22, FLOT1-HSP90B1, FLOT1-GNG2, FLOT1-PTGES3, FLOT1-HSP90AA1, FLOT1-P4HB, FLOT1-CYCS, FLOT1-PLSCR3, FLOT1-VAMP1, FLOT1-TSPAN2, FLOT1- ATP 9A, FLOT1-NTRK2, FLOT1-Elavl4, FLOT1-NAG6, FLOT1-Hcn2, FLOT1-Dnaja2, FLOT1-VAPA, FLOT1-SLC2A13, FLOT1-CD47, ATP2A2-FBXO2, ATP2A2-YWHAH, ATP2A2-RPL22, ATP2A2-HSP90B1, ATP2A2-GNG2, ATP2A2-PTGES3, ATP2A2-HSP90AA1, ATP2A2-P4HB, ATP2A2-CYCS, ATP2A2-PLSCR3, ATP2A2-VAMP1, ATP2A2-TSPAN2, ATP2A2-ATP9A, ATP2A2-NTRK2, ATP2A2-GTP2 Hcn2, ATP2A2-Dnaja2, ATP2A2-VAPA, ATP2A2-SLC2A13, ATP2A2-CD47, ATP2A2-FLOT1, FLOT2-FBXO2, FLOT2-YWHAH, FLOT2-RPL22, FLOT2-HSP90B1, FLOT2-PTGES3, FLOT2-HSP90AA FLOT2-P4HB, FLOT2-CYCS, FLOT2-PLSCR3, FLOT2-VAMP1, FLOT2-TSPAN2, FLOT2-ATP9A, FLOT2-NTRK2, FLOT2-Elavl4, FLOT2-NAG6, FLOT2-Hcn2, FLOT2-Dnaja2, FLOT2-VAPA, FLOT2- SLC2A13, FLOT2-CD47, FLOT2-FLOT1, FLOT2-ATP2A2, EPB41L1-FBXO2, EPB41L1-YWHAH, EPB41L1-RPL22, EPB41L1-HSP90B1, EPB41L1-GNG2, EPB41L1-PTGES3, EPB41L1-HSP90AA1, EPB41L1 EPB41L1-PLSCR3, EPB41L1-VAMP1, EPB41L1-TSPAN2, EPB41L1-ATP9A, EPB41L1-NTRK2, EPB41L1-Elavl4, EPB41L1-NAG6, EPB41L1-Hcn2, EPB41L1-Dnaja2, EPB41L1-VAPA, EPB41L1-SLC2A13, EPB41L1-CD47, EPB41L1-FLOT1, EPB41L1-ATP2A2, EPB41L1-FLOT2, RGS7-FBXO2, RGS7-YWHAH, RGS7-RPL22, RGS7-HSP90B1, RGS7-GGE2, RGS7-PT3 HSP90AA1, RGS7-P4HB, RGS7-CYCS, RGS7-PLSCR3, RGS7-VAMP1, RGS7-TSPAN2, RGS7-ATP9A, RGS7-NTRK2, RGS7-Elavl4, RGS7-NAG6, RGS7-Hcn2, RGS7-Dnaja2, RGS7-VAPA RGS7-SLC2A13, RGS7-CD47, RGS7-FLOT1, RGS7-ATP2A2, RGS7-FLOT2, RGS7-EPB41L1, PLD3-FBXO2, PLD3-YWHAH, PLD3-RPL22, PLD3-HSP90B1, PLD3-GNG2, PLD3-PTGES3, PLD3- HSP90AA1, PLD3-P4HB, PLD3-CYCS, PLD3-PLSCR3, PLD3-VAMP1, PLD3-TSPAN2, PLD3-ATP9A, PLD3-NTRK2, PLD3-Elavl4, PLD3-NAG6, PLD3-Hcn2, PLD3-Dnaja2, PLD3-VAPA, PLD3-SLC2A13, PLD3-CD47, PLD3-FLOT1, PLD3-ATP2A2, PLD3-FLOT2, PLD3-EPB41L1, PLD3-RGS7, ENC1-FBXO2, ENC1-YWHAH, ENC1-RPL22, ENC1-HSP90B1, ENC1-GNG2, ENC1- PTGES3, ENC1-HSP90AA1, ENC1-P4HB, ENC1-CYCS, ENC1-PLSCR3, ENC1-VAMP1, ENC1-TSPAN2, ENC1-ATP9A, ENC1-NTRK2, ENC1-Elavl4, ENC1-NAG6, ENC1-Hcn2, ENC1-Dnaja2, ENC1-VAPA, ENC1-SLC2A13, ENC1-CD47, ENC1-FLOT1, ENC1-ATP2A2, ENC1-FLOT2, ENC1-EPB41L1, ENC1-RGS7, ENC1-PLD3, SYP-FBXO2, SYP-YWHAH, SYP-RPL22, SYP-HSP90B1, SYP-GNG2, SYP-PTGES3, SYP-HSP90AA1, SYP-P4HB, SYP- CYCS, SYP-PLSCR3, SYP-VAMP1, SYP-TSPAN2, SYP-ATP9A, SYP-NTRK2, SYP-Elavl4, SYP-NAG6, SYP-Hcn2, SYP-Dnaja2, SYP-VAPA, SYP-SLC2A13, SYP-CD47, SYP-FLOT1, SYP-ATP2A2, SYP-FLOT2, SYP-EPB41L1, SYP-RGS7, SYP-PLD3, SYP-ENC1, SLC2A3-FBXO2, SLC2A3-YWHAH, SLC2A3-RPL22, SLC2A3-HSP90B1, SLC2A3-GNG2, SLC2A3- PTGES3, SLC2A3-HSP90AA1, SLC2A3-P4HB, SLC2A3-CYCS, SLC2A3-PLSCR3, SLC2A3-VAMP1, SLC2A3-TSPAN2, SLC2A3-ATP9A, SLC2A3-NTRK2, SLC2A3-Elavl4, SLC2A3-NAG3A2, SLC2A3-NAG3A2, SLC2A3-VAPA, SLC2A3-SLC2A13, SLC2A3-CD47, SLC2A3-FLOT1, SLC2A3-ATP2A2, SLC2A3-FLOT2, SLC2A3-EPB41L1, SLC2A3-RGS7, SLC2A3-PLD3, SLC2A3-ENP1, SLC2A3-BPF, STX2A3-BP YWHAH, STXBP1-RPL22, STXBP1-HSP90B1, STXBP1-GNG2, STXBP1-PTGES3, STXBP1-HSP90AA1, STXBP1-P4HB, STXBP1-CYCS, STXBP1-PLSCR3, STXBP1-VAMP1, STXBP1-TSPAN2, STXBP1-ATP9RK, STXBP1-ATP9RK STXBP1-Elavl4, STXBP1-NAG6, STXBP1-Hcn2, STXBP1-Dnaja2, STXBP1-VAPA, STXBP1-SLC2A13, STXBP1-CD47, STXBP1-FLOT1, STXBP1-ATP2A2, STXBP1-FLOT2, STXBP1-EPB41L1, STXBP1-RGS7, STXBP1-PLD3, STXBP1-ENC1, STXBP1-ENC1, SYP, STXBP1-SLC2A3, GAP43-FBXO2, GAP43-YWHAH, GAP43-RPL22, GAP43-HSP90B1, GAP43-GNG2, GAP43-PTGES3, GAP43-HSP90AA1, GAP43-P4HB, GAP43-CYCS, GAP43-PLSCRV1, GAP43-PLSCRVAMP GAP43-TSPAN2, GAP43-ATP9A, GAP43-NTRK2, GAP43-Elavl4, GAP43-NAG6, GAP43-Hcn2, GAP43-Dnaja2, GAP43-VAPA, GAP43-SLC2A13, GAP43-CD47, GAP43-FLOT1, GAP43-ATP2A2, GAP43-ATP2A2, GAP43 FLOT2, GAP43-EPB41L1, GAP43-RGS7, GAP43-PLD3, GAP43-ENC1, GAP43-SYP, GAP43-SLC2A3, GAP43-STXBP1, ATP1B1-FBXO2, ATP1B1-YWHAH, ATP1B1-RPL22, ATP1B1-TP1G1, ATP1B1-PTGES3, ATP1B1-HSP90AA1, ATP1B1-P4HB, ATP1B1-CYCS, ATP1B1-PLSCR3, ATP1B1-VAMP1, ATP1B1-TSPAN2, ATP1B1-ATP9A, ATP1B1-NTRK2, ATP1B1-Elavl4, ATP1B1-B6 Dnaja2, ATP1B1-VAPA, ATP1B1-SLC2A13, ATP1B1-CD47, ATP1B1-FLOT1, ATP1B1-ATP2A2, ATP1B1-FLOT2, ATP1B1-EPB41L1, ATP1B1-RGS7, ATP1B1-PLD3, ATP1B1-ENC1, ATP1B1-SYP, ATP1B1-SLC2A3, ATP1B1-STXBP1, ATP1B1-GAP43, MAOB-FBXO2, MAOB-YWHAH, MAOB-RPL22, MAOB-HSP90B1, MAOB-GNG2, MAOB-PTGES3, MAOB-HSP90AA1, MAOB-P4HB CYCS, MAOB-PLSCR3, MAOB-VAMP1, MAOB-TSPAN2, MAOB-ATP9A, MAOB-NTRK2, MAOB-Elavl4, MAOB-NAG6, MAOB-Hcn2, MAOB-Dnaja2, MAOB-VAPA, MAOB-SLC2A13, MAOB-CD47, MAOB-FLOT1, MAOB-ATP2A2, MAOB-FLOT2, MAOB-EPB41L1, MAOB-RGS7, MAOB-PLD3, MAOB-ENC1, MAOB-SYP, MAOB-SLC2A3, MAOB-STXBP1, MAOB-GAP43, MAOB-ATP1B1, CHRM1- FBXO2, CHRM1-YWHAH, CHRM1-RPL22, CHRM1-HSP90B1, CHRM1-GNG2, CHRM1-PTGES3, CHRM1-HSP90AA1, CHRM1-P4HB, CHRM1-CYCS, CHRM1-PLSCR3, CHRM1-VAMP1, CHRM1-TSPAN2, CHRM1-ATP9A, CHRM1-NTRK2, CHRM1-Elavl4, CHRM1-NAG6, CHRM1-Hcn2, CHRM1-Dnaja2, CHRM1-VAPA, CHRM1-SLC2A13, CHRM1-CD47, CHRM1-FLOT1, CHRM1-ATP2A2, CHRM1-FLOT2, CHRM1-EPB41L1, CHRM1- RGS7, CHRM1-PLD3, CHRM1-ENC1, CHRM1-SYP, CHRM1-SLC2A3, CHRM1-STXBP1, CHRM1-GAP43, CHRM1-ATP1B1, CHRM1-MAOB, SLC7A14-FBXO2, SLC7A14-YWHAH, SLC7A14-RPL22, SLC90A14H SLC7A14-GNG2, SLC7A14-PTGES3, SLC 7A14-HSP90AA1, SLC7A14-P4HB, SLC7A14-CYCS, SLC7A14-PLSCR3, SLC7A14-VAMP1, SLC7A14-TSPAN2, SLC7A14-ATP9A, SLC7A14-NTRK2, SLC7A14-Elavl4, SLC7A14-LC7ALC7ALC7A14 VAPA, SLC7A14-SLC2A13, SLC7A14-CD47, SLC7A14-FLOT1, SLC7A14-ATP2A2, SLC7A14-FLOT2, SLC7A14-EPB41L1, SLC7A14-RGS7, SLC7A14-PLD3, SLC7A14-SLC7A14-ENC1, SLC7A14-ENC1, ALC7 SLC7A14-GAP43, SLC7A14-ATP1B1, SLC7A14-MAOB, SLC7A14-CHRM1, NDUFS7-FBXO2, NDUFS7-YWHAH, NDUFS7-RPL22, NDUFS7-HSP90B1, NDUFS7-GNG2, NDUFS7NPT7HPT7H CYCS, NDUFS7-PLSCR3, NDUFS7-VAMP1, NDUFS7-TSPAN2, NDUFS7-ATP9A, NDUFS7-NTRK2, NDUFS7-Elavl4, NDUFS7-NAG6, NDUFS7-Hcn2, NDUFS7-Dnaja2, NDUFS7-VAPA, NDUFS7-LCA, NDUFS7-LC NDUFS7-FLOT1, NDUFS7-ATP2A2, NDUFS7-FLOT2, NDUFS7-EPB41L1, NDUFS7-RGS7, NDUFS7-PLD3, NDUFS7-ENC1, NDUFS7-SYP, NDUFS7-SLC2A3, NDUFS7-STXBP1, NDUFS7-B4343 MAOB, NDUFS7-CHRM1, NDUFS7-SLC7A14, GPRIN1-FBXO2, GPRIN1-YWHAH, GPRIN1-RPL22, GPRIN1-HSP90 B1, GPRIN1-GNG2, GPRIN1-PTGES3, GPRIN1-HSP90AA1, GPRIN1-P4HB, GPRIN1-CYCS, GPRIN1-PLSCR3, GPRIN1-VAMP1, GPRIN1-TSPAN2, GPRIN1-ATP9A, GPRIN1-NTRK2, GPRIN1-Elavl4, GPRIN1-NAG6 GPRIN1-Hcn2, GPRIN1-Dnaja2, GPRIN1-VAPA, GPRIN1-SLC2A13, GPRIN1-CD47, GPRIN1-FLOT1, GPRIN1-ATP2A2, GPRIN1-FLOT2, GPRIN1-EPB41L1, GPRIN1-RGS7, GPRIN1-PLD3, GPRIN1-ENC1, GPRIN1-ENC1, SYP, GPRIN1-SLC2A3, GPRIN1-STXBP1, GPRIN1-GAP43, GPRIN1-ATP1B1, GPRIN1-MAOB, GPRIN1-CHRM1, GPRIN1-SLC7A14, GPRIN1-NDUFS7, ATP8A1-FBXO2, ATP8A1-YWHAH, ATP8A90B1H ATP8A1-GNG2, ATP8A1-PTGES3, ATP8A1-HSP90AA1, ATP8A1-P4HB, ATP8A1-CYCS, ATP8A1-PLSCR3, ATP8A1-VAMP1, ATP8A1-TSPAN2, ATP8A1-ATP9A, ATP8A1-NTRK8, ATP8A1-TP6ATP4 Hcn2, ATP8A1-Dnaja2, ATP8A1-VAPA, ATP8A1-SLC2A13, ATP8A1-CD47, ATP8A1-FLOT1, ATP8A1-ATP2A2, ATP8A1-FLOT2, ATP8A1-EPB41L1, ATP8A1-RGS7, ATP8A1, PLD3EN, CTP8A1-PLD3EN ATP8A1-SLC2A3, ATP8A1-STXBP1, ATP8A1-GAP43, ATP8A1-ATP1B1, ATP8A1-MAOB, ATP8A1-CHRM1, ATP8A1-SLC7A14, ATP8A1-NDUFS7, AT P8A1-GPRIN1.
Furthermore, three or more types of combinations can be used by further combining one type of different protein with the above two types of combinations.
The combination of the two or more proteins or the two or more genes corresponding thereto can be similarly applied to the screening methods (Ia), (Ib), (II) and (III) described later. Each of which can screen for compounds that suppress the expression or function of two or more proteins.
When screening by combining two or more proteins, it is preferable to use a single cell that produces all of the two or more proteins. For example, the mammalian cells, for example, HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, simian Vero cells, CHO cells, CHO (dhfr ^-) cells, mouse L cells, mouse AtT- Using 20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells, etc., the two or more proteins can be transformed by co-transformation with expression vectors containing DNAs encoding the two or more proteins. Cells that are expressed simultaneously can be produced and used to screen for compounds that suppress the expression or function of two or more proteins.

  Examples of 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.

  Since the activity of the protein Pn is preferably measured using Aβ production inhibition as an index, when the protein Pn or a partial peptide thereof or a cell having the ability to produce it is contacted with a test substance, an Aβ production system is used. Preferably coexist. As the Aβ production system, cells expressing APP, which is a precursor of Aβ, are preferably used. Here, when 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. The 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. On the other hand, when using the isolated protein Pn or a partial peptide thereof, 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. Can be implemented. The concentration of the added protein Pn or a partial peptide thereof is appropriately selected in the range of, for example, about 0.1 to about 100 μg / ml for each protein or peptide.

  When 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. In particular, when it is desired to examine the effect of the test substance on the human protein Pn, a knock-in animal in which the endogenous gene Gn of the animal is replaced with the human gene Gn can also be used. In this case, 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. There are no particular restrictions on the breeding conditions of the animals to be used, but it is preferable that the animals are raised in an environment of SPF grade or higher. Contact of the test substance with the cells is carried out by administering the test substance to the animal individual. 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. . Although there is no restriction | limiting in particular, for example, about 0.5-20 mg / kg as a single dose can be administered 1 to 5 times a day for about 1 day to 6 months.

The protein Pn activity in the above screening method can be measured by measuring the production amount of Aβ 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 a partial peptide thereof, or cells that have the ability to produce it (cells that express APP). When 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, when the test substance is brought into contact with the test substance, 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 Aβ is the amount of Aβ (eg, Aβ38, Aβ40, Aβ42, etc., preferably Aβ40 and / or Aβ42) in the test sample, for example, various immunological methods such as ELISA using anti-Aβ antibody and Western blotting. It can be performed by measuring using a technique, mass spectrometry or the like.

  For example, in the above screening method, the activity of protein Pn in the presence of the test substance (for example, the amount of Aβ in the test sample) is, for example, about 10% or more compared to the activity in the absence of the test substance. When 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 Aβ production inhibitory action. Can be selected as a candidate.

Screening method (Ia)
Alternatively, in a non-human animal introduced with at least one gene selected from (G1) to (G12), 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).
(A) a step of administering a test substance to a non-human animal into which at least one gene selected from (G1) to (G12) has been introduced; (b) at least one phenotype reflecting AD pathology in the animal; A step of evaluating (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is not administered, and the step (a) is a screening method using the activity of the protein Pn as an index The method can be carried out in the same manner as in the case of administering a test substance to a non-human mammal individual producing proteins Pn and APP.
In the step (b), phenotypes that reflect the pathology of AD include, for example, (1) Aβ amount in tissues such as brain tissue, cerebrospinal fluid, blood, (2) neuronal cell death, (3) central nervous system Inflammatory reactions of the system, (4) reduced cognitive ability, (5) accumulation of amyloid plaques, (6) deterioration of cerebral blood flow, (7) reduction of cerebral glucose metabolism, etc., but are not limited thereto . For the evaluation of these phenotypes, 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).

Screening method (Ib)
In another preferred embodiment, the activity of protein Pn can be measured using the increased activity of γ-secretase as an indicator. Specifically, the screening method includes:
(A) contacting the protein Pn and γ-secretase with a test substance;
(B) measuring γ-secretase activity, and (c) selecting a substance that reduces or modulates γ-secretase activity compared to when measured in the absence of the test substance. .

As γ-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 γ-secretase itself.
For γ-secretase activity, APP or Notch as a substrate is added, and APP metabolite fragments (Aβ38, Aβ40, Aβ42, AICD, etc.) or Notch metabolites (NICD) are subjected to ELISA, Western blotting, and reporter gene assay. It can be measured by detecting using mass spectrometry or the like.

  For example, in the screening method described above, the γ-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 Aβ production-inhibiting substance.

  In any one of the above screening methods, as 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 Aβ production inhibitor can also be selected by measuring and comparing the activity of the protein Pn.

Screening method (II)
The present invention also relates to a method for screening an Aβ 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. I will provide a. The cells used in this method, the type of the 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 the protein Pn is a nucleic acid that can hybridize with the DNA encoding the protein Pn under highly stringent conditions, that is, the base sequence represented by SEQ ID NO: n or a base sequence complementary thereto and a 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 also be measured at the protein level by detecting these proteins using an antibody against the protein Pn (hereinafter sometimes referred to as “the detection antibody of the present invention”). .

Therefore, more specifically, 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 Aβ production inhibitor characterized by measuring and comparing, and (b) culturing cells having the ability to produce protein Pn in the presence and absence of a test substance, under both conditions Provided is a method for screening an Aβ production inhibitor, characterized in that the amount of the protein is measured and compared using the detection antibody of the present invention.

For example, the measurement of the amount of mRNA or protein of protein Pn can be specifically performed as follows.
(I) Normal or disease (for example, AD) model non-human mammals (for example, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) After that, 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. On the other hand, the amount of protein Pn can be quantified using Western blot analysis or various immunoassay methods described in detail below.
(Ii) 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.

Screening for substances that alter the expression level of protein Pn
(I) A certain time before giving a drug or the like to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour to 6 hours) Before) or after a certain time (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later) After time has elapsed (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later), mRNA that encodes protein Pn contained in cells isolated from the animal Quantitatively analyze the amount of protein or protein Pn, or (ii) add a test substance to the medium or buffer when culturing the transformant according to a conventional method, and incubate for a certain period of time (after 1 day) ~ 7 days later, preferably 1 day ~ 3 days later, more preferred After days 2 days ~ 3), mRNA amount encodes a protein Pn included in the transformant, or the amount of protein Pn quantification can be performed by analyzing.

Specifically, 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 is reacted competitively with the sample solution and the labeled protein Pn, 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, the detection antibody of the present invention insolubilized on the carrier, and another labeled detection antibody of the present invention are reacted simultaneously or successively, 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).
In the above quantification method (ii), it is desirable that the two types of antibodies recognize different portions of the protein Pn. For example, if one antibody recognizes the N-terminal part of protein Pn, the other antibody can react with the C-terminal part of the protein.
As 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. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can also be used for binding of an antibody or antigen and 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.

  In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, 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. Further, in the immunoassay by the sandwich method, 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.
In the competition method, protein Pn in a sample solution and labeled protein Pn are reacted competitively with an antibody, and then an unreacted labeled antigen (F) and a 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 protein Pn in the sample solution. In this reaction method, 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), and 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).
In the immunometric method, the protein Pn in the sample solution and the immobilized protein Pn are subjected to a competitive reaction 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. The protein Pn is reacted with an excess amount of labeled antibody, and then the immobilized protein Pn is added to bind the unreacted labeled antibody to the solid phase, and then 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.
In nephrometry, the amount of insoluble precipitate produced as a result of the 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 small and only a small amount of precipitate can be obtained.

  In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of protein Pn, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.

For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequential Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (End of academic program , Inc. issued) and the like can be referred to.
As described above, the amount of protein Pn in cells can be quantified with high sensitivity by using the detection antibody of the present invention.

  For example, in the screening methods (a) and (b) above, 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. When the test substance is inhibited by 10% or more, preferably about 20% or more, more preferably about 30% or more, and further preferably about 50% or more, the test substance is expressed as a protein Pn expression-suppressing substance, and thus Aβ production-suppressing action Can be selected as a candidate for a substance having

  Alternatively, in the above screening method, 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. Such 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. When such cells are used, the expression level of protein Pn can be evaluated by measuring the expression level of the reporter gene using a conventional method.

  In the above screening method, as a control, 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 is used. An Aβ production inhibitor can also be selected by measuring and comparing the expression level of Pn.

Screening method (III)
The present invention also provides a method for screening an Aβ production inhibitor comprising the following steps (a) to (c).
(A) A step of contacting protein Pn and γ-secretase with a test substance (b) A step of measuring the binding activity of protein Pn and γ-secretase (c) When measured in the absence of the test substance The step of selecting a substance that decreases the binding activity as compared with the above can 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 γ-secretase (the former Can be carried out according to the above screening method (Ib)).
More specifically, the screening method includes
(1) The amount of labeled protein Pn binding to γ-secretase when the labeled protein Pn and γ-secretase are contacted in the presence and absence of the test substance is measured and compared. Or
(2) The labeled protein Pn and the cell or membrane of the labeled protein Pn when the γ-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. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.

  For example, in the screening methods (1) and (2) above, the amount of label (protein Pn) that binds to γ-secretase in the presence of the test substance is greater than that in the absence of the test substance. When it is inhibited by about 10% or more, preferably about 20% or more, more preferably about 30% or more, more preferably about 50% or more, the test substance suppresses the binding activity between protein Pn and γ-secretase. The substance can be selected as a candidate of a substance having an Aβ 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 Aβ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer). ) Is useful as a preventive and / or therapeutic agent.

  When 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.

(4) Use as a diagnostic agent In addition, the present invention relates to a disease involving Aβ (eg, AD) or Notch protein cleavage characterized by measuring the expression level of protein Pn in a sample collected from a test animal. Provides a method for determining the onset or risk of developing a disease (eg, cancer) associated with. The method includes the following steps (a) to (c).
(A) Providing a test animal-derived sample (b) From the expression level of at least one gene selected from gene Gn (n = 1 to 12) or protein Pn (n = 1 to 12) in the sample A step of measuring the activity of at least one selected protein (c) a test animal having an increased expression level or activity compared to a case of measuring in a sample derived from a normal animal, a disease involving Aβ (eg, , AD) or a disease that involves the cleavage of Notch protein (eg, cancer), or is determined to have a high risk of developing in the future

  Examples of test animals include humans and other mammals, preferably humans or mice, rats, rabbits, dogs, monkeys and the like that are widely used as experimental animals. Examples of 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.
As a result of the above measurement, the expression level of gene Gn or protein Pn in the sample collected from the test animal was significantly higher than the expression level of gene Gn or protein Pn in the sample collected from the normal animal. In this case, it is possible to determine that the test animal has developed a disease involving Aβ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer) or has a high risk of developing in the future. . Alternatively, 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 develops a disease involving Aβ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer) or has a high risk of developing in the future It can also be determined.

  In the above diagnosis of a non-human animal, an animal in which the gene Gn is 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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

Example 1 Identification of brain γ-secretase binding protein In brain tissue, γ-secretase activity was strongly detected in the microsomal fraction. Therefore, this fraction was prepared, and the γ-secretase complex was purified by inhibitor pull-down method. went.

(1) Preparation of fraction containing brain γ-secretase Rat brain was obtained from Funakoshi.
After homogenizing rat brain tissue in buffer A (20 mM Hepes, 50 mM KCl, 2 mM EGTA, pH 7.5 with complete protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA)), After removing cell debris and nuclear components by centrifugation at 1,000 xg and 10,000 xg, 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 a solubilized γ-secretase complex was prepared by centrifugation at 100,000 × g. For the binding experiment, a solubilized γ-secretase complex prepared in 0.5% (w / v) CHAPSO was used.
(2) Purification of brain γ-secretase-containing fraction Commercially available affinity resin is a compound in which a PEG linker is introduced into a commonly used γ-secretase inhibitor (DAPT) and a compound in which a PEG linker is introduced into cholesterol (Cholesterol). Using Affigel-DAPT + Cholesterol resin (Affigel-DAPT + Cholesterol resin) immobilized on Affi-Gel-102Gel (BIO-RAD, cat. No; 153-2401) (FIG. 1) (International Publication No. 2010/053115) The following experiment was conducted.

This method, the existing membrane environment is the target of DAPT .gamma.-secretase complex, to construct artificially on affinity resins, approach to pull down efficiently membrane protein (T arget Or iented A ffinity C hromatography (TOrAC method)) (International Publication No. 2010/053115).
DAPT as a raw material is also known as LY-374973, N- [N- (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester, and has the molecular formula C ₂₃ H ₂₆ F ₂ N ₂ O ₄ , a known compound having a molecular weight of 432.46, J. Neurochem. 2001, 76, 173, Org. Biomol. Chem., 2005, 3, 2450-2457, Bioorganic & Medicinal Chemistry Letters., 2004, 14, 1983- Can be produced by the method described in 1985. In addition, commercial products (Sigma, Cat. No. D5942, Calbio, Cat. No. 565770, Tocris, Cat. No. 2634, Alexis, Cat. No. 270-416-M005, Pepnet, Cat. No. IGS-3641-PI) is also available.

Affigel-DAPT + Cholesterol resin (FIG. 1) (equivalent to 1.2 μmol) was accurately weighed and a binding experiment was conducted with the rat brain lysate (200 μl) obtained in (1) above.
Antagonism (+) was previously added to Lysate in 0.2 μl of 100 mM DAPT (final concentration 100 μM) and incubated for 30 minutes before being used for binding experiments. After completion of the binding experiment, the resin was centrifuged at 12,000 × g, the supernatant was discarded, and the remaining resin was washed 3 times with 800 μl of buffer B (20 mM Hepse pH 7.5, 50 mM KCl, 2 mM EGTA + protease inhibitor, 0.5% CHAPSO). Washed (15 min, 3 times). SDS loading buffer was added to the resin and incubated. The sample solution thus obtained was separated with a commercially available SDS gel (BioRad ready Gel J, 5-20% SDS, cat. No; 161-J371V), and the SDS gel was analyzed.

(3) Specificity confirmation by Western blotting
Proteins separated by SDS-PAGE were transferred to PVDF Membrane Filter Paper Sandwich 0.2um Pore Size, 20 / pk. (Invitrogen cat. No; LC2002). The PVDF membrane was transferred to a tray, 50 ml of blocking agent Blocking One (nakalai) was added, and the mixture was shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 minutes, 3 times).

The primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO, cat. No; NKB-101) and shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 minutes, 3 times). The secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO, cat. No; NKB-101) and incubated at room temperature for 1 hour. Washed 3 times with TBS-T (10 minutes, 3 times). The detection reagent was ECL Plus western Blotting Detection System (GE Healthcare cat. No; RPN2132).
Detection was performed using a Lumino Image Analyzer LAS-1000 (Fuji Film) as a detection device.

  As a result, as shown in FIG. 2, the lane (−) in which DAPT, which is a γ-secretase inhibitor, was not added in advance to the brain extract was γ-secretase (nicastrin (NCT), presenilin 1 (PS1-NTF, PS1- CTF), Ahu-1 (APH-1), pen-2 (PEN-2) complex) recognizes and binds DAPT on the resin, but lanes in which DAPT, which is an antagonist, is added to the brain extract in advance Since (+) is already bound to the target γ-secretase of free DAPT, it does not bind to DAPT on the resin, and the Nct, PS1, Aph-1, and Pen-2 bands disappear. . The disappearance of the band indicates that these four components are specifically bound to DAPT as a complex.

  Therefore, the Affigel-DAPT + Cholesterol resin in FIG. 1 binds to components of γ-secretase such as Nct, PS-1, Aph-1, and Pen-2, which are DAPT binding proteins, and this binding is antagonized by DAPT. It was found to be specific.

(4) Identification of γ-secretase complex component
Proteins separated by SDS-PAGE were cut into strips from the top to the bottom of the antagonist (-) lane and the antagonist (+) lane, respectively. The peptide obtained by in-Gel trypsin digestion of the cut gel piece was measured with LTQ-Orbitrap (Thermo Fisher Science) to identify the protein. The protein group obtained by antagonism (+) was subtracted from the protein group obtained by antagonism (−). As a result, a plurality of novel γ-secretase components that specifically bind to γ-secretase and are expressed in the brain were found (Table 1).

Example 2 Inhibition of Aβ production by siRNA knockdown of a novel binding factor For those identified by the above method, the effect on Aβ production was evaluated by the RNAi method.

  The human embryonic kidney cell line (HEK-APP), which constitutively expresses the amyloid precursor protein (APP), which was used for evaluation, was obtained from Dr. Eirikur Benedikz of Karolinska Institute (Nilsson et al., Biochem Biophys Res Commun, 341, 1294-9.). 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 Designed by the method described in Patent Publication 2006-236153, synthesized by Gene Design Co., Ltd. (Table 2).

siRNA was introduced into HEK-APP cells using the siRNA introduction reagent Lipofectamine ^™ RNAiMAX Transfection Reagent (Invitrogen). 72 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 siRNA showing cytotoxicity of 30% or more was excluded from the test. The knockdown efficiency of the target gene was confirmed by real-time PCR. Pre-Developed TaqMan (registered trademark) Assay Reagents from Applied Biosystems was used as the target gene primer, and the GAPDH gene was used as an internal standard. The amount of Aβ42 in the culture supernatant was measured by ELISA (Wako Chemicals, Osaka, Japan). AllStars Negative Control siRNA (qiagen 1027280) was used as a control for the evaluation system, and the inhibition rate was calculated with the value (Neg. Con) when Negative Control siRNA was introduced as 100%. As a result, the amount of Aβ42 was reduced by reducing the expression level of the new factor, as in the case of decreasing the expression level of presenilin 1 (PS1), which is a constituent factor of γ-secretase used as a test system control. The knowledge to be obtained was obtained (Fig. 3).

  Since 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 γ-secretase, the substance obtained by this screening method is Aβ production and / or Notch cleavage inhibitor or modulator is expected as a prophylactic and therapeutic drug for diseases such as AD and cancer. Alternatively, since these novel proteins may not affect the Notch cleavage activity of γ-secretase, the substance obtained by this screening method does not inhibit the cleavage of other physiological substrates such as Notch, There is a possibility that only APP cleavage activity of γ-secretase activity can be inhibited, and it is also expected as a safe preventive and therapeutic drug for diseases such as AD with few side effects.

Claims (24)

The following proteins:
(P1) F-box only protein 2;
(P2) 14-3-3 protein eta;
(P3) 60S ribosomal protein L22;
(P4) Endoplasmin;
(P5) Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2;
(P6) Prostaglandin E synthase 3;
(P7) Heat shock protein HSP 90-alpha;
(P8) Protein disulfide-isomerase;
(P9) Cytochrome c;
(P10) Phospholipid scramblase 3;
(P11) Vesicle-associated membrane protein 1; and
(P12) Tetraspanin-2;
An Aβ production inhibitor comprising one or more substances that suppress the expression or function of a protein Pn selected from (n is an integer of 1 to 12). The agent according to claim 1, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) FBXO2;
(G2) YWHAH;
(G3) RPL22;
(G4) HSP90B1;
(G5) GNG2;
(G6) PTGES3;
(G7) HSP90AA1;
(G8) P4HB;
(G9) CYCS;
(G10) PLSCR3;
(G11) VAMP1; or
(G12) TSPAN2;
(B) Ribozyme nucleic acid for gene Gn transcript (c) Nucleic acid having RNAi activity for gene Gn transcript or precursor thereof The agent according to claim 1, wherein the substance that suppresses the function of the protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) An antibody that binds to protein Pn (b) A low molecular compound that binds to protein Pn (c) A compound that inhibits the binding activity between protein Pn and γ-secretase   The agent according to any one of claims 1 to 3, wherein Aβ is Aβ40 or Aβ42.   The agent in any one of Claims 1-4 for the treatment or prevention of Alzheimer's disease or cancer. In mammals, the following proteins:
(P1) F-box only protein 2;
(P2) 14-3-3 protein eta;
(P3) 60S ribosomal protein L22;
(P4) Endoplasmin;
(P5) Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-2;
(P6) Prostaglandin E synthase 3;
(P7) Heat shock protein HSP 90-alpha;
(P8) Protein disulfide-isomerase;
(P9) Cytochrome c;
(P10) Phospholipid scramblase 3;
(P11) Vesicle-associated membrane protein 1; and
(P12) Tetraspanin-2;
A method for inhibiting Aβ production in the mammal, comprising inhibiting the expression or function of a protein Pn selected from (n is an integer of 1 to 12).   7. The method of claim 6, comprising administering to the mammal an effective amount of one or more substances that suppress the expression or function of protein Pn. The method according to claim 7, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) FBXO2;
(G2) YWHAH;
(G3) RPL22;
(G4) HSP90B1;
(G5) GNG2;
(G6) PTGES3;
(G7) HSP90AA1;
(G8) P4HB;
(G9) CYCS;
(G10) PLSCR3;
(G11) VAMP1; or
(G12) TSPAN2;
(B) Ribozyme nucleic acid for gene Gn transcript (c) Nucleic acid having RNAi activity for gene Gn transcript or precursor thereof The method according to claim 7, wherein the substance that suppresses the function of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) An antibody that binds to protein Pn (b) A low molecular compound that binds to protein Pn (c) A compound that inhibits the binding activity between protein Pn and γ-secretase   The method according to any one of claims 6 to 9, wherein Aβ is Aβ40 or Aβ42.   11. A method according to any of claims 6 to 10 for the treatment or prevention of Alzheimer's disease or cancer.   Aβ, characterized in that the expression level of at least one gene selected from (G1) to (G12) or a substance that reduces the activity of at least one protein selected from (P1) to (P12) is selected. Screening method for production inhibitory substance. A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) a reporter gene under the control of the transcriptional regulatory region of (a1) gene Gn (n is any integer from 1 to 12) or (a2) gene Gn (n is any integer from 1 to 12) 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 Selecting a substance that reduces the expression level as a candidate for an Aβ production inhibitor compared to A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) contacting a cell expressing amyloid precursor protein with at least one protein selected from (P1) to (P12) and a test substance (b) measuring the activity of the protein (c) A step of selecting a substance that reduces the activity of the protein as compared to the case where it is measured in the absence of the test substance.   15. The method of claim 14, wherein the protein is provided by the cell itself.   The method according to claim 14 or 15, wherein the activity of the protein is measured using the production amount of Aβ as an index. A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) contacting at least one protein selected from (P1) to (P12) and a test substance with a cell expressing γ-secretase or a cell membrane fraction thereof; and (b) measuring γ-secretase activity. Step (c) A step of selecting a substance that reduces or modulates γ-secretase activity as compared with the case where it is measured in the absence of the test substance.   The method of claim 17, wherein the protein is provided by the cell itself.   A method for screening an Aβ production inhibitor, which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer of 1 to 12) and γ-secretase. A screening method for an Aβ production inhibitor comprising the following steps (a) to (c).
(A) a step of bringing protein Pn (n is an integer from 1 to 12) and γ-secretase into contact with a test substance (b) a step of measuring the binding activity of protein Pn and γ-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.   A non-human animal introduced with at least one gene selected from the above (G1) to (G12), wherein a substance that improves the phenotype that reflects the pathology of Alzheimer's disease is selected, or treatment of Alzheimer's disease or Prophylactic drug screening or efficacy evaluation method. 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 (G12) has been introduced; (b) at least one expression reflecting the pathology 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 to the case where the test substance is evaluated when not administered.   The phenotype is brain tissue, cerebrospinal fluid, Aβ amount in tissues such as blood, neuronal cell death, central nervous system inflammatory reaction, cognitive ability, amyloid plaque accumulation, cerebral blood flow and cerebral glucose 23. The method of claim 22, wherein the method is selected from the group consisting of metabolic amounts. 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 test animal-derived sample (b) Expression level of at least one gene selected from (G1) to (G12) in the sample or selected from (P1) to (P12) 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

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