WO2005106473A1

WO2005106473A1 - Method of screening therapeutic agent for neurodegenerative disease

Info

Publication number: WO2005106473A1
Application number: PCT/JP2005/008471
Authority: WO
Inventors: Yasuki Yoshimoto; Taiji Shimoda
Original assignee: Ono Pharmaceutical Co., Ltd.
Priority date: 2004-04-28
Filing date: 2005-04-27
Publication date: 2005-11-10

Abstract

A method of screening a substance capable of inhibiting any binding between protein with S-100β binding capacity and S-100β, comprising bringing a protein with S-100β binding capacity into contact with S-100β in the presence and absence of an analyte, measuring the levels of binding between protein with S-100β binding capacity and S-100β in two instances, and comparing them to each other. The substance obtained by this screening method, partial peptide from the protein of the invention, antibody capable of inhibiting any binding between the protein and S-100β and polynucleotide comprising the base sequence, or part thereof, complementary to the polynucleotide coding for the protein are useful in the treatment and diagnosis of, for example, neurodegenerative diseases, such as Alzheimer's Disease, Parkinson's disease and amyotrophic lateral sclerosis.

Description

Screening method for therapeutic drugs for neurodegenerative diseases

The present invention relates to a method of screening for a substance that inhibits the binding between S-100J3 binding protein and S-100 / 3. More specifically, a method for preventing neurodegenerative diseases using the S—100] 3 binding protein and a screening method for Z or a therapeutic agent, a substance obtained by the screening method, The present invention relates to a 0/3 binding protein, an antibody thereof, a polynucleotide encoding the S-1003 binding protein or a polynucleotide complementary to the polynucleotide, a pharmaceutical composition containing the substance, and the like. Background art

The cells that make up the parenchyma of the central nervous system include neurons and glial cells, and the number of cells is much greater in glial cells than in neurons. Glial cells play a role in supporting the function of nerve cells. Astrocytes, a type of glial cell, maintain extracellular ionotropic neurotransmitter homeostasis as supporting cells for neurons (Pharmacology and function, pp. 193-228, Academic Press, Inc., (1993 )) And the supply of neurotrophic factors (Hmrmacology and function, pp. 267-308, Academic Press, Inc., (1993)), which play an important role in controlling brain function. It is thought to be acting.

Conventionally, neurodegenerative diseases (Alzheimer's disease, amyotrophic lateral sclerosis, etc.) have been thought to be mainly due to abnormalities in nerve cells. However, in recent years, the idea that glial cells surrounding nerve cells, especially astrocytes, are functionally abnormal has become prominent.

S—100 J3 (protein having the amino acid sequence of SEQ ID NO: 37) is Astrocyte-specific protein that has a calcium-binding site and regulates intracellular calcium concentration, binds to various cytoskeletal proteins, and is involved in changes in cell morphology and release of neurotransmitters (Trends Biochem) Sci, 13, 437-443, (1988)). S-103 is produced by activation of astrocytes and released outside the cell. The released S-103 exhibits trophic and traumatic effects on nerve cells (Progress in Neurobiology, 46, 71-82, (1995)), and also exerts an effect on astrocytes. In addition, it has been reported that it has a growth promoting action and an inducing action of inducible nitric oxide synthase (J. Biological Chemistry, 271.2543-2547, (1996)).

It is also known that S-100] 3 is overexpressed in activated astrocytes during brain injury. The amount of S—100β increased in the brain of Alzheimer's patients (Proc. Natl. Acad. Sci. USA, 86, 7611-7615, (1989)), and the increase was in the brain of Alzheimer's patients. It has been reported that there is a correlation with senile plaque density (J. Neurosci. Res., 398-404, (1994)). In addition, activated astrocytes with overexpression of S-100i3 exist near the senile plaques in the brain of the Alhaima patient, and the intensity of the expression is correlated with abnormal neuronal process extension. Is reported (J. Neuropathol. Exp. Neurol., 55, 273-279, (1996)). In addition, stroke (Stroke, 30, 1190-1195, (1999)), head injury (Neurosurgery, 45, 477-483, (1999)), multiple sclerosis (Acta Neurol Scand, 96, 142-144) , (1997)), Down syndrome (Pro Natl. Acad. Sci. USA, 86, 7611-7615, (1989)). Can be

Thus, the association between S-1000 J3 and neurodegenerative diseases has been strongly suggested. Also, various proteins such as the late glycation reaction product receptor (RAGE) are known as S-10 binding proteins (Mol Cell Biol, 23, 4870-81, (2003)). The detailed mechanism of action, such as molecules involved in the intracellular signal transduction system by the specific binding protein S-103, is not yet clear. 1

Disclosure of the invention

As described above, various proteins are known as S-100 binding proteins. And a method for screening for a prophylactic and / or therapeutic agent for a neurodegenerative disease using an S—100] 3 binding protein that is a specific signaling molecule for S—100] 3. A substance obtained by the method, an S-1000 binding protein, an antibody thereof, and a polynucleotide containing a polynucleotide encoding the S-100 binding protein or a polynucleotide complementary to the polynucleotide. Drug compositions and the like are not known at present, and it has been desired to provide screening methods and pharmaceutical compositions.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, identified S-100 / 3 binding proteins by the West-Western method, and furthermore, these proteins were identified as S-1000 J3. They found that they were involved in information transmission, and completed the present invention.

That is, the present invention

1. A screening method for a substance that inhibits the binding between S—100 binding protein and S—100 β,

2. The screening method according to the above 1, wherein the amount of binding between the S-100 // 3 binding protein and S-100 is compared in the absence and presence of the test substance.

3. Contact the S—100 / 3 binding protein with S—100 in the absence and presence of the test substance, and in each case the S—100 / 3 binding protein and S—100 (3) measuring the amount of binding of 3, and comparing

4. An amino acid sequence in which the S—100 binding protein is one amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102 and 126, or substantially the same as the amino acid sequence The screening method according to the above 1, which is a protein or a partial peptide thereof,

5. The S—100] 3 binding protein also has the amino acid sequence represented by SEQ ID NO: 126. 1

Or the protein having an amino acid sequence substantially identical to the sequence thereof or a partial peptide thereof,

6. The screening method according to the above 1, using ELISA, immunoprecipitation assay, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, luciferase assay, reporter assay or protein reconstituted assay.

7. Contact the S-100 binding protein with S-100 in the absence and presence of the test substance, and determine the amount of binding between the S-1003 binding protein and S-100] 3 in each case by ELISA and immunization. 4. The screening method according to the above 3, wherein measurement and comparison are performed with a sedimentation assay, a fluorescence resonance energy transfer, a bioluminescence resonance energy transfer, a luciferase assay, a reporter assay or a protein reconstruction assay.

8. A screening method for a substance that alters the amount of factor production induced by binding of S-100 binding protein to S-100] 3,

9. The screen-junging method according to the above item 8, wherein the amount of factor production induced by the binding of S-100 binding protein and S-100 J3 is compared in the absence and presence of the test substance.

10. The screening method according to the above 9, wherein the S-100 binding protein and S-100 are brought into contact with each other in the absence and presence of the test substance, and the amount of the factor produced in each case is measured and compared.

1 1. The S-100-binding protein has an amino acid sequence selected from one of SEQ ID NOs: 1 to 36, 84 to 102 and 126, or a protein or a partial peptide thereof having an amino acid sequence substantially identical to the amino acid sequence. The screening method according to the above 8, which is

12. The screening according to the above item 8 or 11, wherein the S-100 / 3 binding protein is an amino acid sequence represented by SEQ ID NO: 126, a protein having substantially the same amino acid sequence as that sequence, or a partial peptide thereof. Method, 1 3. The screening method according to the above 8 using an ELISA,

14. Contacting S-100β with a cell having an amino acid sequence represented by SEQ ID NO: 126 or a protein having an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof, 10. The screening method according to 10 above, wherein the amount of JV1CP-1 production induced in the absence and presence of is measured by ELISA and compared.

1 5. A substance that inhibits the binding between S- 100 3-binding protein and S- 100 j8,

1 6. A substance that changes the amount of factor production induced by the binding of S-100 3 to S- 100 3 binding protein,

1 7. The factor induced by the binding of S- 100 j3 binding protein to S- 100 J3 is MCP-1, IL-6, IL-18, Aβ, or Ca ^2+. The substance described in 6,

1 8. A protein having an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102 and 126, or a protein having an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof, 16 Substances described in 6,

1 9. The protein which has an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or an amino acid sequence substantially identical to the amino acid sequence or a partial peptide antibody thereof. The substance described in 15 or 16;

20. S- 100] substance that suppresses expression of 3-binding protein,

2 1. A protein having an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or a protein having an amino acid sequence substantially identical to the amino acid sequence, or a polypeptide encoding a partial peptide thereof 21. The substance according to the above 20, which is a polynucleotide comprising a nucleotide sequence complementary to a nucleotide or a part thereof,

22. The substance according to 21 above, wherein the polynucleotide is S iRNA or antisense DNA; '23. The substance according to 15 to 16 and / or 20 above as an active ingredient 2005/008471

A pharmaceutical composition,

24. The pharmaceutical composition according to the above 23, which is an agent for preventing and / or treating a neurodegenerative disease.

2 5. Neurodegenerative diseases include Parkinson's disease, Parkinson's syndrome, striatal substantia nigra, Huntington's disease, chorea-ataxia, progressive supranuclear palsy, diffuse Lewy body disease, basal ganglia Degeneration, Alzheimer's disease, dementia (dementia), spinocerebellar degeneration, motor neuropathy, demyelinating disease, cerebrovascular disorder · brain tumor · hypovolemic shock · traumatic shock · head injury and Z 24. The pharmaceutical composition according to the above item 24, which is a neurological dysfunction due to cerebrospinal cord injury, a cerebral spinal cord disease due to an infectious disease, a neurological dysfunction due to toxic radiation, a mental illness, epilepsy, Mage syndrome, dystonia, Down syndrome, or a sleep disorder. object,

26. A screening kit for a substance that inhibits the binding between S—100] 3 binding protein and S—100 j3,

27. A method for producing a substance that inhibits the binding between S-100jS-binding protein and S-100; 3, which comprises the screening method described in 1 or 8 above. 27.A method for inhibiting the binding between S-100 // 3 binding protein and S-100 // 3, which comprises using the substance obtained by the production method according to 27.

29. Prevention and prevention of neurodegenerative diseases, characterized by administering to mammals an effective amount of a substance that inhibits the binding between S- 100] 3 binding protein and S- 100] 3. And Z or treatment method, and

30. Use of a substance that inhibits the binding between S—100 03 binding protein and S—100 ^ for the manufacture of a prophylactic and / or therapeutic agent for neurodegenerative diseases. The amino acid sequence substantially identical to S-1000j8 according to the present invention includes about 85% or more, preferably about 90% or more, more preferably about 9% or more of the amino acid sequence represented by SEQ ID NO: 37. Amino acid sequences having a homology of 5% or more, and most preferably about 98% or more, can be mentioned. Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 37 include, for example, SEQ ID NO: 37 5 008471

And a protein having substantially the same activity as the protein having the amino acid sequence described in SEQ ID NO: 7 and having the same amino acid sequence as described in SEQ ID NO: 7. Examples of substantially the same activity include signal transduction activity, receptor binding activity, and epitope activity. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, the signal transduction activity or the receptor binding activity is equivalent (for example, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Although it is preferable, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

Further, as the protein of the present invention, for example, in addition to the protein consisting of the amino acid sequence of SEQ ID NO: 37, some of the amino acids (preferably 1 to 10, more preferably 1 to 5) are used. Deletion, substitution with an arbitrary amino acid, or addition or insertion of an arbitrary amino acid at several positions (preferably 1 to 10 positions, more preferably 1 to 5 positions) in the amino acid sequence And those containing an amino acid sequence obtained by combining them. Here, the position of deletion, substitution, addition, or insertion of an amino acid is not particularly limited.

The partial peptide of S-100 according to the present invention is the partial peptide of S-100 according to the present invention described above, which is substantially the same as the partial peptide S-100j according to the present invention. Any peptide may be used as long as it has the same activity. For example, a peptide having at least 10 or more, preferably 30 or more, more preferably 100 or more continuous amino acid sequences of the amino acid sequence of S-100; 3 is included. Further, the partial peptide according to the present invention may be a partial peptide in which some amino acids (preferably 1 to 10, more preferably 1 to 5) in the amino acid sequence have been deleted or substituted with any amino acid. An amino acid sequence in which an arbitrary amino acid is added or inserted in several places (preferably 1 to 10 places, more preferably 1 to 5 places) in the amino acid sequence, and a combination thereof. Those containing a amino acid sequence are also included. Here, the position of amino acid lack, substitution, stress [1], or insertion is not particularly limited.

The [S-100] 3-binding protein may be any amino acid sequence as long as the protein has an activity of binding to S-100.

The S-1Ο-binding protein is preferably an amino acid sequence substantially identical to the amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or substantially the same as the amino acid sequence. And more preferably a protein containing an amino acid sequence identical to or substantially identical to the amino acid sequence shown in SEQ ID NO: 126.

An amino acid sequence identical to or substantially identical to an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 contains the amino acid sequence ^ Protein (hereinafter, referred to as the protein of the present invention) Any amino acid sequence may be used as long as it has an activity of binding to Is 100.

In addition, the protein according to the present invention includes, for example, the amino acid sequence of SEQ ID NOS: 1 to 36, 84 to 102, and 126, and a part of the amino acids (preferably 1 ~ 10, more preferably 1-5, and more preferably 1-3, and some amino acids (preferably 1-10, more preferably 1-5) , More preferably 1 to 3) substituted with another amino acid, and the amino acid sequence thereof has several amino acids (preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3). ) Is added or inserted, and those having an amino acid sequence obtained by combining them are also included. 'When the amino acid sequence is deleted, substituted, added or inserted as described above, the position is not particularly limited.

As a protein having an amino acid sequence substantially identical to the amino acid sequences represented by SEQ ID NOs: 1 to 36, 84 to 102, and 126, SEQ ID NO: 1 To about 85%, preferably about 90% or more, more preferably about 95% or more, and most preferably about 95% or more. Amino acid sequences having a homology of 98% or more. Examples of proteins having an amino acid sequence substantially identical to the amino acid sequences described in SEQ ID NOs: 1 to 36, 84 to 102, and 126 include, for example, SEQ ID NOs: 1 to 36, 84 to It has an amino acid sequence substantially identical to the amino acid sequence described in 102 and 126, and has the amino acid sequence described in SEQ ID NOs: 1 to 36, 84 to 102, and 126. A protein having substantially the same activity as the protein having the same is preferred. Substantially the same activity is as defined above.

The partial peptide of the protein according to the present invention (hereinafter, referred to as the partial peptide of the present invention) is the partial peptide of the protein of the present invention, and is substantially the same as the protein of the present invention. Any peptide may be used as long as it has the above activity. For example, a peptide having at least 10 or more, preferably 30 or more, more preferably 100 or more continuous amino acid sequences in the amino acid sequence of the protein according to the present invention is included. The partial peptide according to the present invention may be a partial peptide in which some amino acids (preferably 1 to 10, more preferably 1 to 5) in the amino acid sequence are deleted, or substituted with any amino acid. The amino acid sequence of which amino acid is added or inserted at several positions (preferably 1 to 10 positions, more preferably 1 to 5 positions) of the amino acid sequence, and an amino acid obtained by combining them. Those containing sequences are also included. Here, the position of lack, substitution, addition, or insertion of the amino acid is not particularly limited. Substantially the same activity is as defined above.

In the S-100 of the present invention, the protein of the present invention, and the partial peptides thereof, the constituent amino acids may have a carboxyl group, and the carboxyl group is amidated or esterified. May be. The ester groups include, for example, alkyl such as methyl, ethyl, propyl, and butyl. 08471

And esterified by a cycloalkyl group such as cyclopentyl or cyclohexinole, or a phenyl group such as phenyl // naphthinole. Further, the amino group of the constituent amino acids may be protected with a formyl group, an acetyl group or the like. In addition, those having a sugar chain bonded thereto are also included in the protein and the partial peptide thereof according to the present invention.

As the salt of S-100 according to the present invention, the protein according to the present invention, and their partial peptides, salts with an acid (such as an inorganic acid or an organic acid) or a base (such as an alkali metal salt) are used. Physiologically acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, etc.) or organic acids (formic acid, acetic acid, propionic acid, fumaric acid, citric acid, oxalic acid, apple) Acid, methanesulfonic acid, benzenesulfonic acid, succinic acid, maleic acid, tartaric acid, benzoic acid, etc.).

Factors induced by the binding of S-100 binding protein to S-100] 3 include chemokines, cytokins, peptides, or second messengers. Specific examples include IL-8, IL-6, MCP_1, Α | 8, Ca ^{2+ and the} like. ■.

The polynucleotide (DNA, RNA or DNAZRNA chimera) encoding the protein or its partial peptide according to the present invention includes a nucleotide sequence encoding the protein or its partial peptide according to the present invention. Anything can be used as long as it is available.

The polynucleotide encoding the protein or its partial peptide according to the present invention may be any of genomic DNA, cDNA, synthetic DNA, RNA, cRNA, or DNA-RNA hybrid.

The polynucleotide encoding the protein of the present invention or a partial peptide thereof (hereinafter, referred to as the DNA of the present invention) has a sequence number selected from SEQ ID NOs: 38 to 73, 103 to 121, and 127. Base sequence shown In addition to the polynucleotide having a sequence, it has a nucleotide sequence that hybridizes under stringent conditions with the polynucleotide or a polynucleotide having a complementary sequence thereof, and has substantially the same properties as the protein of the present invention. Any polynucleotide may be used as long as it is a polynucleotide encoding a protein having the same. As such a polynucleotide, the nucleotide sequence represented by SEQ ID NO: 38 to 73, 103 to 121, and a nucleotide sequence selected from the group consisting of 127, and about 90% or more, 'Preferably, a polynucleotide having a base sequence having a homology of about 95% or more, and the like can be mentioned. Hybridization is carried out by a known method, for example, Molecular Cloning (Sambrook, J., Fritsch, EF and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or Current Protocol in Molecular Biology (FM Aus bel et al., published by John Wiley & Sons, Inc.). Stringent conditions include, for example, hybridization at 65 ° C and a washing treatment at 65 ° C with a buffer containing 0.1 XSSC, 0.1% SDS.

The DNA according to the present invention may be amplified by chemical synthesis or by a PCR method using a synthetic DNA primer encoding a part of the protein of the present invention or a partial peptide thereof, or It can be obtained by a hybridization method using a synthetic DNA encoding the protein of the present invention or a part of its partial peptide as a probe. Recently, cDNA libraries of various human tissues have been marketed and can be used in accordance with the attached instructions. Examples of the thread used for obtaining the DNA according to the present invention by the PCR method or the hybridization method include, for example, a tissue of human in which S-100 is considered to act. , Preferably the brain. The hybridization method can be performed, for example, according to the method described above or the method described in Gene, 10, 63 (1980). The DNA obtained in this manner was obtained by converting a vector DNA containing the DNA. The required amount can be obtained by introducing it into an appropriate host and growing it.

As a method for obtaining the protein or a partial peptide thereof according to the present invention,

(1) a method for purification and isolation from living organisms or cultured cells,

(2) a method for peptide synthesis, or

(3) a method of producing using genetically modified technology,

Etc., but the method (3) is preferable industrially.

Examples of an expression system (host-vector system) for producing a protein or peptide using a genetic recombination technique include, for example, an expression system for bacteria, yeast, insect cells, and mammalian cells.

For example, when expressing in E. coli, an initiation codon (ATG) is added to the 5 'end of the polynucleotide encoding the mature protein portion, and the resulting cDNA is converted to an appropriate promoter (eg, trp promoter, 1 ac The promoter is connected downstream of the LPL promoter, T7 promoter, etc.) and inserted into a vector that functions in E. coli (eg, pBR322, pUC18, pUC19, etc.) to produce an expression vector. Next, Escherichia coli (eg, E. ColiDHl, E. ColiJM109, Ε · Coli HB101, etc.) transformed with the expression vector is cultured in an appropriate medium, and the protein or peptide of interest is extracted from the cells. You can get children. In addition, the use of a bacterial signnanolepeptide (eg, a signal peptide of pe1B) allows secretion of a target protein or peptide during periplasm. In addition, it can produce fusion proteins with other polypeptides.

When expressed in mammalian cells, the DNA according to the present invention may be expressed in an appropriate vector (for example, a retrowinoresetter, papillomawinores vector, vaccinia virus vector, SV40-based vector, etc.). An expression vector is prepared by inserting downstream of a promoter (eg, SV40 promoter, LTR promoter, meta-mouth thionein promoter, etc.). Then get Transform appropriate mammalian cells (eg, monkey COS-1 cells, COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.) with the obtained expression vector, and culture the transformants in an appropriate medium. As a result, the protein of the present invention or its partial peptide is expressed. Furthermore, it is also possible to produce a fusion protein by linking with another polypeptide, for example, a cDNA fragment encoding the constant region (Fc portion) of the antibody.

In the case of expression in insect cells, the DNA of the present invention is inserted downstream of an appropriate promoter (polyhedrin promoter, P10 promoter, etc.) and inserted into a viral vector that functions in insect cells. To produce an expression vector. As the insect cell, for example, when the virus is Ac NPV, a cell line (S f cell) derived from the larva of night roth moth is used. When the virus is BmNP V, a cell line derived from silkworm (BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn cell). , JL, InVivo, 1977, Vol. 13, p213_217) As the insects, silkworm larvae, etc. are used. Technology (Bio / Technology), 1988, Vol. 6, p. 47-55.

When expression is performed in yeast, the DNA of the present invention is connected downstream of an appropriate promoter (PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc.), and a vector (pSH19, p SH15) to produce an expression vector. Next, the yeast transformed with the expression solid (eg, Saccharomyces 'Celebiche AH22, AH22R-, 20B-12, Schizosaccharomyces ■ bomb NC YC 1913, Pichia' Pastoris KM71, etc.) The desired protein or peptide can be obtained by culturing in an appropriate medium. The protein or peptide obtained as described above must be isolated and purified by general biochemical methods. 8471

Can do.

As a method for screening a substance that inhibits the binding of the protein or its partial peptide to S-100β according to the present invention, for example, the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 126 is used. By comparing the binding amount of a protein having an amino acid sequence of the above or its partial peptide in the presence and absence of the test substance by ELISA (Enzyme-Linked Immunosorbent Assay). .

Further, the screening method of the present invention can be performed, for example, by the following method. That is, a protein according to the present invention or a partial peptide thereof, or a fusion protein obtained by adding Tag (eg, His-Tag, GST, etc.) to the protein or an Atsushi well on which an antibody against the protein is immobilized, in the presence of the test substance Alternatively, after adding S-100 / 3 labeled with biotin or the like in the absence of the protein (however, when the above antibody is used, the protein of the present invention or its partial peptide or T After adding the fusion protein with ag, add S-100 labeled with biotin, etc.), and a commercially available kit for detecting free S-100 / 3 labeled substance such as biotin Alternatively, a method of detecting or quantifying the antibody using an antibody against S-100β may be used. In such a case, the substance that releases S-100] 3 can be selected as a substance that inhibits the binding of the protein of the present invention or a partial peptide thereof to S-100β. Similarly, the method of adding the protein according to the present invention, a partial peptide thereof, or a fusion protein obtained by adding Tag thereto to an Atsushi well having S-100) 3 immobilized thereon is also carried out. be able to. The immobilization of the protein according to the present invention, the partial peptide thereof, the fusion protein having Tag added thereto, or S-100] 3 can be carried out, for example, by immobilizing the protein labeled with biotin and an at-sealing gel. It can be performed by reacting with avidin.

The screening method of the present invention is carried out by a method using Biacore. 5008471

be able to. Specifically, the protein according to the present invention or its partial peptide

Prepare a tag attached tag and capture it on a sensor chip on which an anti-Tag antibody is immobilized to prepare a sensor surface. To this, S-1'000 and a test substance are added, and the binding amount and force kinetics of the protein or partial peptide of the present invention and S-IOOJS are measured using Biacore with an interaction sensorgram. From a quantitative kinetic study, a substance that reduces the binding ability of the protein of the present invention or its partial peptide to a substance that inhibits the binding of the protein or its partial peptide to S-100 according to the present invention is used. Select as

The screening method of the present invention can be carried out by a method using a chemically amplified luminescence assay. Specifically, biotinylated S-100 and a protein or a partial peptide thereof according to the present invention to which Tag (for example, His-Tag, GST, etc.) is added are prepared by a known method. In a mixture of an Axceptor bead obtained by complexing a Tag antibody and a protein or a partial peptide thereof according to the present invention with Tag, in the presence or absence of a test substance, biotinylated S-100j8 and Streptavidin-donor beads are added, and the fluorescence intensity of each is detected or quantified, and the substance whose fluorescence intensity decreases is selected as a substance that inhibits the binding of the protein of the present invention or its partial peptide to S-1003. .

The screening method of the present invention can be carried out by a method using Lucifuslase Atsushi. Specifically, an expression vector having a DNA encoding the GAL4 DNA binding domain of yeast or an expression vector having a VP16 activation domain of a viral simplex virus, S-100] 3 and the protein or the protein of the present invention, Each partial peptide was introduced, and a fusion protein consisting of a GAL4 DNA binding domain and S-100] 3, a VP16 activation domain and a protein according to the present invention or a fusion protein consisting of the partial peptide were Create a vector to express. Mammalian cells were screened using a reporter containing these expression vectors, a GAL4 binding region and a sequence encoding firefly luciferase. The luminescence intensity in the presence or absence of the test substance is detected or quantified by various luciferase quantification reagents. A substance that reduces the luminescence intensity as compared to that in the absence of the test substance is selected as a substance that inhibits the binding between the protein of the present invention or its partial peptide and S-100.

The screening method of the present invention can be performed by a method using immunoprecipitation. Specifically, a transformant transformed with DNΑ according to the present invention tagged with Tag is prepared by the above-described method, and the transformant (for example, U373MG cells) is cultured on a culture plate. And proliferate. A test substance is added to the cell extract of the transformant, immunoprecipitation is performed with a Tag antibody, and the amount of co-precipitated S-100 is detected or quantified by a method such as Western blotting. A substance that reduces the amount of co-precipitated S-100 compared to that in the absence of the test substance is selected as a substance that inhibits the binding of the protein of the present invention or its partial peptide to S-100. .

The screening method of the present invention is based on a method using a fluorescence resonance energy transfer (FRET), a method using a bioluminescence resonance energy transfer (BRET). This can be performed in the separate volume of Experimental Medicine, Handbook for Protein Experiments, Yodosha (2003)). Specifically, a reporter protein (eg, lactamase, galactosidase, etc.) that converts a dye / fluorescence-producing substrate or a fluorescently labeled protein is fragmented into two, one of which is S-100] 3, and the other is one. Vectors each expressing the protein of the present invention or a partial peptide thereof in a fused form are produced. In the cultured cells transformed by both expression vectors, the substance whose absorbance or fluorescence intensity decreases compared to the absence of the test substance is defined as the protein of the present invention or its partial peptide and S-100. Is selected as a substance that inhibits the binding of.

In addition, a vector that expresses a protein obtained by fusing S-100 / 3 to either end of the luciferase gene, A transformed cell is produced by a vector expressing the protein poor by fusing the protein according to the above or a partial peptide thereof. A test substance and then a substrate are added to the cell extract to form a color, and a substance whose fluorescence intensity decreases as compared to the absence of the test substance is determined by combining the protein of the present invention or its partial peptide with S-100. Select as a substance that inhibits binding. .

The screening method of the present invention can also be carried out by a method using a protein reconstitution assay (PCA); Takeshibashi et al., Experimental Medicine Separate Volume, Protein Experiment Handbook, Yodosha (2003)). it can.

The method for screening a substance that changes the amount of factor production induced by the binding of the protein or its partial peptide to S-100 / 3 according to the present invention includes, for example, SEQ ID NO: 1 26. Produce a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by 26, or a partial peptide thereof, in a cell, and in the presence or absence of the test substance, It can be performed by comparing the production amounts of factors induced, for example, MCP-1 and IL-16 by ELISII.

Furthermore, the screening method of the present invention can be performed, for example, by the following method. That is, a transformant transformed with the DNA according to the present invention is prepared by the above method. The transformant (eg, U373MG cells) is spread on a culture plate and grown. The test substance and S-100β (for example, carboxymethylated S-100β) are added and cultured. The culture supernatant after 24 hours is collected, and for example, MCP-1 produced as a result of S-1000 stimulation is quantified using a commercially available MCP-1 measurement kit or the like. The substance that decreases the amount of MCP-1 is selected as a substance that changes the amount of factor production induced by the binding of the protein of the present invention or its partial peptide to S-100 / 3. ·

The substance obtained by the screening method of the present invention is a test substance, for example, A substance or a salt thereof selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc .; A substance that inhibits the binding of the partial peptide to S-103 and / or a factor induced by the binding of S-white matter or its partial peptide according to the present invention to S-103] 3 It is a substance that changes the amount of production. '

The substance obtained by the screening method of the present invention is converted to a salt by a known method. As the salt, a pharmaceutically acceptable salt is preferable.

Examples of the salt include an alkali metal salt, an alkaline earth metal salt, an ammonium salt, an amine salt, and an acid addition salt.

The salt is preferably water-soluble. Suitable salts include salts of alkali metals (such as potassium and sodium), salts of alkaline earth metals (such as calcium and magnesium), ammonium salts, and pharmaceutically acceptable organic amines (tetramethylammonium, triethylamine, Methylamine, dimethylamine, cyclopentylamine, benzyl ^ / amine, phenetamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) aminomethane, lysine, arginine, and Ν-methyl-D-glucamine. Can be

The acid addition salt is preferably water-soluble. Suitable acid addition salts include, for example, inorganic salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, or acetate, lactate, tartrate, Organic salts such as benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, and dalconate.

The substance obtained by the screening method of the present invention, that is, the substance that inhibits the binding of the protein of the present invention to S-103 and the protein of the present invention or the protein of the present invention that binds to S-; Substances that alter the friction of induced factors 5 008471

Examples include the protein of the present invention or a partial peptide thereof, and an antibody that inhibits the binding of the protein of the present invention to S-100. .

An amino acid sequence preferably selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 as a substance that inhibits the binding of the protein of the present invention or a partial peptide thereof to S-100 J3 A protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as described above, more preferably an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 126. It is a protein or a partial peptide thereof.

'As a substance that changes the amount of factor production induced by the binding of the protein or its partial peptide of the present invention to S-100J3, preferably SEQ ID NOS: 1 to 36, 84 to 1 A protein having an amino acid sequence identical or substantially identical to an amino acid sequence selected from 02 and 126, or a partial peptide thereof, and more preferably an amino acid sequence represented by SEQ ID NO: 126 Or a partial peptide thereof containing the same or substantially the same amino acid sequence as

The antibody that inhibits the binding of the protein according to the present invention to S-100 is an antibody against the protein according to the present invention or a partial peptide thereof or S-100] 3, and the binding between the two. Those having an inhibitory activity are mentioned. Such an antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody of the present invention or a partial peptide thereof may be used as an antigen in a known manner (Takeshida et al., Experimental Medicine Supplement, Protein Experiment Handbook, Sheep Tsuchiya (2003)).

An antibody that inhibits the binding of the protein of the present invention to S-100 is preferably an amino acid sequence selected from SEQ ID NOS: 1 to 36, 84 to 102, and 126. An antibody against a protein containing the same or substantially the same amino acid sequence or a partial peptide thereof, and a protein according to the present invention and S- An antibody that inhibits the binding of 100β.

Examples of the substance that suppresses the expression of the S—100] 3 binding protein include a nucleotide sequence complementary to a polynucleotide encoding a protein having a binding activity to S—100 jS or a part thereof. Included polynucleotides include, for example, antisense DNA, antisense RNA, SiRNA and the like. The polynucleotide comprising the nucleotide sequence complementary to the polynucleotide encoding the protein according to the present invention or a part thereof may have the DNA according to the present invention inserted into the antisense region of the vector as described above. (Sasaki et al., Separate Volume on Experimental Medicine, Revised Procedures for Biological Experiments, Yodosha (2003)).

The amino acid sequence preferably selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 as a substance that suppresses the expression of the protein according to the present invention, which is an S—100] 3 binding protein A nucleotide sequence complementary to a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the above, or a polynucleotide comprising a part thereof, such as antisense DNA, antisense RNA, S i RNA and the like.

Substance Obtained by the Screening Method of the Present Invention, Partial Peptide of the Protein of the Present Invention, Antibody that Inhibits the Binding of the Protein of the Present Invention to S-100 / 3, and Antisense of the DNA of the Present Invention DNA, antisense RNA or Si RNA is useful for the prevention and / or treatment of diseases involving S-100] 3.

Diseases involving S-100 ^ include neurodegenerative diseases and the like. As used herein, neurodegenerative diseases include nerve cells (central nerves (eg, cranial nerves, spinal nerves, etc.)) and Z or peripheral nerves (eg, autonomic nervous system (eg, sympathetic nerves, parasympathetic nerves, etc.), motor nerves, etc. System, sensory nervous system)), including all diseases accompanied by degeneration, and is not limited by its etiology. 471

Parkinson's disease, Parkinson's syndrome, striatum substantia nigra, Huntington's disease, chorea-ataxia, progressive supranuclear palsy, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's Disease, dementia (dementia) (for example, cerebral vascular dementia (cerebrovascular dementia), Alzheimer's dementia (Alzheimer's dementia), senile dementia (senile dementia), Pick's disease, Frontotemporal dementia (frontotemporal dementia), familial dementia (familial dementia), etc., spinocerebellar degeneration (eg, Oliveb cerebellar atrophy, primary cerebellar cortical atrophy) Syndrome, familial spinocerebellar ataxia (eg, Matsucarde-Joseph's disease, etc.), dentate nucleus pallidum pallidus, atrophic atrophy, familial paraplegia, Friedreich's disease, etc.) For example, amyotrophic lateral sclerosis Familial amyotrophic lateral sclerosis, etc., demyelinating diseases (eg, multiple sclerosis, general sclerosis, acute sporadic encephalomyelitis, acute cerebellar inflammation, transverse myelitis, Guillain-Barre syndrome, etc.) ) As well as cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombosis, cerebral embolism, etc.), transient ischemic attacks, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, subarachnoid) Hemorrhage etc.) 'brain tumor (eg astroglioma, brain abscess etc.) · hypovolemic shock · traumatic shock · head injury and Z or cerebrospinal trauma (eg cerebral contusion · penetration · shear · Nervous dysfunction associated with compression lacerations, trauma at birth, infant whip-shatter syndrome, etc., cerebrospinal disease associated with infectious diseases (eg, meningitis, influenza encephalopathy, Kreutz-Erdyakov disease, dementia due to AIDS, etc.),Objects (arsenic, cadmium, organic mercury, sarin, soman, tabun, VX gas, etc.) 神経 neurological dysfunction due to radiation, mental illness (eg, neurosis, psychosomatic disorder, anxiety, schizophrenia, manic depression, etc.) And epilepsy, Mage syndrome, dystonia, Down syndrome, sleep disorders (eg, hypersomnia, narcolepsy, sleep apnea syndrome, etc.). Preferred as neurodegenerative diseases are, for example, Alzheimer's disease, Down's syndrome, multiple sclerosis, neurological dysfunction associated with stroke or cerebral infarction, Parkinson's disease, cerebrospinal disease associated with infection, epilepsy or amyotrophic lateral sclerosis It is.

In addition, the protein or its partial peptide according to this effort, The body, or the DNA of the present invention or its antisense DNA can be used for diagnosis of the above-mentioned diseases.

A substance obtained by the screening method of the present invention, a partial peptide of the protein of the present invention, an antibody that inhibits the binding of the protein of the present invention to S-100, or an antisense DNA of the DNA of the present invention The antisense RNA or siRNA is usually administered systemically or locally, generally orally or parenterally. Preferred are oral administration, intravenous administration and intraventricular administration. ,

The dosage varies depending on the age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is generally within the range of 100 g to 100 mg per adult per dose. It may be administered once to several times a day or parenterally once per adult per day, in the range of 10 / ig to 100 mg per day. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or may be required beyond the range. .

A substance obtained by the screening method of the present invention, a partial peptide of the protein of the present invention, an antibody that inhibits the binding of the protein of the present invention to S-100, or an antisense DNA of the DNA of the present invention; When administering antisense RNA or siRNA, solid compositions, liquid compositions and other fibrous compounds for oral administration, injections for parenteral administration, external preparations, suppositories Used as etc.

Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. Capsules include soft capsules and hard capsules.

In such solids or compositions, the one or more active substances may contain at least one inert diluent (e.g., ratatose, mannitol, dulose, hydroxypropylcellulose, microcrystalline cellulose, starch). , Po JP2005 / 008471

Rivier pyrrolidone, magnesium aluminate metasilicate, etc.). According to a conventional method, the composition pharmacologically excipients other than an inert diluent, for example, a lubricant (eg, magnesium stearate), a disintegrant (eg, calcium cellulose dalycholate), a stabilizer (eg, human serum albumin) , Ratatos, etc.) and solubilizing agents (arginine, aspartic acid, etc.).

Tablets or pills may be coated with a gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc., if necessary, or with two or more layers. You may. Also included are capsules of absorbable materials such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents (eg, purified water, ethanol). Etc.) may be included. Such compositions may contain, in addition to the inert diluents, wetting agents, suspending agents such as suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. For aqueous or non-aqueous solutions and suspensions, one or more active substances are mixed with at least one inert diluent. Examples of the aqueous diluent include distilled water for injection and physiological saline. Examples of the non-aqueous diluent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 '(registered trademark), and the like.

Such compositions may also contain preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents (eg, human serum albumin, ratatose, etc.), solubilizing agents (eg, arginine, aspartic acid, etc.). Auxiliaries may be included.

External preparations for parenteral administration include, for example, ointments, gels, creams, compresses, patches, liniments, sprays, inhalants, sprays, aerosols, eye drops, and Nasal drops and the like can be mentioned. These can be one or more 5 008471

It contains the above active substances and is produced by known methods or commonly used formulations.

Other compositions for oral administration include sprays which contain one or more active substances and are formulated in a manner known per se. The composition contains, in addition to the inert diluent, a stabilizer such as sodium bisulfite to provide isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citrate. It may be. The method for producing sprays is described in detail, for example, in US Pat. Nos. 2,868,691 and 3,095,355. '

Other compositions for parenteral administration include suppositories for rectal administration and pessaries for vaginal administration, which contain one or more active substances and are prescribed in a conventional manner. .

When the antisense DNA, antisense RNA or siRNA of the DNA according to the present invention is used for prevention or treatment of a disease associated with S—100. NA, antisense RNA or SiRNA can be administered to humans or mammals according to a standard method after inserting alone or into an appropriate vector such as a retrovirus vector or an adenovirus vector. They can be used as they are, or formulated as physiologically acceptable carriers such as auxiliary ribosomes (ribosomes, HVJ ribosomes, etc.) for promoting their introduction into cells.

The DNA according to the present invention, or a polynucleotide comprising a sequence complementary thereto or a part thereof, for example, is used as a probe to detect the DNA or mRNA according to the present invention. Therefore, the above-mentioned S-100 can be used as a reagent for examining related diseases. When a genetic factor is suspected as a cause of a disease associated with S-100; 3, genetic diagnosis can be performed using these factors.

The antibody against the protein or the partial peptide thereof according to the present invention relates to the present invention. Since S100 can detect such a protein, the above-mentioned S-100] 3 can be used as a reagent for testing a related disease.

The screening method of the present invention can be carried out in a kit comprising the necessary reagents. In particular,

a) Measurement buffer and washing buffer,

b) a sample of the protein or its partial peptide according to the present invention,

c) the choice ligand, and

d) A kit containing a ligand standard solution.

Here, the protein or preparation of a partial peptide thereof according to the present invention may be used as immobilized on the test vessel, the labeled ^{^{ligand, [3 H], [1}} 2 5 1], [1 4 C ], ^{[3 5} S] labeled present invention S-l 0 0 like;. 8 a suitable solvent or buffer may ,, ligand standard solution be used after dissolved in 0 1% © shea serum albumin Alternatively, a solution obtained by dissolving S-1000j3 in PBS containing may be used. As a measurement method, a buffer for measurement is added to a sample of the protein or partial peptide according to the present invention, and a test substance and a labeled ligand are added and reacted. After removing the reaction solution and washing with a washing buffer, the radioactivity is measured using a liquid scintillation counter.

The screening kit of the present invention contains the protein of the present invention or a partial peptide thereof, but may also contain the protein of the present invention or an antibody against the partial peptide thereof. In addition, the screening kit of the present invention includes a cell capable of producing the protein of the present invention or a partial peptide thereof.

(Preferably, those containing a transformant (for example, an animal cell or the like) transformed with the DNA of the present invention are also included.

[The invention's effect]

The screening method of the present invention comprises a substance which can be used for prevention and / or treatment of a disease associated with S-100] 3, a partial peptide of the protein of the present invention, an antibody against the same, or Antisense DNA of DNA according to the invention, 5008471

Development of Thisense RNA or Si RNA Can be used. Brief Description of Drawings

Fig. 1 shows the mRNA expression level of S-100 binding protein FLJ12587 in healthy subjects and Parkinson's disease tissues (N: healthy subjects, PARK: Parkinson's disease)

Figure 2 shows the mRNA expression level of the S-100] 3-binding protein DKFZ p76 1 HO421 in healthy subjects, amyotrophic lateral sclerosis and Alzheimer's disease tissue (N: healthy subjects, AL S: amyotrophic lateral sclerosis, A 1 z: Alzheimer's disease).

FIG. 3 shows the mRNA expression level of the S-100 / 3 binding protein POGZ1 in healthy human and Alzheimer's disease tissues (N: healthy human, Alz: Alzheimer's disease).

FIG. 4 shows the expression level of: mRNA of S-100 / 3-binding protein MGC15730 in a healthy human Alzheimer's disease tissue (N: healthy human, A1z: Alheimer's disease).

Fig. 5 shows the mRNA expression level of the S-100] 3-binding protein FKRP in healthy subjects, amyotrophic lateral sclerosis and Alzheimer's disease tissues (N .: healthy subjects,. ALS: amyotrophic lateral cord). Sclerosis, _A1Z : Alzheimer's disease).

FIG. 6 shows the binding between the BAP28.2 protein and the S ^ 1003 protein (Mock: negative control). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1: Identification of S-100 binding protein by West-Western method. 5008471

Separate volume 8 New Cell Engineering Experiment Protocol (1993, Shujunsha, Cancer Research Division, Institute of Medical Science, The University of Tokyo). That is, human brain-derived mRNA was prepared, cDNA was synthesized using reverse transcriptase, and then second-strand synthesis was performed using Escherichia coli-derived DNA polymerase, DNA ligase, and RNAs. After the completion of the synthesis, blunting was performed using {4 DN} polymerase. After completion of the blunting, EcoRI linker ligation was performed, and EcoRI and lambda gt11 that had been treated with allyl phosphatase were ligated with T4 ligase. Library 1 was amplified according to a known method.

Identification of the binding protein by the West-Western method was performed as follows. After the library was induced with isopropyl-1-D-galactopyranoside, 5 × 10 ⁴ plaques were expressed per 15 Omm petri dish and transferred to a PVDF membrane. After blocking, the plate was incubated for 1 hour at room temperature in a Tris buffer containing 10 μg / mL of S-100] 3 and 1 mM. Thereafter, the PVDF membrane was washed with a phosphate buffer, and further incubated with an S-100] 3 antibody at room temperature for 1 hour. The cells were washed again with a phosphate buffer and incubated with an iminog-mouth purine antibody conjugated to horseradish peroxidase. The PVDF membrane was washed again with a phosphate buffer, and positive clones were detected using a known chemiluminescent reagent. Positive clones were isolated in SM buffer and identified by sequence. Positive clones are shown below in the form of positive clone names (NCB IID or DDBJID, identified amino acid sequence, identified base sequence). RP 3—495010 (NCB IID: AL031 121, SEQ ID No. 1, SEQ ID No. 38), RP 11—160O5 (NCB IID: AC 01 5821, SEQ ID No. 2, SEQ ID No. 39), RP 11-67 G 7 (NCB IID: AC 01 1291, SEQ ID NO: 3, SEQ ID NO: 40), RP 1 -287021 (NCB IID: AL 136319, SEQ ID NO: 4, SEQ ID NO: 41), RP 3-509 I 19 (NCB IID : AL 12 JP2005 / 008471

1834, SEQ ID NO: 5, SEQ ID NO: 42), CTC-286 C20 (NCB IID: AC0273 14, SEQ ID NO: 6, SEQ ID NO: 43), RP11-444D3 (NCB IID: AL087322, SEQ ID NO: 7, Sequence) No. 44), ZNF 140-1 ikerotein (NCB IID: AF 1 556 56, column No. 8, SEQ ID No. 45), RP 11 1-4—45 1 G1 (NCB IID: AC 01 3727, SEQ ID No. 9, SEQ ID No. 46) ), RP 4-550H1 (NCB IID: AL 035420, SEQ ID No. 10, SEQ ID No. 47), FLJ 12991 (NCB IID: AK023053, SEQ ID No. 11, SEQ ID No. 48), FL J 22382 (NCB IID: AK026035) , SEQ ID NO: 12, SEQ ID NO: 49), IMAGE4423 166 (NCB IID: BC033161, SEQ ID NO: 13, SEQ ID NO: 50), I MAGE 52612 13 (NCB IID: BC036485, SEQ ID NO: 14, SEQ ID NO: 51), FL J 41109 (NCB IID: AK123 104, SEQ ID NO: 15; SEQ ID NO: 52), FLJ 42709 (NCB IID: A 124699, SEQ ID NO: 16, SEQ ID NO: 53), SEC 14-1 ike 2 (NCB IID: BC04833. 7, SEQ ID NO: 17, SEQ ID NO: 54), DKF Zp31 3 P0 1 1 (NCB IID: AL 83,2467, SEQ ID NO: 18, SEQ ID NO: 55), I MAGE 2823852 (NCB I ID: BC001 102, SEQ ID NO: 19, 'SEQ ID NO: 56), FL J 34134 (NCB IID: AKO 914 53, SEQ ID NO: 20, SEQ ID NO: 57), Precerebellin (NC BIID: M58583, SEQ ID NO: 21, SEQ ID NO: 58), FL J13673 (NCB IID: AKO 2375 SEQ ID NO: 22, SEQ ID NO: 59), Smallglutam ine—richtetratr l-copeptide (NCB IID: BC000390, SEQ ID NO: 23, SEQ ID NO: 60), NT2R I 2006071 (NCB IID: AKO 56262, SEQ ID NO: 24, SEQ ID NO: 61), FL J20542 (NCB IID: NM-017871, SEQ ID NO: 25, SEQ ID NO: 62), KI AA0627 (DDB JID: AB 014527, SEQ ID NO: 26, SEQ ID NO: 63), DKFZ p

451 G 202 (NCB IID: AL 83 1955, SEQ ID NO: 27, SEQ ID NO: 64), Z incfingerrotei n. 3 1 15 BP (NCB I ID: AJ 1 32592, SEQ ID NO: 28, SEQ ID NO: 65), FLJ 43968 ( NCB I ID: AK125956, SEQ ID NO: 29, SEQ ID NO: 66), CG I-043 (NCB IID: AF 15 1801, SEQ ID NO: 30, SEQ ID NO: 67), DKFZ p 761 HO 421 (NCB IID: AL 831813, Sequence No. 31, SEQ ID NO: 68), 'POGZ 1 (NCBIID: NM-01 5100, SEQ ID NO: 32, SEQ ID NO: 69), CUB / Sushi 1 mu ltipledomain 1 rotein (NCB IID: AF 333704 _N SEQ ID NO: 33, SEQ ID NO: 70) (hereinafter CUB

Call it 5 u sh i 1. ), FL J 12587 (DDB J ID: BD 286

600, SEQ ID NO: 34, SEQ ID NO: 71), Cateninbinding protein (NCB IID: NM—014412, SEQ ID NO: 35, SEQ ID NO: 72) (hereinafter referred to as force-binding protein), MGC 15730 (NCCB IID: NM) — 032880, SEQ ID NO: 36, SEQ ID NO: 73), MG C40157 (NCB IID: BC 027986, SEQ ID NO: 84, SEQ ID NO: 103), FL J 351 13 (NCB I ID: AK092432, SEQ ID NO: 85, SEQ ID NO: 104) ), SNO oncogene (NCB IID: X15219, SEQ ID NO: 86, SEQ ID NO: 105), FLJ4223 (NC BIID: AK124207, SEQ ID NO: 87, SEQ ID NO: 106), FLJ35109 (NCB IID: AK092428, SEQ ID NO: 88, arrangement number 107), Basictranscriptionfact or 3 (NCBΓ ID: BC008062, SEQ ID NO: 89, SEQ ID NO: 108), CI 1 orf 23 (NCB IID: NM-0183 12, SEQ ID NO: 90, SEQ ID NO: 109) ), NAC transcriptionfactor (NC BIID: AK090650, SEQ ID NO: 91, SEQ ID NO: 1 10), Seq PT / JP2005 / 008471

uence 23787 fr om P atent WO 02068579 (NCB IID: CQ 737853, SEQ ID NO: 92, SEQ ID NO: 1 1 1), -S terilealha mo tifd oma in 6 (NCBI ID: BC01 2981, SEQ ID NO: 93, SEQ ID NO: 1 12) , CG I—024 (NCB IID: AF 1 32958, SEQ ID NO: 94, SEQ ID NO: 113), TR IB 2 homo 1 og 2 (NC BIID: NM—0-21643, SEQ ID NO: 95, SEQ ID NO: 1 14 ) _N Fu ku tinrelated protein (NCB IID: NM-024301, SEQ ID NO: 96, SEQ ID NO: 115) (hereinafter referred to as FKRP;), ZNF 37A (NCB ID: AJ 492195, SEQ ID NO: 97, SEQ ID NO: 1) 16), FL J 4,5802 (NCB IID: AK 1 27702, SEQ ID NO: 98, SEQ ID NO: 117), KI AA0293 (NCB IID: AB 006631, SEQ ID NO: 99, SEQ ID NO: 18), Transcriptionfctor 20 (NCB IID: NM—005650, SEQ ID NO: 100, SEQ ID NO: 119), Tetratricopeptiderepeat 3 (NCB IID: D.830 77, SEQ ID NO: 101, SEQ ID NO: 120), BAP28 (NCB IID: XM—375853 1, SEQ ID NO: 102, SEQ ID NO: 121) . BAP 28. 1 and referred) and BAP 28 (NCB IID: XM one 3758 53 1.2, SEQ ID NO: 126, SEQ ID NO: i 27) (hereinafter, referred to as BAP 28. 2).. These clones were identified as S-100] 3-binding proteins.

Example 2: Functional analysis of S-100 binding protein using S i RNA

U373 MG cells cultured in a modified Danorebecco's medium containing 10% fetal serum were seeded at 7.5 × 10 ⁴ cells / well in a 24-well plate, and cultured at 37 ° C. at 37 ° C. Next, the negative controls: DKlFZ p45 1 G202, force chain binding protein, DKFZ p 761H0421, CG I-043, POGZl, CUB / S ushil, FL J 12587, and MGC 15730 Si RNA was introduced into cells according to a known method. Sense of S i RNA used The sequence of the chain is shown below.

Negative subjects:

5'-GCG CGC UUU GUA GGA UU C G d T d T- 3 '' (SEQ ID NO: 74)

Catenin binding protein:

5 'GAG UAC GUG AUG CCC UUA C dTdT— 3' (SEQ ID NO: 75)

CG I-043:

5'-GAC UGG ACA GCA UCG UUG GdTdT— 3 '(SEQ ID NO: 76)

DKFZ 761H042

5'-GUA CUA CCA UGC UAA GAA C dT dT- 3 '(SEQ ID NO: 77)

POGZ 1:

5'-GCU GCC ACU GUG GAA UCU GdT dT-3 '(SEQ ID NO: 78)

CUB / Sushi1:

5 '-GCU CCA UGU GGU GGA CAU C dT dT— 3' (SEQ ID NO: 79)

FL J 12587:

5'-GGA CAC ACU GCU GGA CCU C dTdT-3 '(SEQ ID NO: 80)

MGC 15730:

5 ⁵ -GUG UAA CUU CAA GAC AGA UdT dT—3, (SEQ ID NO: 81)

D FZp 451G202

5 'A GGA GCC UAA UGU CCA GUU GdT dT— 3 '(SEQ ID NO: 128)

After the completion of gene transfer, the medium was replaced with Dulbecco's modified medium containing 1.0% fetal bovine serum, and the cells were cultured at 37 ° C for 24 hours. Then, the medium was replaced with a serum-free Dulbecco's modified medium, cultured for 24 hours, and then replaced with a serum-free Dulbecco's modified medium containing 10 / zmo 1ZL carboxymethylated _100β. The culture supernatant of was recovered, and MCP-1 was quantified by ELISA. The results of measuring the amount of MCP-1 produced by stimulating carboxymethylated S-1H0] 3 with a negative control or S-100 j3 signaling protein SinRNA introduced are shown. Shown in 1. The catenin binding protein, DKFZ p76 1H042 l, POGZl, CUB / Sushi, and CG I-043 inhibited the production of MCP-1. On the other hand, DKFZ p 451G202, FLJ 12587, and MGC 15730 enhanced MCP-1 production. table 1

(The production amount of MCP-1 is shown in% when the negative control is 100%. (N = 4)) Example 3: Functional analysis of S-100 / 3 binding protein using forced expression system

U373MG cells cultured in Dulbecco's modified medium containing 10% fetal bovine serum were seeded at 7.5 × 10 ⁴ cells / well in a 24-well plate, and cultured at 37 ° C. at 37 ° C. Next, a full-length open reading frame of BAP28.2 (SEQ ID NO: 127, base numbers 1 to 5190) was incorporated into the expression vector. 2005/008471

The prepared vector was introduced into cells by lipofection.

After completion of the gene transfer, the medium was replaced with Dulbecco's modified medium containing 10% fetal calf serum, and cultured at 37 ° C for 24 hours. Next, the medium was replaced with a serum-free Dulbecco's modified medium, and after culturing for 48 hours, the medium was replaced with a serum-free Dulbecco's modified medium containing 10 / zmo 1 ZL carboxymethylated S-100] 3. . The culture supernatant after 24 hours was collected, and M-CP-1, IL-16, and IL-8 in the culture supernatant were quantified by ELISA.

Stimulation with carboxylmethylated S-100; 3 in a negative control or in a state in which the S-100iS fusion protein was forcibly expressed, and the amount of MCP-1, IL-16, and IL-18 produced was measured. Are shown in Tables 2-5. FL J 12587 and MG C 15730 suppressed the production of MCP-1. BAP28.2 also enhanced the production of MCP-1, IL-6, and IL-8.

Table 2

(When the negative control was 100%, the production of IL- 6 shown in ^{_{0/0. (N = 4}} )) Table 5

(The production amount of IL-8 is shown in% when the negative control is 100%. (N = 4)) Example 4: S-100 / 3 binding in diseased tissue using RT-PCR Analysis of protein expression

CDNA was prepared from 1 g of human total RNA according to a known method. The total RNA used was human substantia nigra (Ambion), human substantia nigra (Ambion), human normal human cerebral cortex (Ambion), human amyotrophic lateral sclerosis Cerebral cortex (Ambion) and human Alzheimer's disease cerebral cortex (Biochain). After preparation of the cDNA, the catenin-binding protein, CG 1-043, POGZl, CUB / Su shi 1, DKFZ p761H0421 _s FL J12587, and MGC 15730 are subjected to PCR of the cDNA region and electrophoresis on agarose gel. Performed semi-quantitatively. The primers used for PCR are shown below.

Catenin binding protein:

5 'ATG GCT TCA GAA GAG CTA CAG AA A GAT CTA-3 (SEQ ID NO: 129)

5'-TCA AAA TTC CGT GTC TC C TTT GG C TTG CTT— 3 (SEQ ID NO: 130)

CG I—043:

5 '1 TTC AAC ACT ATG GAA GAT TTC TT A C CG GAC—3 (SEQ ID NO: 131)

5'-CCT CTT CAT TTA CTT CAA TCA GG T TTG CAT-3 (SEQ ID NO: 132)

POGZ 1: 2005/008471

5 '1 TGT GTC GCT ATA GCA CCT GCT GT T CTC GAG—3 (SEQ ID NO: 82)

Five, -

—3, (SEQ ID NO: 83)

CUB / Sushi1:

5 'One GGA GGA GAT TCC. AGT CGC TGC T C C TGC TTC One 3' (SEQ ID NO: 133)

5 '1 GTG GCC TTC CAA GAT GTA GCC AG G GAG GCA-3' (SEQ ID NO: 134)

DKF Z p 761 HO 42

5'-CTT TGC TGT TAG CCA GTT TGG TT G TGC CAC- 3 '(SEQ ID NO: 135)

5'-AGA TCT ACA GGG AGG CTA AAC TT G AGG CTG- 3 '(SEQ ID NO: 136)

FL J 12587:

5 'ACC CCT CGG AGA ACA GGT GGA CG A TCA AGG-3' (SEQ ID NO: 137)

5 '—TGC TCC T'CA CGC GGG CAG GTG CT T CCA G G T-3' (SEQ ID NO: 138)

MGC 15730:

5 '1 CTG GAG GAA AAC CAG CAC CCA TG G TTT ATT—3, (SEQ ID NO: 139)

5 '1 TCA CTG TCA GGG CGA GCA CCA AG G TGA GCC-3' (SEQ ID NO: 140)

Comparison of mRNA expression levels of S-100 / 3 binding protein in various diseased tissues revealed a decrease in FLJ12587 mRNA level in substantia nigra of human Parkinson's disease. In human amyotrophic lateral sclerosis cerebral cortex, JP2005 / 008471

An increase in the mRNA level of DKFZ p 761H0421 was observed. Furthermore, in the cerebral cortex of the Hartzheimer's disease, an increase in mRNA levels of POGZl, DKFZp761HO421, and MGC15730 was observed (FIGS. 1-4).

Example 5: Functional analysis of S_100 J3 binding protein BAP 28.2 and FKRP using SIRA

U373MG cells cultured in Dulbecco's modified medium containing 10% fetal bovine serum were seeded at ⁴ × 10 ⁴ cells / well in a 24-well plate, and cultured at 37 ° C. once. Next, SiRNA was introduced into cells according to a known method. The sequence of the sense strand of the siRNA used is shown below.

Negative subjects:

5'-GCG CGC UUU GUA GGA UUC GdTdT— 3 '(SEQ ID NO: 74)'

BAP 28.2:

5'-GCA GAU UGA GAA GAA CUG GdTdT— 3 '(SEQ ID NO: 122)

FKRP:

5 'One GCA AC C ACU UGC ACG UGG AdTdT—3,' (SEQ ID NO: 123)

After completion of the gene transfer, the medium was replaced with a Dulbecco's modified medium containing 10% fetal bovine serum, and the cells were cultured at 37 ° C for 24 hours. Then, serum-free Dulbecco's modification: After replacing with if ground and culturing for 24 hours, 1Ό / zmo 1ZL of carboxymethylated S- 100 j8 or 0.3 ng / niL recombinant IL-1 1 ] 3 was added to a serum-free Dulbecco's modified medium. After 24 hours, the culture supernatant was collected, and MCP-1 in the culture supernatant was quantified by ELISA.

Stimulation with carboxymethylated S-100] 3 with negative control or S-100] 3 binding protein S RNA introduced, and the amount of MCP-1 produced T / JP2005 / 008471

Table 6 shows the measurement results. BCP 28.2 and FKRP suppressed the production of MCP-1. Table 7 shows the results of measuring the amount of MCP-1 produced by puncturing with recombinant IL-1 [3] 3 in a negative control or in a state in which S-100β binding protein S RNA was introduced. BAP 28..2 did not inhibit MCP-1 production upon IL-1; 8 stimulation.

(Recombinant IL-1] 3 stimulation (n = 4)) Example 6: Expression analysis of S-100] 3-binding protein FKR.P in diseased tissues using RT-PCR

CDNA was prepared from 1 μg of human total RNA according to a known method. 'The RNAs used were human healthy human cerebral cortex (Ambion), human amyotrophic lateral sclerosis cerebral cortex (Ambion), and human Alzheimer's disease cerebral cortex (Biochhanin). After cDNA preparation, PCR was performed, and semiquantitative determination was performed by electrophoresis on an agarose gel. The following primers were used for FKRP PCR.

5 ⁵ -GGT GCA GCT GCT GGA CTT GAC CT TC— 3, (SEQ ID NO: 124)

5'-TCA GCC GCT TCC CGT CAG ACT CAGC 1-3, (SEQ ID NO: 125) JP2005 / 008471

Comparison of the mRNA levels of S-100i3 binding protein in various diseased tissues showed that FKRP mRNA levels increased in human amyotrophic lateral sclerosis cerebral cortex and human Alzheimer's disease cerebral cortex. (Figure 5). Example 7: Evaluation of binding between BAP28 protein and S-100] 3 protein '' After CHO cells were cultured in Dulbecco's modified medium containing 10% fetal calf serum and hypoxanthine thymidine: 7.5 × 10 ^Four seeds were seeded on a 24-well plate. 37. After culturing in C, an expression vector incorporating the full-length open reading frame of BAP28.2 (SEQ ID NO: 127: 1 to 5190) was introduced into the cells by ribofusion.

After the completion of gene transfer, the medium was replaced with Dulbecco's modified medium containing 10% fetal bovine serum and hypoxanthine thymidine, and cultured at 37 ° C for 72 hours. After washing the cells with a phosphate buffer, the cells were collected with a zel scraper. The collected cells were concentrated by centrifugation, disrupted by sonication, suspended in lO / igZmL of Tris buffer containing S-100i3 and lmM calcium, and incubated at 4 ° C for 2 hours. . Next, it was added to protein G agarose and incubated at 4 ° C for 2 hours. After centrifugation, the supernatant was recovered to remove non-specifically adsorbed proteins to protein G agarose. They were then incubated with the S-100] 3 antibody at 4 ° C for 16 hours. Subsequently, protein G agarose was added and incubated at 4 ° C for 2 hours. Protein G agarose was washed with Tris buffer containing 1 mM calcium, and then subjected to SDS-PAGE. As a result of silver staining of the gel, it was confirmed that BAP28.2 was co-precipitated with the S-100 antibody. Industrial applicability

The protein according to the present invention has a binding activity to S-100 / 3, and is involved in S-100 signal transduction. Useful for screening of substances that inhibit 100/3 signal transmission. 05 008471

The Also, a substance obtained by the screening method of the present invention, a partial peptide of the protein of the present invention, an antibody that inhibits the binding of the protein of the present invention to S-1OOiS, and a protein encoding the protein of the present invention. The polynucleotide comprising a nucleotide sequence complementary to the polynucleotide or a part thereof is useful for the prevention and / or treatment of a disease associated with S-100J3. Further, the antibody against the protein or the partial peptide thereof according to the present invention, or the DNA or the antisense DNA thereof according to the present invention is useful for diagnosis of the above-mentioned diseases.

Claims

The scope of the claims

1. A method for screening a substance that inhibits the binding of an S-100] 3-binding protein to S-100β.

2. The screening method according to claim 1, wherein the amount of binding between the S-100 binding protein and S-100 | 3 is compared in the absence and presence of the test substance.

3. Contact S-100 J3 food protein and S-100 in the absence and presence of the test substance, and in each case, the binding of S-100.i3 binding protein to S-100; 3 The method according to claim 1, wherein the amount is measured and compared.

4. The S-100] 3 binding protein has one amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102 and 126, or an amino acid sequence substantially identical to the amino acid sequence 2. The screening method according to claim 1, which is a protein or a partial peptide thereof.

5. The S-1 00] 3 binding protein according to claim 1 or 4, wherein the protein is an amino acid sequence represented by SEQ ID NO: 126, a protein having an amino acid sequence substantially identical to the amino acid sequence, or a partial peptide thereof. The described screen Jung method.

6. The screening method according to claim 1, wherein ELISA, immunoprecipitation assay, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, luciferase assay, reporter assay or protein reconstitution assay is used.

7. S-100] 3 binding protein and S-100 | 3 in the absence and presence of the test substance The amount of binding between S-100) 3 binding protein and S-10Qβ in each case was determined by ELISA, immunoprecipitation assay, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, luciferase assay, reporter 4. The screening method according to claim 3, wherein the screening method is measured and compared in a protein assay or a protein reconstruction assay.

8. A screening method for a substance that alters the amount of factor production induced by binding of S-100 / 3 binding protein to S-100 / 3.

9. The screening method according to claim 8, wherein the amount of factor production induced by the binding of the S-100] 3 binding protein to the S-100] 3 is compared in the absence and presence of the test substance.

10. The method according to claim 9, wherein the S-100] 3-binding protein is brought into contact with S-100β in the absence and presence of the test substance, and the amount of factor production induced in each case is measured and compared. Screen Jung method.

1 1. The S-100 binding protein is an amino acid sequence selected from SEQ ID NOS: 1 to 36, 84 to 102, and 126, or a protein having an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof. The screening method described in claim 8.

12. The script according to claim 8 or 11, wherein the S-100 binding protein is a protein having the amino acid sequence represented by SEQ ID NO: 126 or an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof. One Jung way.

13. The screening method according to claim 8, wherein the ELISA method is used.

14. Contact S-100 with a cell capable of producing a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 126 or the sequence thereof or a partial peptide thereof, 11. The screening method according to claim 10, wherein the amount of MCP-1 production induced in the presence and in the presence is measured by ELISA and compared.

15. Substance that inhibits the binding between S-100 binding protein and S-100β.

16. A substance that changes the amount of factor production induced by the binding of S-100 jS binding protein to S-100 J3.

17. The factor induced by the binding of S-100] 3 binding protein to S-100] 3 is MCP-1, IL-6, IL-18, Α] 3, or ^{Ca2 +} . Scope 16 substance. ,

18. The method according to claim 15, which is a protein having an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof, or a partial peptide thereof. Substance.

19. An antibody of a protein having an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof. The listed substances.

20. A substance that suppresses the expression of S-100 J3 binding protein.

21. Encodes a protein having an amino acid sequence selected from SEQ ID NOs: 1 to 36, 84 to 102, and 126 or an amino acid sequence substantially identical to the amino acid sequence or a partial peptide thereof 20. The substance according to claim 20, which is a polynucleotide comprising a base sequence complementary to a polynucleotide to be expressed or a part thereof.

22. The substance of claim 21 wherein the polynucleotide is a siRNA or an antisense DNA. .

23. A pharmaceutical composition containing the substance according to claims 15, 16 and / or 20 as an active ingredient.

24. The pharmaceutical composition according to claim 23, which is a prophylactic and / or therapeutic agent for a neurodegenerative disease.

25. Neurodegenerative diseases include Parkinson's disease, Parkinson's syndrome, striatal substantia nigra degeneration, Huntington's disease, chorea-ataxia, progressive supranuclear palsy, diffuse Lewy body disease, cerebral cortex Basal ganglia degeneration, Alzheimer's disease, Dementia (dementia), Spinocerebellar degeneration, Motor neuropathy, Demyelinating disease, Cerebrovascular disorder ■ Brain tumors · Hypovolemic shock · Traumatic shock · Head injury and Claims 24 to 24 include neurological dysfunction due to nervous or cerebral spinal cord trauma, cerebrospinal dysfunction due to infectious disease, toxic neurological dysfunction, mental illness, epilepsy, Mage syndrome, dystonia, Down syndrome, and sleep disorder. Pharmaceutical composition.

26. Screening kit for substances that inhibit the binding between S—100] 3 binding protein and S—100] 3.

27. A method for producing a substance that inhibits the binding between S—100] 3 binding protein and S—100—3, comprising the screening method according to claim 1 or 8. .

28. A method for inhibiting the binding between S-100 binding protein and S-100 J3, comprising using the substance obtained by the production method according to claim 27.

29. Prevention of neurodegenerative diseases characterized by administering to mammals an effective amount of a substance that inhibits the binding of S—100] 3 binding protein to S—100; Or treatment method.

30. Use of a substance that inhibits the binding of S—100 0 binding protein to S—100 0 ^ for the manufacture of a prophylactic and / or therapeutic agent for neurodegenerative disease.