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WO2005073395A1 - Procede d’examen de la capacite a reguler l’expression de l’activite de la neprilysine - Google Patents

Procede d’examen de la capacite a reguler l’expression de l’activite de la neprilysine Download PDF

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Publication number
WO2005073395A1
WO2005073395A1 PCT/JP2005/001869 JP2005001869W WO2005073395A1 WO 2005073395 A1 WO2005073395 A1 WO 2005073395A1 JP 2005001869 W JP2005001869 W JP 2005001869W WO 2005073395 A1 WO2005073395 A1 WO 2005073395A1
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androgen receptor
expression
activity
ability
substance
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Japanese (ja)
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Kozo Motonaga
Koichi Saito
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a method for assaying the ability of a substance to control the expression of neprilysin activity.
  • Alzheimer's disease As pathological features of the brain of patients with Alzheimer's disease, formation of senile plaques and accumulation of neurofibrillary tangles are known in addition to neuronal loss. Among these, the earliest pathological change in Alzheimer's disease is the formation of senile plaques, and the main component is the amyloid] 3 protein (hereinafter sometimes referred to as Ai3). Or, it is thought that abnormal decomposition is deeply related to the onset and progression of Alzheimer's disease.
  • senile plaques In the relationship between Alzheimer's disease and senile plaques, the appearance of senile plaques is highly disease-specific, and is observed earlier than neurofibrillary tangles. It has been noted that it occurs in a common pathway located upstream of a complex cascade leading to onset.
  • a i3 is an insoluble peptidic protein consisting of about 40 amino acid residues, and is composed of 3-secretase and R-secretase from precursor protein (hereinafter, sometimes referred to as; 3APP). It is caused by cutting by both. 3-secretase has been identified as a novel aspartate protease (Neuron, 27, 419-422, 2000). In addition, it has been clarified that fasciotic Alzheimer's disease (FAD) -causing gene, presenilin or a complex containing presenilin is involved in the expression of ⁇ -secretase. Neuron, 27, 419—422, 2000).
  • FAD fasciotic Alzheimer's disease
  • neprilysin one of the neutral endopeptidases expressed in various tissues, has already been identified as a major degrading enzyme. Therefore, a method for increasing neprilysin gene expression or a method for increasing neprilysin enzyme activity (hereinafter sometimes referred to as a method for generally increasing neprilysin activity expression) in neuronal cells is one of the treatment methods for Alzheimer's disease. It is considered. For example, in cultured neurons, treatment with neuropeptides (somatosustin) It has been reported that synthase activity has been increased about two-fold, suggesting that drugs targeting the somatostatin receptor may be an effective causal treatment for Alzheimer's disease. , P51-54, 2003, published in Medical and Dental Medicine).
  • neprilysin is regulated by many transcription factors.However, the regulation of gene expression of neprilysin in neurons, which are target cells for neurodegenerative diseases, is still not yet understood. Not revealed
  • the substance By assaying the neprilysin activity expression control ability of the substance, if the substance having the ability can be searched for and identified, the substance can be used as an agent for treating or preventing a disease associated with amyloid 0 protein (ie, specifically, Can be used, for example, as a therapeutic or prophylactic agent for Alzheimer's disease), and can treat or prevent the above-mentioned disease. Disclosure of the invention
  • An object of the present invention is to provide a method for assaying the ability of a substance to control the expression of neprilysin activity, which is simple and effective for searching for a substance having the ability to control the expression of neprilysin, and the like.
  • the present inventors have found that a substance having an ability to regulate the expression of androgen receptor activity significantly affects the control of the expression of neprilysin activity, and that the ability of the substance to regulate the expression of androgen receptor activity is closely related to the ability to regulate the expression of neprilysin activity.
  • an androgen receptor agonist increases neprilysin gene expression in a nerve cell
  • an androgen receptor agonist increases neprilysin enzyme activity in a nerve cell
  • an androgen receptor agonist increases amyloid protein (neurolysin) in a nerve cell.
  • the present inventors have found new findings, such as suppressing the amount of extracellular secretion of ii), and have led to the present invention.
  • the androgen receptor in the contact system between the androgen receptor and the test substance A first step of measuring the ability of the test substance to regulate the expression of androgen receptor activity by an expression index generated according to the binding state between the scepter and the test substance; and (2) the ability measured in the first step A second step of evaluating the ability of the test substance to control the expression of neprilysin activity based on the difference obtained by comparing
  • a method for assaying the ability to control the expression of neprilysin activity of a substance characterized by having the following hereinafter sometimes referred to as the assay method of the present invention
  • an androgen receptor consisting of an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1
  • an androgen receptor comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1
  • composition comprising the substance selected by the search method according to the preceding clause 7, or a pharmaceutically acceptable salt thereof;
  • An agent for controlling the expression of neprilysin which comprises, as an active ingredient, a substance selected by the search method described in 7 above or a pharmaceutically acceptable salt thereof;
  • a neprilysin activity expression controlling agent comprising a substance having an androgen receptor activity expression controlling ability or a pharmaceutically acceptable salt thereof as an active ingredient (hereinafter referred to as the neprilysin activity expression controlling agent of the present invention). is there. ) ;
  • a method for controlling the expression of neprilysin which comprises the step of contacting a nerve cell with a pharmacologically effective amount of a substance having an ability to control the expression of androgen receptor 1 activity in order to control the expression of neprilysin;
  • a method for controlling the expression of neprilysin which comprises the step of promoting the binding between an androgen receptor and a substance having an ability to control the expression of an androgen receptor.
  • a method for preventing or treating an amyloid ⁇ protein-related disease comprising a step of controlling neprilysin activity expression by the method for controlling neprilysin activity expression according to the above item 12 or 13; Brief description of the drawings
  • FIG. 1 shows the results of dihydrotestosterone (hereinafter referred to as DHT) by the repo overnight GeneAssy method using cells prepared by introducing the plasmid pGL3-MMTV-BSD DNA and plasmid pRC / RSV-MR Kozak DNA.
  • Fig. 4 is a view showing the results of measuring the ability of the compound having androgen agonist activity) to regulate the activity of androgen receptor.
  • the columns in the figure are, from left to right, a section to which only DMSO used as a solvent for DHT was added (DMS0), a section to which DHT was added so as to have a final concentration of IpM (IpM DHT), and a final concentration of 1 OpM.
  • FIG. 4 is a view showing the results of measuring the ability to regulate androgen receptor activity of a compound having a gonist activity).
  • the columns in the figure are, from left to right, a section to which only DMSO used as a solvent for test substance A was added (DMS0), and a section to which test substance A was added so that the final concentration was ⁇ ( ⁇ test substance A) , Test substance A added to a final concentration of ⁇ ⁇ (test substance A), test substance A added to a final concentration of 1 M (IM test substance A), final concentration of 10 xM The test substance A was added (10 zM test substance A), the test substance A was added to a final concentration of 50 M (50 M test substance A), and the final concentration was 100 M As shown in the figure, the measurement results in the section where the test substance A was added (100 M test substance A) are shown.
  • Figure 3 shows that test substance B (Androgenagonist) was prepared by the repo overnight Gene Atsie method using cells prepared by introducing DNA of plasmid pGL3-MMTV-BSD and DNA of plasmid pRC / RSV-MR Kozak. Compounds with Active Activity) Androgen Receptor It is a figure showing the result of having measured activity regulation ability. The columns in the figure are, from left to right, a section to which only DMSO used as the solvent for test substance B was added (DMS0), and a section to which test substance B was added so that the final concentration was InM (InM test substance B).
  • test substance B The group to which test substance B was added to a final concentration of 10 riM ( ⁇ test substance B), the section to which test substance B was added to a final concentration of ⁇ ( ⁇ 'test substance B), and a final concentration of 1
  • final concentration of 502 M The results of the measurement in the section where the test substance B was added (50 ⁇ M test substance B) are shown.
  • FIG. 4 is a diagram showing the results of a comparison of the change in the expression level of the neprilysin gene in primary cultured hippocampal cells by the RT-PCR method with various substances having the ability to regulate the expression of steroid and hormone receptor 1 activities.
  • the lanes in the figure are, in order from the left, 1) a section to which only DMSO was added, 2) a section to which DHT was added, 3) a section to which progesterone was added, 4) a section to which dexamethasone was added, and 5) a section to which aldosterone was added.
  • the results obtained by agarose gel electrophoresis of the RT-PC amplification product of the neprilysin gene in the added group and in the added group are shown. It is confirmed that only the amount of DNA in lane 2 increases significantly.
  • FIG. 5 is a diagram showing the results of a comparison between the expression levels of the androgen receptor gene and the neprilysin gene in a neural cultured cell line (CHP212 cells, fraction 2 cells, and PC12 cells) by the RT-PCR method.
  • the lanes in the figure are, from left to right, the RT-product of the neprilysin gene (NEP) in CHP212, IMR32, and PC12 cells, and the RT-product of the androgen receptor gene (AR) in CHP212, IMR32, and PC12 cells.
  • Figure 6 shows the results of agarose gel electrophoresis of the PCR amplification product.
  • Figure 6 shows the change in the expression level of the neprilysin gene in neural culture cells (IMR32 cells) caused by DHT (a compound having androgen agonist activity) (RT-PCR ( (Quantification of neprilysin mRNA by Taqman).
  • RT-PCR (Quantification of neprilysin mRNA by Taqman).
  • the columns in the figure show the measurement results for the section to which only DMSO was added and the section to which DHT was added, in order from the left.
  • Fig. 7 shows changes in the expression level of the neprilysin gene in neural culture cells (CHP212 cells) due to DHT (a compound having androgen agonist activity) (quantification of neprilysin mRNA by RT-PCR (T aqman)). is there.
  • the columns in the figure show the measurement results for the section to which only DMSO was added and the section to which DHT was added, in order from the left.
  • FIG. 8 is a graph showing the results of measurement of the change in the expression level of neprilysin activity in nerve cultured cells (CHP212 cells) by a substance having the ability to regulate the expression of androgen receptor activity.
  • the columns in the figure show the measurement results in the section to which DHT was added and the section to which test substance B was added, in order from the left.
  • FIG. 9 is a graph showing the results of measurement of the change in the expression level of neprilysin activity in neuronal cultured cells (CHP212 cells) by a substance having an ability to regulate the expression of various steroid hormone receptors other than androgen receptor Yuichi.
  • the columns in the figure are measured in order from the left in the section to which DHT was added, the section to which DHT and HFT were added, the section to which progesterone was added, the section to which dexamethasone was added, and the section to which aldosterone was added. The results are shown.
  • FIG. 10 is a graph showing the results of measurement of the change in the amount of amyloid protein (A / 342) secretion in nerve cultured cells (CHP212 cells) by DHT.
  • the columns in the figure show, in order from the left, the measurement results in the section to which only DMSO was added, the section to which DHT and HFT were added, and the section to which DHT was added.
  • FIG. 11 is a graph showing the results of measurement of the change in the amount of secreted amyloid j8 protein (A / 342) in nerve culture cells (CHP212 cells) by a substance capable of regulating the expression of androgen receptor activity.
  • the columns in the figure show, from left to right, the measurement results for the section to which only DMSO was added, the section to which DHT was added, the section to which test substance A was added, and the section to which test substance B was added.
  • Neprilysin is a neutral endopeptida present in various animal tissues. —EC (EC 3.4.24.11) and an extracellular enzyme. It has been rediscovered as enkephalin-degrading beptidase in rat brain and is named enkephalinase. It is known that there are many substrates for neprilysin in in vivo, for example, enkephalin, substance P, atrial natriuretic peptide (ANP), gastrin releasing peptide (GRP), endothelin, etc. .
  • the present invention provides a method for assaying whether or not a substance has the ability to control the expression of neprilysin activity, particularly the ability to promote neprilysin.
  • the androgen receptor (AR) as referred to in the present invention is a protein that is a key in the mechanism of action of androgen (a typical ligand of AR), and is a type of steroid hormone receptor. By recognizing the presence of androgens, the androgen receptor can control various in vivo reactions.
  • an androgen when an androgen binds to an androgen receptor in a cell, the receptor is activated and binds to an androgen receptor response element (ARE) present in the transcriptional regulatory region of the target gene on the chromosome, and binds to the target gene. Promotes transcription.
  • ARE androgen receptor response element
  • any androgen receptor that has an effect in the assay method of the present invention (including a protein having an effect equivalent to the androgen receptor) is used.
  • preferred examples thereof include androgen receptors derived from mammals. More specifically, for example, any of the following androgen receptors can be preferably mentioned.
  • an androgen receptor comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1
  • amino acid sequences having a sequence identity of 80% or more include, for example, the processing of a protein having an amino acid sequence represented by SEQ ID NO: 1 in a cell, the species difference of an organism from which the protein is derived, Includes naturally occurring mutations due to individual differences, differences between tissues, etc., and artificial amino acid mutations.
  • an amino acid sequence represented by SEQ ID NO: 1 is used.
  • site-directed mutagenesis is performed on the DNA to be encoded, and then the DNA is expressed by a conventional method.
  • the site-directed mutagenesis method include a method using an amber mutation (gapped duplex method, ucleic Acids Res., 12, 9441-9456 (1984)), and a method using a mutagenesis primer. And the like.
  • the number of amino acids modified as described above is at least one residue, specifically, one or several, or more.
  • the number of such modifications may be within a range in which androgen receptor activity can be found.
  • substitutions are particularly preferable.
  • the substitution is more preferably an amino acid having similar properties such as hydrophobicity, charge, pK :, and steric structure.
  • Such substitutions include, for example, (1) dali'sine, alanine; (2) palin, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; (5) lysine; And arginine; (6) Substitution within the group of phenylalanine and tyrosine.
  • sequence identity refers to the identity of sequences between two DNAs or two proteins. Refers to homology and homology. Said "sequence identity" is determined by comparing two optimally aligned sequences over the region of the sequence to be compared.
  • the DNA or protein to be compared may have an addition or a deletion (for example, a gap or the like) in the optimal alignment of the two sequences.
  • sequence identity can be determined, for example, by preparing an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GEN ETYX-MAC or a public database. Is generally available, for example, at the website address http://www.ddbj.nig.ac.jp.
  • sequence identity in the present invention may be 80% or more, preferably 90% or more, and more preferably 95% or more.
  • hybridize under stringent conditions in the above (d), the hybridization used herein is, for example, the one described in Molecular Cloning No. 2 by Sambrook J., Frisch EF, Maniatis T. Edition (Molecular Cloning 2nd edition), Cold Spring Harper Laboratory Press (Cold Spring Harbor Laboratory press), and the like.
  • “Stringent conditions” refers to, for example, a solution containing 6XSSC (a solution containing 1.5M NaCK 0.15M trisodium citrate is 10XSSC) and a solution containing 50% formamide. Conditions for forming a hybrid at 45 ° C and washing at 50 ° C with 2XSSC (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6) Can be.
  • the salt concentration in the washing step can be selected, for example, from a condition of 50 ° C at 2XSSC (low stringency condition) to a condition of up to 50 ° C at 0.2XSSC (high stringency condition).
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
  • a gene having a nucleotide sequence encoding the amino acid sequence of an androgen receptor can be obtained, for example, from mammalian tissues such as human ⁇ rat by J. Sambrook, EF Frisch, T. Maniat is; Molecular Cloning, 2nd Edition (Molecular Cloning) Cloning 2nd edition) and Cold Spring Harbor Laboratory (published by Cold Spring Harbor Laboratory, 1989).
  • RNA from mammalian tissues is prepared. Specifically, tissue such as mammalian liver is ground in a solution containing a protein denaturant such as guanidine hydrochloride or guanidine thiosinate, and phenol, black mouth form, etc. are added to the ground material. More denature the protein. After removing the denatured protein as a precipitate fraction by centrifugation etc., total RNA is extracted from the collected supernatant fraction by a method such as guanidine hydrochloride phenol method, SDS-phenol method, guanidine thiosyanate / CsCl method, etc. I do. As a commercially available kit based on these methods, for example, IS0GEN (manufactured by Tsutsubon Gene) can be used.
  • a protein denaturant such as guanidine hydrochloride or guanidine thiosinate
  • phenol, black mouth form, etc. are added to the ground material. More denature the protein.
  • kits based on these methods include, for example, TaKaRa RN A LA PGR "Kit (AMV) Ver.1.1 (Takara Shuzo) and TaKaRa RNA PCR Kit (AMV) Ver. 2.1 (Takara Shuzo).
  • the custom primer is, for example, an oligonucleotide having a length of about 20 bp to about 40 bp, and specifically, for example, a part of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
  • the synthesized single-stranded cDNA is used as a type II, for example, a double-stranded DNA using Escherichia coli DNA polymerase I as a primer with RNA obtained by nicking and gapping the RNA chain using E. coli RNase H as a primer. Synthesize cDNA. Both ends of the resulting double-stranded cDNA are blunt-ended with T4 DNA polymerase. The double-stranded cDNA having blunt-ended ends is purified and recovered by ordinary methods such as phenol-chloroform extraction and ethanol precipitation.
  • the recovered double-stranded cDNA is ligated to a vector such as plasmid PUC118 ⁇ Phase Agt10 using ligase to obtain a cDNA library. May be made.
  • a vector such as plasmid PUC118 ⁇ Phase Agt10 using ligase
  • commercially available double-stranded cDNA or cDNA library may be used.
  • Using the double-stranded cDNA or cDNA library obtained as described above as type I for example, using an oligonucleotide having a partial nucleotide sequence of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 as a primer
  • PCR polymerase chain reaction
  • the primer used in the PCR is, for example, an oligonucleotide having a length of about 20 bp to about 40 bp, which is selected from the 5'-terminal region of the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1. And an oligonucleotide having a base sequence complementary to a base sequence selected from the 3'-terminal region of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. Specifically, for example, as the forward primer, an oligonucleotide having a base sequence represented by base numbers ⁇ to 25 of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 can be mentioned.
  • examples of the reverse primer include oligonucleotides having a base sequence complementary to the base sequence represented by base numbers 2734 to 2754 of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
  • PCR conditions include, for example, a mixture of 10 X LA PCR buffer II (without Mg 2 + ) (manufactured by Takara Shuzo) 5, 25 mM MgCl 2 5 K 2.5 mM dNTP in 50 l of the reaction solution (each (Includes 2.5 mM dATP, dGTP, dCTP, and dTTP.) 8 1 (final concentration of dATP, dGTP, dCTP, and dTTP is 0 • 4 mM), 10 zM primer each 11 (final concentration of 0.2 M), After incubating for 2 minutes at 94 ° C and then at 50 ° C for 5 minutes in a reaction solution containing 0.1-0.5 / ig of type 1 single-stranded cDNA and 2.5 units of TaKaRa LA Taq (Takara Shuzo) One cycle at 94 ° C for 1 minute, then at 50 ° C for 30 seconds, and another 72 ° C for 2.5 minutes as one
  • a DNA having a partial nucleotide sequence of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is prepared.
  • the androgen receptor gene can also be obtained by the hybridization method using lobes.
  • the hybridization conditions include stringent conditions, specifically, for example, 6XSSC (0.9M NaCK 0.09M sodium citrate), 5X Denhardt solution (0.1% (w / v) Ficoll 400, 0.1% ( w / v) polyvinylpyrrolidone, 0.1% (w / v) BSA), 0.5 (w / v) SDS and in the presence of 100 g / ml denatured salmon sperm DNA, or containing 100 g / ml denatured salmon sperm DNA Incubate in a DIG EASY Hyb solution (Boehringer Mannheim) at 65 ° C, then in the presence of 1 XSSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.5% (w / v) SDS at room temperature. Insulate for 15 minutes twice, and further incubate at 68 ° C for 30 minutes in the presence of 0.1XSSC (0.01M NaCK 0.0015M sodium citrate) and 0.5%
  • the androgen receptor gene thus obtained is described in, for example, LSambrook, EFFrisch, T, Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory Vectors can be cloned according to the genetic engineering method described in Publication, 1989 and the like. Specifically, for example, it can be cloned using a commercially available plasmid vector such as a TA cloning kit (Invitrogen) or pBluescriptll (Stratagene).
  • a commercially available plasmid vector such as a TA cloning kit (Invitrogen) or pBluescriptll (Stratagene).
  • the androgen receptor gene is based on, for example, the phosphite-triester method (Hunka piller, M. et al., Nature, 310) based on a known nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1. , 105, 1984) and the like, and can be prepared by chemically synthesizing nucleic acids.
  • the nucleotide sequence of the obtained androgen receptor Yuichi gene was determined by the Maxam Gilbert method (for example, described in Maxam, A.M. & W. Gilbert, Proc. At 1. Acad. Sci. USA, 74, 560, 1977, etc.). Sanger, F. & ARCoulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977, etc.).
  • an androgen receptor element may be manufactured and obtained according to a general genetic engineering method. in this way To prepare an androgen receptor.
  • a vector that can be used in a host cell into which the gene is introduced (hereinafter, referred to as a basic vector), such as a gene that can replicate independently in a host cell,
  • a vector containing an androgen receptor gene (hereinafter referred to as a vector) can be isolated and purified from a host cell and integrated into a vector having a detectable marker according to ordinary genetic engineering techniques. This vector may be referred to as this vector.)
  • Escherichia coli When Escherichia coli is used as a host cell, examples of the basic vector that can be used for the construction of this vector include plasmid pUC119 (manufactured by Takara Shuzo) and phagemid pBluescriptII (manufactured by Stratagene). be able to.
  • plasmids pGBT9, pGAD424, pAC manufactured by Clontech
  • plasmids such as pRc / RSV and pRc / CMV (manufactured by Invitrogen), pipapi orchid virus plasmid pBPV (manufactured by Amersham Pharmacia Biotech) or EB virus Examples include a vector containing an autonomous replication origin derived from a virus such as plasmid pCEP4 (manufactured by Invitrogen), and a virus such as vaccinia virus. Furthermore, when an insect animal cell is used as a host cell, an insect virus such as a baculovirus can be used.
  • this vector is constructed using a vector containing an autonomous origin of replication, for example, the above-described yeast plasmid PACT2, or the sipper lipovirus virus plasmid pBPV, or the EB virus plasmid pCEM, the vector will be transformed into host cells. When introduced, it is retained in the cell as an episom.
  • an autonomous origin of replication for example, the above-described yeast plasmid PACT2, or the sipper lipovirus virus plasmid pBPV, or the EB virus plasmid pCEM
  • a transfer vector containing a base sequence homologous to the genome of the virus to be used may be used.
  • transfer vectors include, for example, pVL1392, pVL1393 (Smith, GE, Summers MD et al .: Mol. Cel 1. Biol., 3, 2156-2165 (1983) available from Pharmingen. )), pSFB5 (Funahashi, S. et al .: J. Virol., 65, 5584-5588 (1991)) and the like.
  • the androgen receptor gene is inserted into the transfer vector as described above, and When the fur vector and the viral genome are simultaneously introduced into host cells, homologous recombination occurs between the transfer vector and the viral genome, and a virus in which the androgen receptor gene has been integrated into the genome can be obtained.
  • virus genome genomes such as Baculovirus, Adenovirus, and Vaccinivirus can be used.
  • the androgen receptor gene when the androgen receptor gene is incorporated into a baculovirus, first, the androgen receptor gene is inserted into the multicloning site of the transfer vector PVL1393, pVL1392, etc.
  • the DNA obtained above and Baculovirus genome DNA (Baculogold; manufactured by Pharmingen) are introduced into insect cell strain Sf21 (available from ATCC) by the calcium phosphate method or the like, and the resulting cells are cultured.
  • virus particles containing the genome of the virus into which the androgen receptor gene has been inserted are collected from the culture solution by centrifugation or the like, and the collected virus particles are deproteinized with phenol or the like to remove the androgen receptor.
  • a genome of a virus containing one gene can be obtained. Further, the obtained virus genome is introduced into a host cell having virus particle-forming ability, such as insect cell Sf2 vermilion, by a calcium phosphate method or the like, and the obtained cell is cultured. In this way, the number of virus particles containing the androgen receptor gene can be increased.
  • the androgen receptor gene can be directly integrated without using a transfer vector.
  • the viral vector DC (X) (El Gilboa et al., BioTechniques, 4, 504-512 (1986)) and the like incorporate the androgen receptor gene into the closing site on the vector.
  • the resulting androgen receptor 'Yuichi gene-incorporated viral vector is introduced into a packaging cell such as, for example, Ampli-GPE (J. Virol., 66, 3755 (1992)), thereby obtaining an androgen.
  • Virus particles containing the genome of the virus into which the receptor gene has been introduced can be obtained.
  • a promoter operable in the host cell is introduced upstream of the androgen receptor gene.
  • ⁇ to operably bind '' means that the androgen receptor is linked to the androgen receptor so that the androgen receptor is expressed under the control of the promoter in the host cell into which the androgen receptor is introduced.
  • Receiving-means combining with a gene examples include a DNA that exhibits a promotor activity in the host cell into which it is introduced.
  • the promoter of the lactose operon of Escherichia coli lacP
  • the promoter of the tryptophan operon trpP
  • the promoter of the arginine operon argP
  • the promoter of the galactone operon Promoter galP
  • tac promoter T7 promoter
  • T3 promoter phage ⁇ promoter ( ⁇ -pL, ⁇ -pR)
  • MMTV mouse papilloma virus
  • the host cell is budding yeast, one example is the ADH1 promoter.
  • an androgen receptor is placed downstream of the promoter so that the promoter and the androgen receptor gene are operably linked to each other. What is necessary is just to insert a gene.
  • the aforementioned plasmids pRc / RSV, pRc / CMV, etc. have a cloning site downstream of a promoter operable in animal cells.
  • the androgen receptor gene can be expressed in the animal cell by introducing a vector obtained by introducing the androgen receptor gene into the closing site into the animal cell.
  • these plasmids contain the SV40 autonomous replication origin (or i) in advance, introducing the plasmid into cultured cells transformed with the SV40 genome lacking the ori, for example, COS cells, etc.
  • the number of copies of the plasmid in the cells is greatly increased, and as a result, the androgen receptor gene incorporated in the plasmid can be expressed in large amounts.
  • the aforementioned yeast plasmid PACT2 has an ADH1 promoter, and if the androgen receptor gene is inserted downstream of the ADH1 promoter of the plasmid or a derivative thereof, an androgen receptor is obtained.
  • This vector can be constructed so that the Yuichi gene can be expressed in a large amount in budding yeast such as CG1945 (manufactured by Clontech).
  • a transformant containing the present vector (hereinafter sometimes referred to as the present transformant) can be obtained.
  • a usual introduction method suitable for the host cell can be applied. For example, when Escherichia coli is used as a host cell, see III. Sambrook, EF Frisch, T.
  • the cells can be introduced into the cell according to a general gene transfer method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method.
  • a general gene transfer method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method.
  • the cells can be introduced using, for example, Yeast trans format ion kit (Clontech) based on the lithium method.
  • the virus genome can be introduced into host cells by the general gene transfer method as described above, and the virus genome containing the androgen receptor Yuichi gene inserted therein is contained. By infecting a host cell with a virus particle, the genome of the virus can be introduced into the host cell.
  • a host cell into which a marker gene has been introduced at the same time as the present vector may be cultured by a method suitable for the nature of the gene.
  • the marker gene is a gene that confers drug resistance to a selected drug showing lethal activity on host cells
  • the vector is introduced using a medium to which the selected drug is added.
  • the cultured host cells may be cultured.
  • the combination include a combination of a neomycin resistance imparting gene and neomycin, a combination of a hygromycin resistance imparting gene and hygromycin, and a combination of a blasticidin S resistance imparting gene and blasticidin S.
  • the marker gene is a gene that complements the auxotrophy of the host cell
  • the cells into which the vector has been introduced are cultured in a minimal medium that does not contain nutrients corresponding to the auxotrophy. Just fine.
  • a detection method based on androgen binding activity can also be used.
  • the present vector and a vector having a marker gene are linearized by digestion with a restriction enzyme or the like. Thereafter, these are introduced into host cells by the method described above. Next, the cells are usually cultured for several weeks, and then a transformant of interest may be selected and obtained based on the expression level of the introduced gene. Further, for example, first, the present vector having the above-described gene imparting drug resistance as a marker gene is introduced into a host cell by the method described above.
  • the cells are subcultured for several weeks or more in a medium to which the selective drug has been added, and the selected drug-resistant clones that have survived in a colony are purified and cultured, whereby the androgen receptor gene is introduced into the chromosome of the host cell.
  • the obtained transformant can be selected and obtained.
  • genomic DNA of the cell was prepared according to ordinary genetic engineering methods, and the prepared genomic DNA was If the presence of the above-mentioned androgen receptor gene is detected by using a method such as PCR or Southern hybridization using DNA having a partial nucleotide sequence of the introduced androgen receptor gene as a primer or probe, Good.
  • the transformant can be cryopreserved and can be used after awakening as needed, so that the effort of preparing the transformant for each experiment can be omitted, and the properties and handling conditions must be determined in advance.
  • the test can be performed using the confirmed transformant.
  • the present transformant obtained as described above is cultured, and the andalus produced from the culture is cultured. By recovering Genrecept, it is possible to produce an androgen receptor.
  • the present transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells.
  • the transformant when the present transformant is a microorganism, the transformant may be prepared by using various media containing a carbon source, a nitrogen source, organic or inorganic salts, and the like, which are used for ordinary culture in general microorganisms. And can be cultured. Culture is performed according to the usual method for general microorganisms, and solid culture, liquid culture (rotary shaking culture, reciprocal shaking culture, JarFermenter culture, tank culture, etc.) are possible. It is.
  • the culture temperature and the pH of the medium can be appropriately selected from the range in which the microorganisms grow.For example, a culture medium having a pH of about 6 to about 8 at a culture temperature of about 15 ° C. to about 40 ° C. It is common to culture in.
  • the culturing time varies depending on various culturing conditions, but is usually about 1 day to about 5 days.
  • the induction time is preferably within one day, usually several hours.
  • the transformant When the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium used for ordinary culture of general cultured cells. When the transformant is prepared using a selective drug, it is preferable to culture the transformant in the presence of the selective drug.
  • the final concentration under conditions of 37 ° C, 5% C0 etc. 2 presence with 1 0% and so as DMEM medium FBS was added (two Ssui Ltd., etc.) What is necessary is just to culture
  • the culture temperature is determined using an insect cell culture medium such as Grace's medium containing 10% (v / v) FBS and 2% (w / v) Yeastlate. Culture at 25 ° C to 35 ° C. At this time, in the case of cells that are easily detached from a petri dish such as Sf21 cells, the cells can be dispersed by pipetting without using a trypsin solution and subculture can be performed.
  • a transformant containing a virus vector such as a baculovirus
  • the culturing time is preferably set to be before cell death due to a cytoplasmic effect, for example, up to 72 hours after virus infection.
  • the androgen receptor can be obtained by combining methods generally used for the isolation and purification of general proteins.
  • the transformants obtained by the above-described culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, if necessary, proteins are solubilized, and ion exchange, hydrophobicity, gel filtration, etc. Purification may be performed by using various chromatography steps alone or in combination. Further, for example, the transformant obtained by the culture may be removed by centrifugation or the like, and the present enzyme may be purified from the culture supernatant in the same manner as described above. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.
  • the collection of the androgen receptor produced by the present transformant from the culture may be appropriately performed by a combination of ordinary isolation and purification methods.
  • the cells of the transformant are collected by centrifugation or the like, and the collected cells are suspended in a buffer such as a normal buffer such as 0 mM HEPES pH7, ImM EDTA, lmM DTT, 0.5 mM PMSF. Crush with a polytron, sonication, dounce homogenizer, etc.
  • the obtained crushed liquid is ultracentrifuged at tens of thousands of xg for several tens of minutes to about one hour, and the supernatant fraction is collected, whereby a fraction containing an androgen receptor can be obtained.
  • a more purified androgen receptor can be recovered.
  • DNA binding using an androgen receptor response element ie, an oligonucleotide having a length of about 15 bp to about 200 bp including a base sequence to which the androgen receptor binds, is used as a probe.
  • ARE androgen receptor response element
  • the assay method of the present invention comprises the following steps: (1) The androgen possessed by the test substance is determined by an expression index generated according to the binding state between the androgen receptor and the test substance in the contact system between the androgen receptor and the test substance. The first step of measuring the ability to regulate the expression of the receptor activity, and (2) a second step of evaluating the ability of the test substance to control the expression of neprilysin activity based on the difference obtained by comparing the ability measured in the first step with the ability in the control.
  • the binding state between the androgen receptor and the test substance in a contact system between the androgen receptor and the test substance (specifically, without directly measuring the ability to control the expression control of neprilysin activity).
  • an expression index generated according to (binding capacity-binding amount) can evaluate the ability of a substance to control the expression of neprilysin activity based on the result of measuring the ability to regulate the expression of androgen receptor activity. It is simple and effective, and is most suitable for primary screening.
  • a contact system in a nerve cell and the like can be preferably mentioned.
  • a more preferred system is a system in which the nerve cells are, for example, (a) a nerve-derived primary cell, (b) a nerve-derived cultured cell line, (c) a CHP212 cell, or (d) an IMR32 cell.
  • the first step of the assay method of the present invention may be, for example, a so-called reporter gene assay using an androgen receptor, a two-hybrid assay using an androgen receptor, or a binding assay using an androgen receptor. Etc. can be implemented. This will be described in more detail below. First, a reporter gene assay using an androgen receptor will be described.
  • the method comprises: (1) a transformant in which a repo overnight gene and an androgen receptor evening gene linked downstream of a transcription control region containing an androgen receptor response element are introduced into a host cell; (2) a step of measuring the expression level of the reporter gene in the transformant or an index value having a correlation with the level, and (3) a step of measuring the measured expression level or Based on an index value that is correlated with the quantity Measuring the ability of the test substance to regulate the expression of androgen receptor activity.
  • Examples of the ability to regulate the expression of androgen receptor activity include agonist activity and antagonist activity for the androgen receptor.
  • Examples of the “reporter gene linked downstream of the transcription control region containing the androgen receptor response element” in the above method include, for example, an LTR-derived transcription control region of mouse papilloma virus (MMT V) containing an androgen receptor response element (MMT V). (Nucleic Acids Research., 19, 1563-1569) or the downstream of a transcription control region containing a consensus sequence of the androgen receptor response element and a base sequence necessary for transcription initiation. And the like, which can be used to monitor the ability to regulate the expression of androgen receptor activity in host cells.
  • MMT V mouse papilloma virus
  • MMT V androgen receptor response element
  • the “androgen response element” is a specific nucleotide sequence present in the transcription regulatory region of the target gene whose expression level is regulated by the androgen receptor. For example, when a complex of an androgen and an androgen receptor recognizes and binds to the sequence, transcription of a target gene located downstream thereof is promoted.
  • Specific examples include the nucleotide sequence in the LTR of mouse papilloma virus (MMT V) (Nucleic Acids Research., 19, 1563-1569).
  • MMT V mouse papilloma virus
  • a base sequence containing a consensus sequence of an androgen response element one or more times can also be used. In order to obtain a sufficient transcription control ability, it is preferable that the consensus sequence as described above is usually tandemly linked in about 2 to 5 times.
  • DNA having such a nucleotide sequence can be prepared by chemical synthesis, or amplification and cloning by PCR or the like.
  • base sequence necessary for transcription initiation examples include a nucleotide sequence having a TATA box and a sequence of a leader for transcription initiation.
  • the nucleotide sequence of the 5 'upstream region of the thymidine kinase gene (tk) derived from the simple herpes virus (HSV), one of the 5' upstream regions of the mouse metamouth thionine I gene The base sequence from the ⁇ ⁇ th base (the transcription start point is the + 1st. The same applies hereinafter) to the 115th base (Genbank A ccession No.
  • DNA having such a base sequence can be prepared, for example, by chemical synthesis based on the base sequence.
  • an oligonucleotide for amplifying DNA encoding the above-described region is designed and prepared based on a known nucleotide sequence, and PCR is performed using the prepared oligonucleotide as a primer. Can also be prepared.
  • reporter gene luciferase gene, secreted alkaline phosphatase gene,] 3 galactosidase gene, chloramphenicol acetyltransferranase gene, growth hormone gene, etc. are used. Genes encoding reporter proteins that are relatively stable in host cells are preferred.
  • a reporter gene and an androgen receptor gene linked downstream of a transcription control region containing an androgen receptor response element are linked to an androgen receptor non-endogenous host cell, specifically, for example, a HeLa cell.
  • Transformants are prepared by introducing the cells into host cells such as CV-1 cells, Hepal cells, NIH3T3 cells, HepG2 cells, C0S1 cells, BF-2 cells, and CHH-1 cells.
  • the androgen receptor gene may be introduced into the host cell in a form incorporated in a basic vector operably linked to a promoter operable in the host cell, for example, as described above.
  • a reporter gene linked downstream of the transcription control region containing the androgen receptor response element may also be used in a form integrated in the basic vector.
  • the repo overnight gene or the androgen receptor night gene to be introduced may be integrated into the chromosome of the host cell.
  • the cell is operably linked to a vector that incorporates a reporter gene that is linked downstream of the transcriptional control region containing the andresponse receptor, and a promoter that can function in the host cell.
  • a vector having an androgen receptor gene and a vector having a marker gene are introduced into a host cell.
  • the cells are usually cultured for several weeks, and the target transformant is selected based on the expression level of the introduced marker gene, whereby the cell is ligated downstream of the transcription control region containing the androgen receptor response element. It is possible to obtain a transformant in which the reporter gene and the androgen receptor gene operably linked to the promoter operable in the host cell are introduced into the chromosome of the host cell.
  • the genomic DNA of the cell is prepared according to a conventional genetic engineering method. From the prepared genomic DNA, the reporter gene or androgen receptor DNA was synthesized by a method such as PCR using a DNA having a partial base sequence of the introduced gene as a primer or a probe, or Southern hybridization. What is necessary is just to detect the presence of one gene.
  • the transformant can be cryopreserved and can be used after awakening as needed, which saves the labor of preparing the transformant for each experiment and also confirms the properties and handling conditions in advance. The test can be performed using the obtained transformant. This is also useful, for example, when performing large-scale screening with automated robots.
  • an androgen receptor endogenous host cell specifically, for example, an LNCaP cell, a CEP212 cell, or an IMR32 cell
  • a reporter gene linked downstream of a transcription control region containing an androgen response element is introduced.
  • a transformant is prepared.
  • an endogenous androgen responsive gene may be used as a reporter gene using an androgen receptor endogenous host cell or a non-endogenous cell into which the androgen receptor gene has been introduced.
  • endogenous androgen-responsive genes include, for example, PSN (Prostate Specific Fictive Antigen) gene, PSMA (prostate Specific Fic membrane ant igen) gene, Probasin gene, hK2 (kal l ikrein 2) gene, Neprilysin Genes and the like can be mentioned.
  • the transformant prepared as described above and a test substance were, for example, indirectly contacted for several hours to several days, and then specifically cultured in a medium to which the test substance was added for several hours to several days. Thereafter, the expression level of the reporter gene in the transformant or an index value having a correlation with the level is measured.
  • the androgen receptor produced by the transformant When the androgen receptor produced by the transformant is activated by the binding of a test substance (androgen-like active substance: agonist), the transcription of the reporter gene is promoted, and the reporter gene is coded.
  • the repo protein that is produced Transformants are accumulated in cells or the like or secreted into the medium.
  • the index value having a correlation with the expression amount of the reporter protein per cell of the transformant or the amount thereof is determined. Measure.
  • luciferase gene when a luciferase gene is used as a reporter gene, luciferin, a luciferase substrate, is added to a crude cell extract prepared from a transformant contacted with a test substance.
  • Light is emitted at an intensity proportional to the amount of luciferase in the crude cell extract. Therefore, by measuring this luminescence intensity with a measuring device such as a luminometer, the amount of luciferase and, consequently, the amount of luciferase gene expression can be determined. Similarly, the amount of reporter gene expression or an index value having a correlation with the amount of the reporter gene under the condition where the transformant is not brought into contact with the test substance was measured, and the condition under which the measured value was brought into contact with the test substance was measured.
  • the ability of the test substance to regulate the expression of androgen receptor activity is determined. Can be evaluated.
  • an androgen such as DHT
  • the repo reaction was performed in the same manner as above. An expression level of one gene or an index value having a correlation with the level is measured.
  • test substance Compare the expression level of the reporter gene under the condition that the transformant was contacted with the androgen or the index value having a correlation with the amount, and compare the reporter gene under the condition that the androgen was contacted with the test substance. If the expression level of one gene or an index value having a correlation with the level is low, the test substance is evaluated as having the ability to regulate the expression of androgen receptor activity (in this case, the antagonist activity against Andorchigen receptor). can do.
  • a substance having neprilysin activity expression control activity can be easily selected based on the androgen receptor 1 activity expression control ability evaluated by such an evaluation method, and further, the substance or a pharmaceutically acceptable salt thereof can be selected.
  • the method uses the ability of a test substance to regulate the expression of androgen receptor activity, and measures the ability to form a complex of two fusion proteins (two-hybrid) and the ability to regulate the transcription of the formed complex.
  • Test system based on the expression level of a reporter gene in a plant or an index value having a correlation with the expression level (two-hybrid system; Nishikaa et al., Toxicol. Appl. Pharmacol., 154, 76-83 (1999)) It is.
  • the method includes, for example, (1) the first step of contacting a test substance with an androgen receptor and a transcriptional coactivator in order to form a complex between the androgen receptor and a transcriptional coactivator (for example, transfection of an androgen receptor complex gene); A first step of bringing the transformant into contact with a test substance), (2) an expression value of a reporter gene whose transcription is controlled by the complex after the first step, or an index value having a correlation with the amount. And (3) evaluating the ability of the test substance to regulate the expression of androgen receptor activity based on the expression level measured in the second step or an index value having a correlation with the expression level. A third step.
  • Examples of the ability to regulate the expression of androgen receptor activity include agonist activity and antagonist activity for the androgen receptor.
  • the complex of the androgen receptor and the transcriptional conjugation factor is composed of one of the following components I (A or B) and one of the following components II (X or Y ) And a protein having the other component (B or A) of the following component I and the other component (Y or X) of the following component II, depending on the ligand.
  • the transcription coupling factor having component (I) recognizes a conjugate of an androgen receptor and a ligand and Transcriptional conjugation factor capable of binding to, specifically, SRCl / NCoAl (Onate, SA et al., Science, 1995, 270, 1354), TIF2 / GRIP1 (Voegel, JJ et al., EMBO, J., 1996, 15, 3667).
  • the androgen receptor having the component I (B) for example, an androgen receptor capable of binding to the above-mentioned transcription coupling factor can be mentioned.
  • the androgen receptor has a region to which the ligand binds in order to form a complex with the ligand.
  • the DNA having the nucleotide sequence encoding the amino acid sequence of such a region is a partial nucleotide sequence of the androgen receptor gene, for example, the ligand binding of the androgen receptor in the nucleotide sequence of the androgen receptor gene. Examples include a DNA comprising a base sequence encoding a region and excluding a base sequence encoding the amino acid sequence of component II (X).
  • transcription factors having (X) as component II include the nucleotide sequence of DNA to which Ga14 protein binds (SEQ ID NO: 2) and the nucleotide sequence of DNA to which LeX protein binds (SEQ ID NO: 3).
  • the nucleotide sequence of the DNA to which the LacI receptor protein binds SEQ ID NO: 4
  • the nucleotide sequence of the DNA to which the tetracycline receptor protein binds SEQ ID NO: 5
  • the nucleotide sequence of the DNA to which the ZFHD-1 protein binds Transcription regulators that bind to DNA consisting of any base sequence such as SEQ ID NO: 6) and that can function in host cells can be mentioned.
  • transcription factors having component II include, for example, Gal4 protein, Lex protein, LacI receptor protein, tetracycline receptor protein, ZFHD-1 protein, B42 protein, androgen receptor.
  • a transcriptional regulator that can function in a host cell such as a transcriptional coupling factor that can bind to the evening transcription coupling factor binding region under the control of a ligand, can be given.
  • a complex composed of the above-mentioned components is produced, for example, by a transgenic transformant having the following components: (1) One of the following components i (a or b) and one of the following components ii (X or y):
  • a transformant wherein is introduced into a host cell
  • component (i) of component i means a DNA having a nucleotide sequence encoding the amino acid sequence of component (A) of component I, and the DNA is a component. What is necessary is just to prepare from the gene of the transcription coupling factor which has I (A) by the usual genetic engineering technique.
  • component (i) (b) means a DNA having a base sequence encoding the amino acid sequence of component (B), and the DNA is It may be prepared from the gene for androgen receptor having (B) by an ordinary genetic engineering technique.
  • Constituent requirement ii) means a DNA having a nucleotide sequence encoding the amino acid sequence of component (X), and the DNA is usually obtained from the gene of a transcriptional regulator having component (X). It may be prepared by the genetic engineering technique described above.
  • (y) of component ii means a DNA having a nucleotide sequence encoding the amino acid sequence of (Y) of component II, and the DNA is a transcriptional regulator having (Y) of component II. May be prepared from the above gene by a conventional genetic engineering technique.
  • Component iii means DNA to which component (X) can bind and DNA of the repo overnight gene connected downstream of the promoter that can be activated by component (Y) .
  • DNA to which (X) of component II can bind include, for example, the nucleotide sequence of DNA to which Ga14 protein binds (SEQ ID NO: 2) and the nucleotide sequence of DNA to which Lex protein binds (SEQ ID NO: 3).
  • nucleotide sequence of DNA binding to the Lac I receptor protein (SEQ ID NO: 4), the nucleotide sequence of DNA binding to the tetracycline receptor protein (SEQ ID NO: 5), the DNA binding to ZF HD-1 protein And a DNA consisting of any one of the nucleotide sequences (SEQ ID NO: 6).
  • one of the promoters that can be activated by the component II (Y) is, for example, when the component II (Y) is derived from the G a14 protein, for example, the minimum TATA derived from yeast. Box arrangement.
  • One gene is used for normal reporter assays such as luciferase gene, secreted alfa phosphatase gene, 3 galactosidase gene, chloramphenicol acetyltransferrase gene, and growth hormone gene.
  • a reporter gene can be used, and a gene encoding a reporter protein having relatively high stability in a host cell is preferable. These components are inserted in a vector while being appropriately combined so that each component is expressed, and introduced into the same host cell using ordinary genetic engineering techniques, whereby the transformation is carried out. A body can be made.
  • the chimeric gene (chimeric gene 1) is prepared by linking y) with the reading frame of the base sequence.
  • the other component (b or a) of the component i and the other component (y or X) of the component ii should be linked to each other by matching the reading frame of the base sequence.
  • a chimeric gene (chimeric gene 2).
  • Each of these chimeric genes 1 and 2 can be a promoter capable of functioning in a host cell. For example, when the host cell is a budding yeast cell, an inducible promoter such as the GAL1 promoter or a constant promoter such as the ADH promoter is used.
  • Component iii is usually located downstream of “DNA capable of binding to a DNA binding region having an amino acid sequence encoded by the base sequence of (X) of component ii” and “component (ii) of (y)
  • the DNA of the reporter gene connected downstream of the promoter that can be activated by the transcription activation region having the amino acid sequence encoded by the nucleotide sequence, It is introduced into the same host cell as the gene. If the host cell has an available endogenous repo overnight gene, it may be used. In this case, the introduction of the reporter gene can be omitted.
  • Examples of host cells used to prepare gene-transformed transformants of each component include budding yeast cells such as budding yeast strain Y190 (manufactured by Clontech) and mammalian cells such as HeLa cells. .
  • the host cell is a cell that is not endogenous to the androgen receptor.
  • the transformant is brought into contact with the test substance, for example, for several hours to several days. Specifically, after culturing in a medium containing the test substance for several hours to several days, It has a correlation with the expression level of the reporter gene that the transformant has or the amount thereof Measure the index value.
  • the androgen receptor produced by the transformant When the androgen receptor produced by the transformant is activated by binding of a test substance (androgen-like active substance), transcription of the reporter gene is promoted, and the reporter protein encoded by the reporter gene is converted to It is accumulated in the cells of the transformant or secreted into the medium.
  • the index value having a correlation with the expression amount or the amount of the repo overnight gene per cell of the transformed cell is obtained. Is measured.
  • luciferin a luciferase substrate
  • a crude cell extract prepared from a transformant contacted with a test substance.
  • light is emitted at an intensity proportional to the amount of luciferase in the crude cell extract. Therefore, by measuring this luminescence intensity with a measuring device such as a luminometer, the amount of luciferase and, consequently, the expression level of the luciferase gene can be known.
  • the expression level of the reporter gene under the condition that the transformant is not contacted with the test substance or an index value having a correlation with the amount is measured, and the condition under which the measured value is brought into contact with the test substance is measured.
  • the ability of the test substance to regulate the expression of the androgen receptor activity in this case, the agonist activity against the androgen receptor).
  • the reporter gene was obtained in the same manner as described above.
  • the expression level or an index value having a correlation with the level is measured.
  • the expression level of the repo gene under the condition of contacting the transformant with the androgen or an index value having a correlation with the amount of the repo gene was compared with that of the repo gene under the condition of contacting the androgen with the test substance. If the expression level of the Yuichi gene or an index value correlated with the amount is low, the test substance should be evaluated as having the ability to regulate androgen activity (in this case, antagonist activity against androgen receptor Yuichi). Can be.
  • neprilysin activity expression regulator based on the ability to regulate the expression of androgen receptor activity evaluated by such an evaluation method. Furthermore, a neprilysin activity expression regulator containing the substance or a pharmaceutically acceptable salt thereof as an active ingredient can be provided. Then androgen I will explain.
  • This method is a test method capable of measuring the binding ability of a chemical substance to the androgen receptor, quantifying the amount of binding, and analyzing the binding specificity and binding strength. For example, when a test substance is allowed to coexist with a labeled ligand (hereinafter referred to as a labeled ligand) previously bound to the androgen receptor recovered from the present transformant as described above, Due to the competition between the test substance and the labeled ligand, the labeled ligand was released from the androgen receptor, and the amount of the labeled ligand bound to the androgen receptor decreased, depending on the affinity of both for androgen receptor. Thus, the amount of label bound to the androgen receptor decreases.
  • a labeled ligand hereinafter referred to as a labeled ligand
  • the binding state between the androgen receptor and the test substance can be indirectly confirmed.
  • androgen receptor Measurement of the binding ability of the test substance to one is possible.
  • the labeled ligand for example, tritium-labeled DHT or the like can be used. Separation of the bound Z-free form of the labeled ligand can be performed by the hydroxyapatite method ⁇ the glycerol density gradient ultracentrifugation method or the like.
  • the reaction system is roughly divided into three groups. The first group is a system in which only the solvent is added to the site where the labeled ligand is bound to the androgen receptor, and corresponds to a system in which the concentration of the test substance added is zero.
  • the labeling amount of the binding type labeled ligand in the system indicates the total binding amount of the labeled ligand to the androgen receptor.
  • the added concentration at which the unlabeled ligand sufficiently saturates the androgen receptor and the labeled ligand cannot be bound for example, the added concentration at which the unlabeled ligand sufficiently saturates the androgen receptor and the labeled ligand cannot be bound (for example, The amount of bound labeled ligand in the system is Is determined as the amount of non-specific binding to Therefore, the specific binding amount of the labeled ligand to the androgen receptor was calculated from the total binding amount to the non-specific binding amount. This is the value obtained by subtracting the combined amount.
  • the test substance was added, for example, to a final addition concentration of 10 M (this concentration may be arbitrarily changed depending on the purpose) where the labeled ligand is bound to the androgen receptor.
  • a final addition concentration of 10 M this concentration may be arbitrarily changed depending on the purpose
  • the amount of the labeled labeled ligand obtained from this system is determined by the amount of the androgen receptor obtained when the concentration of the test substance determined as described above is zero. It becomes smaller than the specific binding amount of the labeled ligand to Sepuichi. In this way, the binding state between the andorogen receptor and the test substance is indirectly confirmed.
  • the binding ability of the test substance to the androgen receptor can be examined. If the test substance contains a plurality of substances, is there a substance showing affinity for the androgen receptor in the plurality of substances? You can also find out. Further, in order to evaluate the binding ability of the test substance to the androgen receptor overnight in more detail, for example, the androgen receptor / bindin guatasse may be similarly performed by changing the concentration of the test substance in the third group. For example, the amount of bound labeled ligand is measured, and based on the measured values, the amount of bound ligand and the amount of free ligand are calculated.
  • Genetic assays using an androgen receptor Genetic assay using an androgen receptor, Genetic assay using an androgen receptor, a hybrid assay using an androgen receptor, and a binding assay using an androgen receptor, etc. can easily produce a substance that controls neprilysin activity expression.
  • the present invention can be used for searching for the substance or a pharmaceutically acceptable salt thereof as an active ingredient of a neprilysin activity expression controlling agent.
  • a substance having the ability to control the expression of neprilysin may be selected based on the ability to control the expression of neprilysin evaluated by the assay method of the present invention (the search method of the present invention).
  • a negative control when used as a control in a system suitable for evaluation as an androgen receptor, androgen receptor activity is used as an indicator for evaluating the ability of the test substance to control the expression of neprilysin activity.
  • a substance having an activity expression regulating ability of 130% or more, more preferably 150% or more, is selected as a substance having a neprilysin activity expression promoting ability.
  • the androgen receptor can be used as a reagent that provides an indicator for evaluating the ability of a substance to control the expression of neprilysin activity.
  • the substance selected by the search method of the present invention or a pharmaceutically acceptable salt thereof has a neprilysin activity expression controlling ability, and such a substance can be used as an active ingredient of a neprilysin activity expression controlling agent.
  • pharmaceutically acceptable salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals), especially physiologically acceptable salts. Acid addition salt strength S is preferred.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, and salts with benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • a composition comprising a substance capable of controlling the expression of androgen receptor activity or a pharmaceutically acceptable salt thereof is useful as an agent for controlling expression of neprilysin activity, and its effective amount is orally or parenterally administered to humans or the like. Can be administered to mammals.
  • neprilysin activity expression controlling agents of the present invention when administered orally, include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups It can be used in usual forms such as agents, emulsions and suspensions.
  • the agent for controlling expression of neprilysin activity of the present invention can be used in the form of ordinary liquid preparations such as solutions, emulsions and suspensions.
  • Examples of the method of parenterally administering the neprilysin activity expression controlling agent of the present invention in the form described above include an injection method, a rectum administration method in the form of a suppository, and the like.
  • a substance having an ability to control the expression of androgen receptor activity or a pharmaceutically acceptable salt thereof is mixed with an acceptable ordinary carrier, excipient, binder, stabilizer, diluent, or the like. It can be manufactured by the following. When used in the form of an injection, an acceptable buffer, solubilizing agent, isotonic agent and the like can be added.
  • the preparation thus obtained is administered, for example, to mammals (for example, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, horses, pests, cats, cats, dogs, monkeys, etc.). be able to.
  • the dose varies depending on the age, sex, body weight, degree of disease, type of the neprilysin activity expression controlling agent of the present invention, dosage form, etc. of the mammal to be administered. (As 60 kg)) and the amount of the active ingredient per day is about 0.1 mg to about lg, preferably about 0.1 mg to lg, more preferably about 1.0 mg to 200 mg, more preferably about 0.1 mg to lg.
  • the above-mentioned daily dose can be administered once or in several divided doses.
  • an adult for example, assuming a body weight of 60 kg
  • the dose can be administered in terms of 60 kg.
  • diseases such as an amyloid 9 protein-related disease (that is, specifically, for example, Alzheimer's disease).
  • amyloid protein-related disease is a disease in which amyloid iS protein is deposited outside of organs and tissue cells to inhibit the function of these organs and tissue cells, and is generally called amyloidosis.
  • amyloidosis is also included as one of the amyloid / 3 protein-related diseases (see below).
  • Amyloidosis is classified into “systemic amyloidosis,” in which amyloid deposits occur in various parts of the body, and “localized amyloidosis,” in which deposition occurs only in some organs.
  • Examples of localized amyloidosis include Alzheimer's disease, mad cow disease (bovine spongiform encephalopathy, BSE), and new Creutzfeld-Jakob disease (vCJD).
  • Examples of systemic amyloidosis include familial amyloid polyneuropathy (FAP) and the like.
  • FAP familial amyloid polyneuropathy
  • senile amyloidosis which occurs non-genetically in the elderly, dialysis amyloidosis caused by amyloid in which proteins that cannot be removed by the dialysis membrane used in the treatment of dialysis patients, and amyloid formed by cutting proteins expressed in rheumatism And secondary amyloidosis.
  • the amount of the amyloid i3 protein such as A / 340 consisting of the amino acid sequence represented by SEQ ID NO: 7, / 342 consisting of the amino acid sequence represented by SEQ ID NO: 8, and 043 consisting of the amino acid sequence represented by SEQ ID NO: 9 varies.
  • a method using an immunochemical method using an antibody specific to the amyloid j3 protein can be preferably mentioned.
  • the method includes immunoprecipitation, western blotting, enzyme immunoassay, sandwich enzyme immunoassay, or a combination thereof.
  • amyloid j8 protein-specific antibody a polyclonal antibody may be used.
  • BAN50, BNT77, BS85, BA27, BC05 (Biochemistry, 34, 10272-10278, 1995) or 6El0 A monoclonal antibody such as 4G8 may be used.
  • BA27 and BC05 are antibodies selective for A / 340 and Aj342 / 43, respectively, if these antibodies or antibodies having similar selectivity are used, the amount of A / 340 protein, A / 340 The amount of 342 Z43 protein or the amount of both A / 340 and AjS 42/43 proteins can be measured. Then, by using such a method, it is possible to verify the assay method of the present invention and the like.
  • Aarrutsuha haimaimaa disease is caused by the accumulation of amymiloroid protein ( ⁇ (AA jj33)) in the brain. In gold and gold, the accumulation and accumulation of protein in the cell vesicles, the response to inflammatory inflammation, and the functional dysfunction of the neuronal vesicles It is understood that this can lead to the onset of dementia dementia through complicated and complicated transit routes.
  • the dominant inherited gene, “Family-affiliated Arlartshaimaima disease” is a combination of amymiloroidoid jj33 protein and protein secretion and secretion. However, the onset of the disease occurs from here and there, and the gene for the causal gene of the cause is also specified.
  • neamiprilorilaisicin has been attracting attention as an enzyme capable of degrading and decomposing enzymes related to protein protein.
  • a Neppuri Liraisin knock knockout mother mouse manufactured and manufactured at Haha Bavard University College, in the brain Amymiloroidoids]] 33 were analyzed in detail in detail.
  • the radioactively labeled amymiloroidoid // 33 protein protein was injected and administered into the brain of the above-mentioned mausus, The decomposition and disintegration process was monitored by a high-speed and high-velocity liquid liquid liquid, Kukuromamatotoguraraffy. . Again, non-very high sensitivity
  • the present invention provides (1) a step of contacting a neural cell with a pharmacologically effective amount of a substance having an ability to control the expression of androgen receptor activity in order to control the expression of neprilysin; Control method, (2) Androgue A method for controlling the expression of neprilysin, characterized by comprising a step of promoting the binding of a substance having the ability to control the expression of an androgen receptor to a neprilysin;
  • the present invention also provides a method for preventing or treating an amyloid 3 protein-related disease, which comprises a step of controlling the expression of neprilysin activity.
  • Example 1 (Preparation of a plasmid containing a responsive reporter gene and a selection marker gene) Plasmid pMSG (Pharmacia) was digested with restriction enzymes Hind III and Smal, and an androgen response element derived from MMTV-LTR was digested. 1463 bp of DNA was obtained. The obtained DNA was treated with a Bluniing kit (manufactured by Takara Shuzo Co., Ltd.) to blunt its ends (hereinafter, the obtained DNA may be referred to as ARE DNA).
  • ARE DNA Bluniing kit
  • the recovered DNA was mixed with the above-mentioned ARE DNA1, and reacted with T4 ligase.
  • Escherichia coli DH5 competent cells were transformed using the obtained reaction solution. From several colonies of Escherichia coli showing ampicillin resistance, the DNA of the plasmid carried by each was purified, digested with the restriction enzymes KpnI and Clal, and the digest was analyzed by agarose gel electrophoresis.
  • plasmid pGL3-Picture TV plasmid pGL3-Picture TV.
  • the plasmid pUCSV-BSD purchased from Funakoshi
  • BamHI the plasmid pUCSV-BSD
  • the prepared DNA was mixed with the DNA obtained by digesting the above plasmid pGL3-MMTV with BamHI and treating with BAP, and reacted with T4 ligase.
  • the resulting reaction solution was used to transform Escherichia coli DH5 competent cells. From several colonies of E. coli showing ampicillin resistance, DNAs of plasmids carried by each were purified, digested with Bam HI, and the digests were analyzed by agarose gel electrophoresis. Based on the obtained analysis results, a plasmid having a structure in which the blasticidin S deaminase gene expression cassette was introduced into the Bam HI site was selected and named plasmid pGL3-MMTV-BSD.
  • Example 2 (Preparation of Androgen Receptor Yuichi Expression Plasmid)
  • a formal primer consisting of the nucleotide sequence of SEQ ID NO: 10 and the nucleotide sequence of SEQ ID NO: 11 were obtained.
  • Human Prostate cDNA 10 ng (Clontech Quick Clone cDNA no.7 123-1) was converted into type III, and the above primers were each 1 Opmol, LA-Taq polymerase (Takara Shuzo) and buffer attached to the enzyme.
  • the PCR was carried out after adjusting the reaction solution volume to 501 using.
  • the PCR was carried out by using PCR System 9700 (manufactured by Applied Biosystems) under the conditions that the temperature was maintained at 95 ° C for 1 minute and then at 68 ° C for 3 minutes as one cycle, and the cycle was 35 cycles.
  • the entire amount of the reaction solution was subjected to agarose gel electrophoresis using low-melting point agarose (agarose L: Futatsubon Gene). After confirming that the DNA of the size expected from the known base sequence was amplified by ethidium mouth mouth staining, the DNA was recovered.
  • a part of the recovered DNA and a dye terminator-Sequence Kit FS (Applied And a sample for direct sequence was prepared using the Auto Sequencer (manufactured by Applied Biosystems, model 3700). As a result, it was confirmed that the recovered DNA had a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
  • PCR was performed using a forward primer having the nucleotide sequence of SEQ ID NO: 12 and a reverse primer having the nucleotide sequence of SEQ ID NO: 13.
  • DNA having a structure in which a nucleotide sequence encoding the amino acid sequence of a human standard androgen receptor was linked immediately after a Kozak consensus sequence was amplified by PCR. That is, 0.1 zg of the DNA to be type III was used as type III, and the amount of the reaction solution was set to 50 using each 1 Opmol of the above primers, LA-Taq polymerase (manufactured by Takara Shuzo) and the reaction buffer attached to the enzyme.
  • PCR was performed.
  • the PCR was carried out by using PCRsystem9700 (manufactured by Applied Biosystems) under the conditions of 1 cycle of incubation at 95 ° C for 1 minute and then at 68 ° C for 3 minutes, under the condition of 20 cycles.
  • the entire reaction mixture was subjected to electrophoresis using a low-melting point agarose gel, and separated and recovered.
  • Approximately 1 g of the recovered DNA was treated with a DNA blunting kit (Takara Shuzo Co., Ltd.) to blunt its ends, and then reacted with a T4 polynucleotide-forced enzyme. The ends were phosphorylated.
  • the obtained DNA was treated with phenol, it was further purified by ethanol precipitation. The entire amount of the purified DNA was used as an insert DNA for preparing the following expression plasmid.
  • Compounds having androgen agonist activity were identified by a reporter gene assay using cells for measuring the ability to regulate androgen receptor activity expression.
  • Plasmid pGL3-MMTV-BSD DNA (see Example 1) having a firefly luciferase gene connected downstream of ARE of mouse papillomavirus to human HeLa cells, and plasmid pRC / RSV-hAR Kozak
  • the cells prepared by introducing the DNA were added to a phenol red-free MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to give a final concentration of 10% of FBS treated with charcoal dextran.
  • the cells were seeded at about 2 ⁇ 10 4 cells / well in a 96-well plate for measuring and cultivating luciferase luminescence and for culture (Yuichi # 3903) and then cultured overnight.
  • DHT ie, androgen receptor agonist
  • DMSO a control group to which only a solvent
  • the agonist activity of the test substance with respect to androgen receptor can be measured by adding the test substance to each test group in place of DHT and performing the test. The results are shown in FIGS.
  • test substance A and test substance B luciferase activity was found to increase with increasing concentration of test substance added to the cells in the cells for measuring the activity of androgen receptor transcriptional activity. That is, since test substance A and test substance B both have androgen receptor transcription activity regulating ability, it can be evaluated that the test substance has neprilysin activity expression controlling ability.
  • test substances can be used as drugs for preventing or treating amyloid / 3 protein-related diseases (that is, for example, therapeutic or prophylactic agents for Alzheimer's disease). Can be treated or prevented.
  • Test substance A and test substance B described in the examples are actually existing organic synthetic compounds, and tests using the compounds have been actually performed. However, since the structure has not been determined at present, the test results are described in the examples based on the present tense expression.
  • Example 4 Variation in Neprilysin Gene Expression Level in Primary Cultured Hippocampal Cells by Various Steroid Hormone Receptor Activity-Controlling Substances
  • the hippocampus was cut from the whole brain aseptically removed from the SD rat 18-day-old fetus, and the hippocampus was minced with a scalpel, and then 0.25% trypsin and 0.02% DNa Enzyme treatment was carried out by incubating at 37 ° C for 20 minutes in phosphate buffered saline containing seI. After the enzyme reaction was stopped by adding fetal calf serum, the operation of sucking up and discharging the cell solution with a pipette fitted with a plastic tip was repeated three times to disperse the cells.
  • the obtained cell dispersion was filtered using a filter on which two lens papers were stacked, and the filtrate was centrifuged at 100 Orpin for 5 minutes.
  • the recovered cells are transferred to Eagle's minimum essential medium (EM EM, Gibco BRL), and then washed with poly-L-lysine (Sigma) containing EMEM medium containing 10% fetal calf serum.
  • EM EM Eagle's minimum essential medium
  • Sigma poly-L-lysine
  • DMS ⁇ , 01 ⁇ 30 dissolved progesterone dissolved in 0111 ⁇ DMSO, dexamethasone dissolved in DMSO, or aldosterone dissolved in DMSO was added to the cultured cells thus obtained.
  • These cells were cultured, and RNA was prepared using the RNeasy Mini Kit (manufactured by QIAGEN) 24 hours after the addition of DMS ⁇ , the DHT, the progesterone, the dexamethasone, or the aldosterone.
  • RNA (11) was purified using Super Scriptll RNaseH "Reverse Transriptase. The RNA extracted after the reaction was stored at 180 ° C. until use.
  • the mixture was mixed with 1 zl of Super Script II RT, heated at 42 ° C for 50 minutes, further heated at 70 ° C for 15 minutes, and cooled to 4 ° C.
  • the cDNA thus obtained was stored at 120 ° C until use.
  • RT-PCR of the rat neprilysin gene was performed using the oligonucleotide represented by SEQ ID NO: 14 and the oligonucleotide represented by SEQ ID NO: 15.
  • RT-PCR for rat neprilysin gene 1 cycle (95 ° C, 5min), 38 eye les (95 ° C, 30sec, 55 ⁇ :, lmin, 72 ° C, lmin), 1 cycle (72 ° C, 5min) (7min, 4 ° C)
  • Nerve cultured cells Human Neuroblastoma CHP212 (hereinafter, also referred to as CHP212 cells), human Neuroblastoma IMR32 (hereinafter, also referred to as IMR32 cells), rat PC12 cells (hereinafter, also referred to as PC12 cells)
  • CHP212 cells Human Neuroblastoma CHP212 cells
  • IMR32 cells human Neuroblastoma IMR32 cells
  • rat PC12 cells hereinafter, also referred to as PC12 cells
  • RNA is extracted using Micro-To-Midi Tota 1 RNA Purification (Invitrogen), and the extracted total RNA (lg / 201) is ready-to-go you-prime first- Reverse transcription reaction was performed using Strand Beads (Amersham Biosciences).
  • RT-PCR of the human neprilysin gene was performed using the oligonucleotide represented by SEQ ID NO: 16 and the oligonucleotide represented by SEQ ID NO: 17.
  • RT-PCR of the rat neprilysin gene was performed using the oligonucleotide represented by SEQ ID NO: 14 and the oligonucleotide represented by SEQ ID NO: 15.
  • RT-PCR of the human and rat androgen receptor genes was carried out using the oligonucleotide represented by SEQ ID NO: 18 and the oligonucleotide represented by SEQ ID NO: 19.
  • the DNA was mixed with a loading buffer, subjected to agarose electrophoresis using a 2% agarose gel, and then the DNA was stained with ethidium umide. The results are shown in FIG.
  • Example 6 Fluctuation of Neprilysin Gene Expression Level in Nerve Cultured Cells by Test Substance Having Ability to Regulate Androgen Receptor Activity Expression: Quantification of Neprilysin mRNA by RT-PCR (Taqman))
  • RNA (lg / 20D was subjected to Super Scriptll RNaseH "Reverse Transriptase The RNA extracted after the reaction was stored at -80 until use.
  • RNA 1 g, oligo (dT) 15 primers (15 ig / ml) l ⁇ K dNTP Mixture 2 A mixture of 0.5 mM each 1 zl and ddH20 (total amount 12 ⁇ ) was heated at 65 ° C for 5 minutes. Next, the mixture was placed on ice, and 5X reverse transcriptase buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl 2 ) 41, RNasin Plus RNase Inhibitor 40u / n ⁇ ( After mixing Promega and 0.1 M dithiothreitol 21, the mixture was heated at 42 ° C for 2 minutes.
  • 5X reverse transcriptase buffer 250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl 2
  • RNasin Plus RNase Inhibitor 40u / n ⁇ After mixing Promega and 0.1 M di
  • the mixture was mixed with 11 of Super Script II RT, heated at 42 ° C for 50 minutes, further heated at 70 ° C for 15 minutes, and cooled to 4 ° C.
  • the cDNA obtained in this way was stored at 120 ° C until use.
  • the quantitative PCR was performed using Applied Biosystems ABI PRISM 7900HT and Optical 96-Well Reaction Plate. The PCR was performed under the following conditions: (1) 50 ° C for 2 minutes, (2) 95 ° C for 10 minutes, (3) 95 for 15 seconds, (4) 60 ° C for 60 seconds, and 40 cycles. It was conducted.
  • the amplified D.NA was quantitatively detected based on the fluorescence intensity.
  • the DNA was mixed with a loading buffer, and then subjected to agarose electrophoresis using a 3% agarose gel. Was stained with ethidium bromide. The results are shown in FIG. 6 (IMR32 cells, 1.29) and FIG. 7 (CHP212 cells, 2.49).
  • Example 7 Measurement of fluctuation of expression level of neprilysin activity in nerve culture cells by substance having androgen receptor activity expression regulating ability
  • cultured neurons human Neuroblastoma CHP212
  • DMEM culture medium manufactured by Nissui Pharmaceutical
  • FBS treated with charcoal dextran to a final concentration of 1%. After that, it was cultured overnight.
  • DMSO or DHT dissolved in DMSO was added to the cells.
  • test substance B that is, a substance that can be evaluated as having the ability to control the expression of neprilysin activity because the ability to regulate androgen receptor transcription activity is recognized in Example 3). Added.
  • FIG. 8 shows the results of the expression level of neprilysin activity in the nerve cultured cells to which DHT or test substance B was added, as a percentage of the activity expression level in the system treated with only DMS ⁇ . It was confirmed that the expression level of neprilysin activity in the system treated with DHT significantly increased. It is also confirmed that the expression level of neprilysin activity in the system treated with test substance B is significantly increased.
  • Example 8 Measurement of change in expression and amount of neprilysin activity in nerve cultured cells by substances having an ability to regulate the expression of various steroid hormone receptor activities other than androgen receptor
  • the cultured neurons (human Neuroblastoma CHP212) were treated with phenol red free DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) in a final concentration of Using a medium added to give a concentration of about 2 ⁇ 10 5 cells per well in a 6-well plate (Falcon), the resulting mixture was cultured for 1 hour. After the culture, the cells were dissolved in DMS ⁇ , DHT dissolved in DMSO, DHT dissolved in DMSO and HFT (the substance is an androgen receptor antagonist), progesterone dissolved in DMSO, and dissolved in DMSO. Dexamethasone or aldosterone dissolved in DMSO was added.
  • neprilysin activity in the nerve culture cells to which the DHT, the DHT & HFT, the progesterone, the dexamethasone, or the aldosterone was added was shown as a percentage of the activity expression level in the system treated with only DMS0. This is shown in Figure 9. It was confirmed that only the neprilysin activity expression level (346%) in the system treated with DHT significantly increased. Incidentally, the expression of neprilysin activity in the progesterone, dexamethasone or aldosterone-treated system was 123%, 116% and 142%.
  • Cultured neurons (human Neuroblastoma CHP212) were plated in a 6-well plate using phenol red-free DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) in a medium in which FBS treated with charcoal dextran was added to a final concentration of 1%. (Falcon) about 2 x 10 5 cells After seeding, the cells were cultured overnight. After the culture, DMSO, DHT dissolved in DMSO or DHT and HFT dissolved in DMSO (the substance is an androgen receptor antagonist) was added to the cells.
  • amyloid] 342 peptide in the culture solution was added.
  • the amount was measured by ELISA (Human Amyloid j31-42 Measurement Kit Code. No. 17711, Institute for Immunobiology). The results are shown in FIG.
  • FIG. 10 shows the results of amyloid secretion in neuronal cultured cells to which DHT or DHT & HFT was added; 6-protein secretion was shown as% of secretion in the system treated with only DMSO. It was confirmed that the amount of amyloid protein secreted (49.0 pg / ml, 59.6%) in the system treated with DHT was significantly reduced. On the other hand, it was confirmed that the amount of secreted amyloid i3 protein (74.7 pg / id, 91.1%) in the system treated with DHT & HFT was not reduced due to the presence of antagonist.
  • Example 10 Measurement of fluctuation of amyloid i3 protein secretion amount in cultured nerve cells by substance having androgen receptor 1 activity regulating ability
  • a culture medium in which cultured neurons human Neuroblastoma CHP212 were added to a phenol-red free DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to a concentration of 1% of FBS treated with charcoal dextran to a final concentration of 1%. After about 2 x 10 5 cells were seeded in a well plate (Falcon), the cells were cultured overnight.
  • test substance was Is a substance that can be evaluated as having the ability to control neprilysin activity expression
  • test substance B dissolved in DMSO
  • the test substance was a substance that can be evaluated as having the ability to control neprilysin activity expression.
  • Fig. 11 shows the results of the amyloid / 3 protein secretion level in nerve cells to which DHT, test substance A or test substance B was added, expressed as% of the secretion level in the system treated with DMSO alone.
  • DHT amyloid / 3 protein secretion level
  • test substance A or test substance B A: 56.0 pg / ml, 62.1%, B: 62.2 pg / l, 69.0%
  • Oligonucleotide primer designed for PCR SEQ ID NO: 13
  • Oligonucleotide primer designed for PCR SEQ ID NO: 14
  • Oligonucleotide primer designed for PCR SEQ ID NO: 15

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Abstract

Procédé d’examen de la capacité d’une substance à réguler l’expression de l’activité de la néprilysine caractérisé en ce qu’il comprend : (1) une première étape consistant à mesurer la capacité d’une substance test à contrôler l’expression de l’activité d’un récepteur androgène grâce à l’utilisation d’une indication d’expression qui se produit en fonction des conditions de liaison du récepteur androgène à la substance test dans un système dans lequel le récepteur androgène est en contact avec la substance test ; et (2) une deuxième étape consistant à comparer la capacité mesurée au cours de la première étape avec la capacité d’un témoin et, sur la base de la différence ainsi obtenue, à évaluer la capacité de la substance test à réguler l’expression de l’activité de la néprilysine.
PCT/JP2005/001869 2004-02-02 2005-02-02 Procede d’examen de la capacite a reguler l’expression de l’activite de la neprilysine Ceased WO2005073395A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126167A1 (fr) 2009-04-30 2010-11-04 住友化学株式会社 Dérivé de thiophène

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