WO2007020853A1 - 薬剤の標的蛋白質を同定する方法及び標的蛋白質を用いた糖尿病治療薬のスクリーニング方法 - Google Patents
薬剤の標的蛋白質を同定する方法及び標的蛋白質を用いた糖尿病治療薬のスクリーニング方法 Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Definitions
- the present invention uses a molecular chaperone protein having a property of recognizing a change in the three-dimensional structure of a protein and binding to the protein, and the target by binding a compound having a pharmacological action to the target protein.
- the present invention relates to a method for identifying a target protein of a compound having a pharmacological action by detecting a change in the three-dimensional structure of the protein.
- the present invention also relates to a screening method for a therapeutic drug for diabetes using the target protein found by the identification method of the present invention.
- a compound whose target protein is clear has a clear mechanism of action on the living body, and the structure of the compound can be modified by using the strength of binding with the protein or the change in enzyme activity of the protein as an indicator. For this reason, it is easy to conduct improved research aimed at improving the pharmacokinetics including absorption and degradation, as well as pharmacological activity, and it is very advantageous for drug development. On the other hand, it is not easy to improve the compound structure for the purpose of improving the activity of a compound whose target protein is unknown even if a clear pharmacological action is found (non-patent literature). 1).
- Non-Patent Document 2 There are actually many drugs that have known significant pharmacological actions but whose target protein is not clear. Typical examples include biguanide agents that have been used for a long time as a therapeutic agent for diabetes (see Non-Patent Document 2) and thalidomide, whose presence has been revised due to its dramatic therapeutic effect on multiple myeloma. (See Non-Patent Document 3). Although the biguanide has a significant hypoglycemic effect and thalidomide has a significant angiogenesis inhibitory activity, none of these drugs has been identified as a direct target protein in vivo. Therefore, while these drugs have useful pharmacological actions, it has been difficult to conduct research to improve their effects.
- Non-Patent Document 4 lactic acidosis
- Non-Patent Document 3 teratogenicity
- a method of detecting and separating a protein directly binding to the compound by physical and Z or chemical means has been common.
- a method of separating and purifying a target protein bound to a compound by a physical force such as gravity by modifying a part of the compound structure and binding to a high molecular weight affinity bead is known.
- a target protein bound to the compound can be chemically detected by attaching a tag as a label to a part of the compound structure (see Non-Patent Document 5).
- Non-Patent Document 6 genes encoding proteins that bind to the target compound by applying molecular biological techniques such as the yeast two-hybrid method (see Non-Patent Document 6) and the phage display method (see Non-Patent Document 7). Attempts have also been made to screen and identify fragments from within a cDNA library.
- modification of a compound structure is essential for finding a true target protein to be applied to a site that does not affect the pharmacological action of the compound, but drugs and compounds with unknown targets generally have a structure and its structure.
- modification of the compound structure requires time, cost, and special techniques, which makes the above method difficult to become a general-purpose research technique.
- Molecular chaperones are a group of proteins that assist in protein structure formation such as protein molecule folding, unfolding, and multimer formation (see Non-Patent Document 9).
- a group of molecules collectively referred to as the Hsp60 family is particularly called “chaperonin” as a typical molecular chaperone.
- chaperones have properties as functional molecules that recognize the non-natural structure of protein molecules that serve as substrates.
- Insulin is secreted from ⁇ -cells of the spleen Langernoens island, and acts mainly on muscle, liver, and fat to take blood sugar into the cell for storage and consumption, thereby lowering blood glucose level .
- Diabetes mellitus is caused by this lack of action of insulin, but there are two types of patients: type 1 which has impaired production or secretion of insulin and type 2 which makes it difficult for insulin to promote glucose metabolism.
- type 1 blood glucose level is higher than that of healthy people, whereas blood type insulin is absolutely insufficient in type 1, whereas type 2 does not promote blood glucose uptake or consumption in spite of the presence of insulin. Insulin resistance is occurring.
- Type 2 diabetes is a so-called lifestyle-related disease caused by overeating, lack of exercise, and stress in addition to genetic predisposition.
- the number of type 2 diabetic patients is increasing rapidly as calorie intake increases.
- Japan 95% of diabetic patients Accounted for. Therefore, there is an increasing need for treatment of type 2 diabetes that promotes glucose metabolism by improving insulin resistance, which is not just a simple hypoglycemic agent for the treatment of diabetes.
- An injection formulation is prescribed.
- sulfonylurea hypoglycemic agents that act on ⁇ cells in the spleen to stimulate insulin secretion in addition to insulin injections
- sugars OC-darcosidase inhibitors that delay the digestion and absorption of quality.
- thiazolidine derivatives have been used more directly as drugs that directly improve insulin resistance. Its action is the uptake of sugar into the cell and the promotion of sugar utilization within the cell. This thiazolidine derivative has been shown to act as a peroxisome proliferator activated receptor gamma (PPAR y) antigen!
- Non-Patent Document 11 thiazolidine derivatives are known to have side effects that cause fat accumulation and edema as well as improving insulin resistance (see Non-Patent Document 12). Since the occurrence of edema is a serious side effect that causes cardiac hypertrophy, a more useful drug target molecule that replaces PPARy is required to improve insulin resistance.
- a biguanide antihyperglycemic agent see Non-Patent Document 13
- a potent drug which has an effect of improving glucose metabolism.
- Biguanides have been reported to have an effect of increasing sugar utilization and gluconeogenesis by inhibiting anaerobic glycolysis, suppressing appetite, and inhibiting intestinal absorption of sugar. Improve insulin sensitivity. Since biguanide does not act on the pancreas and does not increase insulin secretion, it is characterized by not causing obesity and being less prone to hypoglycemia. The action of biguanide is preferred and does not include the action of the thiazolidine derivatives and insulin preparations described above, and in many cases, it is actually prescribed in combination with other antihyperglycemic agents. In recent years, its potent pharmacological action has been reviewed, and biguanides are now in the second place after thiazolidine derivatives as drugs for improving insulin resistance.
- biguanide agents are known to have side effects that increase lactic acid accumulation and cause lactic acidosis (see Non-Patent Document 14).
- Biguanide drugs have a very long history as a medicine Nevertheless, a clear target protein that corresponds to PPAR y of the thiazolidine derivative has not yet been identified. For this reason, biguanide agents have no information on the structure-activity relationship with the target protein, and to date, it is difficult to conduct improved research aimed at increasing the main effect of hypoglycemia, which is merely a study of deviation from side effects such as improvement of lactic acidosis. Met.
- ATP5B protein is a ⁇ subunit of FIFO-ATP synthase that is encoded on the genome and transported to mitochondria to work (see Non-Patent Documents 15 and 16).
- Non-Patent Documents 15 and 16 it has been reported that the abundance of ⁇ 5 ⁇ decreases in both the gene expression level and protein level in the muscles of type 2 diabetic patients compared to that in healthy individuals. (Ref. 7).
- Non-Patent Document 18 and Patent Document 7 ⁇ 5 ⁇ phosphate level and fasting blood glucose level in the muscles of diabetic patients are inversely correlated (see Non-Patent Document 18 and Patent Document 7), and the polypeptide, antibody, It has been reported that a polynucleotide, a compound that binds to a polypeptide (for example, a nucleic acid fragment), etc., becomes a therapeutic agent for diabetes-related diseases (see Patent Document 7).
- Various polypeptides (3025) contained in human heart mitochondrial proteome including ⁇ 5 ⁇ have been disclosed, and there are reports that these are related to screening for therapeutic drugs for diseases related to mitochondrial function (including diabetes) (Patent Document 8) ).
- Patent Document 1 US Pat. No. 5,585,277 Specification
- Patent Document 2 U.S. Pat.No. 5,679,582
- Patent Document 3 US Patent Application Publication No. 2002Z055123
- Patent Document 4 US Patent Application Publication No. 2004Z191835
- Patent Document 5 Patent No. 2952848
- Patent Document 6 European Patent No. 0770876
- Patent Document 7 International Publication No. 03Z020963 Pamphlet
- Patent Document 8 International Publication No. 03Z087768 Pamphlet
- Non-Patent Document 1 “The Journal of Antibiotics” H Hatori et al., 2004, 57-7, p. 456—461.
- Non-Patent Document 2 Japanese Clinical” Y. Yamacaki et al., 2002, No. 60 ⁇ 9 p. 389- 92
- Non-Patent Document 3 “Drug Discovery Today” Teo SK et al., 2005 15-15 (2) p. 107-114.
- Non-Patent Document 4 "Drags” Lalau JD et al., 1999, 58 ⁇ 1, p. 55-60 / 75-82
- Non-Patent Document 5 “Nature Biotechnology” (UK) 2000, N Shimizu et al., No. 18, p.877—881
- Non-Patent Document 6 “Biochemical Pharmacology”, 2002, D Henthorn et al. 63- 9 p. 1619-1628
- Non-Patent Document 7 “Chemistry & Biology” Sche PP et al., 1999, Vol. 6-10: p. 707-716. PMID: 10508685
- Non-Patent Literature 8 “OUTLINES OF BIOCHEMISTRY” 1987, Eric E. CONN et al.
- Non-Patent Document 9 “Pharmacology & Therapeutics”, 2004, A Sreedhar et al., No. 101-3, p. 227-257
- Non-Patent Document 10 “Nature” 1992, Gething MJ, Sambrook J. et al .: No. 355-6355: p. 33-45
- Non-Patent Document 11 “The Journal of Biological Chemistry” (USA), 1995, No. 270, p. 12953-1295
- Non-Patent Document 12 “Diabetes Frontier” (USA), 1999, No. 10, p.811-818
- Non-Patent Document 13 Japanese Clinical” Y. Yamasaki et al., 2002, No. 60-9 p. 389-92
- Non-Patent Document 14 “Drugs” Lalau JD et al., 1999 No. 58-1 p. 55- 60 / 75- 82
- Non-Patent Document 15 “Nature” (USA), 1997, 386th, p. 299-302
- Non-Patent Document 16 “Nature” (US), 1994, 370th (6491) ), P.62 1-628
- Non-patent document 17 “Diabetes”, 2002, 51st, p. 1913—19 20
- Non-Patent Document 18 “The Journal of Biological Chemistory” 2003, 278, p. 10436-10442
- the present invention identifies a target protein of a low molecular weight compound by using a change in the three-dimensional structure of the protein in response to the compound instead of the binding between the compound and the protein as an index, and without requiring the structural modification of the compound. It is an object to provide a method for doing this.
- Another object of the present invention is to provide a novel method for screening for antidiabetic drugs. Means for solving the problem
- the present inventors have found that a molecular chaperone protein, which is known as a functional molecule that recognizes the non-natural structure of a protein molecule that serves as a substrate, changes the three-dimensional structure of a protein due to a compound (drug whose target protein is unknown).
- a compound drug whose target protein is unknown.
- the target protein of a compound drug with unknown target protein was detected and identified using the change in binding between intracellular protein and molecular chaperone protein as an index.
- the present inventors succeeded in detecting an estrogen receptor as a target protein of low molecular weight compound 17 ⁇ estradiol (Example 2), and also detected FKBP12 as a target protein of FK506 and FK1706.
- the present inventors have found that the ⁇ 5 ⁇ protein, which is the ⁇ subunit of the FIFO-ATP synthase present in the mitochondria membrane, has been found to bind to the biguanide agent that is a therapeutic agent for diabetes. It was clarified that the expression of excessive amounts hinders the activity of intracellular AMP kinase (hereinafter referred to as AMPK) by biguanide (Example 5). From these findings, the present inventors have clarified that the ATP5B protein is a target protein involved in the pharmacological action (main action) of the biguanide agent, and have developed a new method for screening an antidiabetic agent using the protein. It was constructed. Certainly, the substance obtained by the screening method of the present invention has a therapeutic effect on diabetes and has no side effects, and a new screening tool and screening method for a therapeutic drug for diabetes and a pharmaceutical composition for treating diabetes are provided. Provided.
- AMPK intracellular AMP kinase
- the present invention provides:
- a method of screening for an antidiabetic agent ⁇ 2> The screening method according to [1], wherein the step [1] is a step of contacting in the presence of a biguanide,
- ⁇ 3> The screening method according to ⁇ 1> or ⁇ 2>, further comprising a step of confirming that AMPK is activated and a step of confirming that it has Z or diabetes therapeutic activity.
- ⁇ 4> (1) The polypeptide according to ⁇ 1>, (2) the polynucleotide encoding the polypeptide according to ⁇ 1>, or (3) the transformed cell according to ⁇ 1> , A screening tool for anti-diabetic drugs that shares a medicinal target with biguanides,
- ⁇ 5> (1) The polypeptide according to ⁇ 1>, (2) the polynucleotide encoding the polypeptide according to ⁇ 1>, or (3) the transformed cell according to ⁇ 1>.
- ⁇ 6> A pharmaceutical composition for the treatment of diabetes containing a substance obtained by the method according to ⁇ 1> to ⁇ 3>,
- a method for treating diabetes comprising administering a substance obtained by the method according to ⁇ 1> to ⁇ 3> to a subject in need of treatment for diabetes in an effective amount,
- a method for identifying a target protein of a test drug comprising the step of comparing the protein detected in (2) with the protein detected in (4),
- amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 26, and Z or SEQ ID NO: 27, the amino acid sequence includes 1 to: LO amino acid deleted, substituted, and Z or inserted amino acid sequence, and a protein A polypeptide that recognizes the three-dimensional structure change and binds to the protein, or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, sequence SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and Z or SEQ ID NO: 27, and a three-
- Non-patent Documents 9 and 10 the properties of molecular chaperones as functional molecules that recognize the non-natural structure of the protein molecules that serve as substrates
- Patent Documents 1 to 6 the method of the method
- Patent Documents 1 to 6 no method for identifying a target protein of a low molecular weight compound using a molecular chaperone has been known at all.
- Non-Patent Documents 17 and 18 and Patent Document 7 the abundance of ATP5B in the muscles of type 2 diabetic patients (see Non-Patent Documents 17 and 18 and Patent Document 7), phosphate levels of ATP5B in the muscles of diabetic patients, and fasting blood glucose It is known that the values are inversely correlated (see Non-Patent Document 18 and Patent Document 7) .
- Patent Document 7 describes that ATP5B itself and antibodies are therapeutic drugs for diabetes-related diseases. If this is controlled, the therapeutic effect of diabetes can be obtained. It was unclear about.
- There are a number of (3025) polypeptides disclosed in the human heart mitochondrial proteome containing ATP5B and these have been reported to be related to screening of therapeutic drugs for many mitochondrial functions including diabetes (Patent Literature).
- a screening method for an antidiabetic agent having an action is an invention that the present inventors have made for the first time.
- the invention's effect is an invention that the present inventors have made for the first time.
- the screening method of the present invention using the screening tool of the present invention (for example, ATP5B, which is a biguanide target protein), in addition to the structurally similar compound of biguanide,
- the compound can be obtained.
- This structurally dissimilar compound can be a novel anti-diabetic agent that has the effects of not causing obesity and causing hypoglycemia, which are characteristic of biguanides.
- By using the binding to the ATP5B protein as an index it is possible to modify the molecular structure while maintaining the main action of the obtained compound, and it is more effective than conventional biguanide agents. Therefore, it is possible to develop a therapeutic drug for diabetes having high activity and reduced side effects.
- FIG. 1 is a diagram showing a band of ER a in which a change is detected depending on the presence of E2 by a pull-down method using a molecular chaperone protein as a probe.
- Lanes 1 and 2 show the results using GST, and lanes 3 to 6 show the results using GST-HSPA4.
- FIG. 2 is a diagram showing the binding of human ATP5B and phenformin.
- “+” indicates the case of MTX-phenformin-added case
- “one” indicates the case of MTX-phenformin-free case.
- “ ⁇ .” Indicates an input.
- FIG. 3 is a graph showing loss of phenformin-binding ability due to human ATP5B mutation.
- WT indicates that wild type ATP5B is used
- E175V indicates that Glul75Val mutant ATP5B is used
- D295V indicates that Asp 295Val mutant ATP5B is used.
- + indicates the case of MTX-phenformin-added case
- one indicates the case of MTX-phenformin-free case.
- Inp Indicates an input.
- FIG. 4 is a graph showing loss of AMPK activation ability of funformin (PF) due to overexpression of human ATP5B.
- the upper panel shows the results using the anti-phosphorylated AMPK antibody, and the lower panel shows the results using the anti-AMPKa antibody.
- “+” indicates the case where phenformin (PF) is added, and “one” indicates the case where phenformin is not added.
- FIG. 5 is a diagram showing the binding of human ATP5B and MTX-phenformin (MTX-PF) depending on the concentration of phenformin (PF).
- the vertical axis represents the count (ATP5B amount).
- FIG. 6 is a diagram showing whether or not the test substance (free phenformin; PF) is brought into contact with a test substance (free phenformin; PF) to determine whether or not it has an influence on the binding.
- the vertical axis shows the count (ATP5B amount).
- Fig. 7 is a diagram showing whether a test substance (compound A or compound B) was brought into contact with a test substance (I compound A or compound B) in an experiment showing the binding between ATP5B and phenformin, and whether or not the binding was affected. .
- the vertical axis shows the count (ATP5B amount).
- FIG. 8 shows that Compound A and Compound B have hypoglycemic action without causing lactic acid accumulation in vivo.
- compound B black square mark
- metformin black circle mark
- solvent black rhombus mark
- Each of A and the above shows the relative value of each measurement time in the solvent-administered group as 100 based on the measured value at 0 minutes, and the vertical axis shows the blood glucose level change rate (%).
- the measured value at 0 minutes is taken as 100, and the relative value is shown, and the vertical axis indicates the rate of change in lactic acid value (%).
- FIG. 9 is a graph showing the expression level of TARDBP in HeLaS3 cells into which pcDNA-TARDBP has been introduced.
- the vertical axis shows the value of TARDBPZ jS —actin.
- FIG. 10 shows the influence of thalidomide (Tha) on the amount of TNF-a produced by okadaic acid (OA) in empty vector-introduced cells or pcDNA-TARDBP-introduced cells.
- the vertical axis shows the value of TNF-H // 8-actin.
- One of the present invention is to use a molecular chaperone protein, which is a protein molecule having a function of recognizing a change in the three-dimensional structure of a protein in a living body, to detect a specific compound (drug whose target protein is unknown).
- a specific compound drug whose target protein is unknown.
- This is a method for identifying a protein (target protein) whose steric structure has changed in response to a compound by selecting a protein whose amount decreases.
- the identification method of the present invention comprises:
- [1] (1) a step of contacting a test drug, a molecular chaperone protein, and a sample intracellular protein;
- a method for identifying a target protein of a test drug comprising the step of comparing the protein detected in (2) with the protein detected in (4)
- the sample intracellular protein means a group of proteins contained in (expressed / expressed) in a cell (hereinafter referred to as a sample cell) that is considered to contain a target protein to be searched.
- a sample cell a group of proteins contained in (expressed / expressed) in a cell (hereinafter referred to as a sample cell) that is considered to contain a target protein to be searched.
- the identification method of the present invention as long as the test drug, molecular chaperone protein, and sample intracellular protein come into contact with each other, the order of contact, the state of the molecular chaperone protein (isolated or expressed in the cell) Or in the extract of the cell) and the state of the sample intracellular protein (force expressed in the living cell, included in the extract of the cell).
- the identification method of the present invention includes a method using the isolated and purified molecular chaperone protein and the sample intracellular protein contained in the sample cell extract (first identification method), and the molecular chaperone protein.
- First identification method Molecular chaperone protein expressed in a sample cell transformed with a vector containing a part or full length of the encoded polynucleotide and a sample intracellular protein expressed in the transformed sample cell (live cell)
- second identification method a method using a molecular chaperone protein contained in the transformed cell extract and a sample intracellular protein contained in the extract.
- a molecular chaperone protein is isolated.
- a partial or full-length region of a molecular chaperone protein or a partial or full-length region of a molecular chaperone protein fused with a tag such as GST, Flag, or His is expressed in bacteria such as E. coli, yeast, or insect cells.
- tag antibodies that are produced in large quantities by chemical synthesis and then fused to molecular chaperone protein antibodies or molecular chaperone proteins, or high affinity beads or affinity tags. It can be purified using a column.
- a molecular chaperone protein can be produced and purified by transcription and translation of a DNA fragment of the molecular chaperone gene in a test tube.
- a purified molecular chaperone protein is added to a test drug-added calorie into a protein mixture extracted from a sample cell (ie, a solution containing a sample intracellular protein).
- a sample cell ie, a solution containing a sample intracellular protein.
- the molecular chaperone protein and the protein binding thereto are concentrated according to the same method as described above.
- Example 2 (2) (2)
- the protein derived from the sample cell that binds to the molecular chaperone protein only when the test drug is not added or the sample cell derived from the sample cell that binds to the molecular chaperone protein only when the test drug is added Protein can be detected.
- the second identification method of the present invention includes:
- a method for identifying a target protein of a test drug comprising the step of comparing the protein detected in (2) with the protein detected in (4)
- the third identification method of the present invention is:
- a test drug a vector containing a polynucleotide encoding a molecular chaperone protein, a molecular chaperone protein in a state contained in a cell extract of a sample cell transformed with one, and contained in the extract Contacting the sample intracellular protein in a state;
- a method for identifying a target protein of a test drug comprising the step of comparing the protein detected in (2) with the protein detected in (4) It is.
- a vector containing a partial or full-length region of a polynucleotide encoding a molecular chaperone protein was searched for! /, Including the target protein A partial or full-length region of a polypeptide that is a molecular chaperone protein, or a partial or full-length region of the polypeptide fused with a tag such as GST, Flag, or His.
- a tag such as GST, Flag, or His.
- a compound hereinafter referred to as a test drug for which a target protein is to be searched is added (contacted) or not added (not added) to the transformed living cells. .
- the molecular chaperone protein, the protein in the sample cell and the sample drug expressed in the transformed sample cell, or the molecular chaperone protein and the sample cell expressed in the transformed sample cell are thereby obtained.
- the inner protein can be contacted.
- a test agent is added to a protein mixture extracted from the transformed cells (ie, a sample cell extract containing a molecular chaperone protein and a sample intracellular protein).
- the molecular chaperone protein contained in the transformed sample cell extract, the sample intracellular protein and the sample drug contained in the extract, or the transformed sample cell The molecular chaperone protein in the state of the extract and the sample intracellular protein in the state of the extract can be brought into contact with each other.
- the protein that binds to the molecular chaperone protein is concentrated according to the same method as the first identification method.
- a known molecular chaperone protein can be used as a molecular chaperone protein that can be used in the identification method of the present invention.
- Hsp90 HtpG; Katsuko shows the name of the enterococcus
- Hsp70 Dnaj
- Hsp60 GroEL
- Hsp40 Dnaj
- Hsp27 IbpAB
- Hspl04 ClpB
- GRP78 DnaK
- Representative proteins belonging to each family are listed (A Sreedhar et al., Pharmacology & Therapeutics, 2004, No. 101-3 p. 227-257; DS Latchman3 ⁇ 4, Cardivascular Research, 2001 51 p. 637-646) .
- Hsp32 low molecular weight sHSPs (small heat shock proteins), etc. can be used as chaperones as well (p Laksanalamai, Extremphiles, 2004 8-1 pi-11).
- the molecular chaperone protein that can be used in the identification method of the present invention is a known molecular chaperone or an amino acid sequence representing a known molecular chaperone protein, preferably 1 to 10 (preferably 1 to 7, more preferably 1). To 5 (more preferably 1 to 3) amino acids are deleted, substituted, and Z or inserted amino acid sequences, and a protein that recognizes a change in the conformation of the protein and binds to the protein. Peptides (hereinafter referred to as functional equivalent variants) are included.
- polypeptides that recognize changes and bind to the proteins (hereinafter referred to as homologous polypeptides) are included.
- the origin of the functional equivalent variant and homologous polypeptide of the present invention is not limited to a specific species.
- a functional equivalent variant or a homologous polypeptide it is not limited to a natural polypeptide, but artificially altered by genetic engineering based on an amino acid sequence representing a known molecular chaperone protein. Also included.
- the “identity” in the present specification means a value Identity obtained by using a parameter prepared by default in a NEEDLE program (J Mol Biol 1970; 48: 443-453) search. .
- the parameters are as follows.
- the molecular chaperone protein used in the identification method of the present invention includes SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6, and the protein shown in Z or 27 (human HSPAlA; RefSeq accession number NP—00 5336, human HSPH1; RefSeq accession number NP 1 006635, human HSPCA; RefSe q accession number NP—005339, human HSPDl; RefSeq accession number NP—95 5472, human DNAJAl; RefSeq accession number NP 001530, human HSPB1; RefSe q accession number NP—001531, human HSPEl; RefSeq accession number NP—00 2148, human HSPA4; RefSeq accession number NP—002145, human HSP90B1; Ref Seq accession number NP—003290, human CCT6B;
- “Recognizes a change in the three-dimensional structure of a protein and binds to the protein” means that a molecular chaperone protein binds to the protein in response to a change in the three-dimensional structure of the protein due to binding to the test drug, or This means that the molecular chaperone protein that was bound to the unaltered protein dissociates in response to a change in the three-dimensional structure of the protein due to binding to the test drug.
- Molecular chaperone proteins respond to changes in protein conformation Whether or not it “binds” can be confirmed in the same manner as in the method of “detecting a protein that binds to a molecular chaperone protein” in the identification method of the present invention.
- biguanides recognitionze changes in the three-dimensional structure of proteins and bind to the proteins
- the molecular chaperone protein that had bound to the unchanged ATP5B protein was altered in conformation with the biguanide. Confirm by divergence in response to conversion.
- SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, and Z or 27 are particularly preferred.
- Each of these proteins belongs to each of the different families of molecular chaperones (Hsp90, Hsp70, Hsp60, Hsp40, Hsp27, Hspl04, GRP78), and each chaperone protein has unique properties. Each is expected to hold.
- the molecular chaperone protein can be easily produced and obtained by a general genetic engineering and Z or biochemical technique using a polynucleotide encoding the molecular chaperone protein.
- the polynucleotide can be easily produced and obtained by a general genetic engineering technique based on the sequence information disclosed in the present specification or known gene sequence information. For example, it can be obtained as follows, but not limited to this method, it can also be obtained by a known operation (“Molecular Cloning J [Sambrook, J et al., Cold Spring Harbor Laboratory Press, 1989, etc.]).
- PCR-based methods (2) conventional genetic engineering methods (ie cDNA libraries) Examples include the ability to use the ability of a transformed strain transformed with the library to select a transformed strain containing the desired polypeptide, or (3) a chemical synthesis method.
- a transformation method can be carried out in the same manner as described in WO01Z34785.
- the "embodiment of the invention” in the above-mentioned patent document 1) a method for producing a protein gene a) a polypeptide encoding a molecular chaperone protein by the procedure described in the first production method
- Nucleotides can be produced.
- tissue power such as human liver, brain, and mammary gland also extracts mRNA.
- the mRNA can then be subjected to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer to synthesize a first strand cDNA.
- a polynucleotide encoding a molecular chaperone protein or a part thereof was used.
- PCR polymerase chain reaction
- a polynucleotide encoding a molecular chaperone protein can be produced by the method described in Example 1.
- a polynucleotide encoding a molecular chaperone protein can be produced. Specifically, it can be produced by peptide synthesis using a liquid phase or solid phase method. The synthesis may be performed by sequentially binding amino acids one by one or after synthesizing a polypeptide fragment having several amino acids.
- the polypeptide of the present invention obtained by these means can be purified according to various known methods.
- Sequence variations can also be made by artificially altering, for example, the forces that may be caused by mutations in nature.
- the present invention does not ask the cause and means of the mutation.
- an artificial means for producing the above-mentioned mutant for example, a base-specific substitution method of a polynucleotide encoding the above-mentioned polypeptide (Methods in Enzymology, (1987) 154, 350, 367-382), etc.
- the phosphate triester method and phosphate phosphate And chemical synthesis means such as the dye method (Science, 150, 178, 1968). By combining them, it is possible to obtain a polynucleotide with a desired base substitution.
- non-specific bases in the polynucleotide molecule can be substituted by repeating the PCR method or by making manganese ions or the like present in the reaction solution.
- the polynucleotide encoding the molecular chaperone protein obtained as described above is ligated downstream of an appropriate promoter by the method described in “Molecular Clonmg, Sambrook, J et al., Cold spring Harbor Laboratory Press, 1989”.
- the molecular chaperone protein can be expressed in a test tube or a test cell. Specifically, by adding a polynucleotide containing a specific promoter sequence upstream of the initiation codon of the polynucleotide obtained as described above, transcription of the gene in a cell-free system using this as a cage, Expression of molecular chaperone protein by translation is possible.
- a polynucleotide encoding a molecular chaperone protein is incorporated into an appropriate vector plasmid and introduced into a host cell in the form of a plasmid, the polypeptide can be expressed in the cell.
- a cell in which such a configuration is incorporated into chromosomal DNA may be obtained and used.
- the fragment containing the isolated polynucleotide can be transformed into eukaryotic and prokaryotic host cells by reincorporation into an appropriate vector plasmid.
- molecular chaperone proteins can be expressed in the respective host cells by introducing appropriate promoters and sequences involved in phenotypic expression into these vectors.
- the host cell is not particularly limited as long as it can realize expression of a sufficient amount of molecular chaperone protein for the purpose of use in the method of the present invention.
- Examples of host cells include monkey COS cells (Gluzman, Y. (1981) Cell, 23, 175-182), Chinese hamster ovary cells (CHO) dihydrofolate reductase deficient strain (Urlaub, G an d Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77, 42 16—4220), HEK293 cells derived from human fetal kidney and the EBNA-1 gene of Epstein Barr Vir us was introduced into the same cells 293 — Examples include EBNA cells (Invitrogen).
- a method for transforming a host cell and expressing a gene can be carried out, for example, by the method described in the method for producing a recombinant protein in "Embodiment of the invention” 2) of the patent document.
- An expression vector (an expression vector for molecular chaperone expression) used for production of a molecular chaperone-expressing cell is not particularly limited as long as it contains a desired polynucleotide.
- an expression vector obtained by inserting a desired polynucleotide into a known expression vector appropriately selected according to the host cell to be used can be mentioned.
- Known expression vectors include, for example, pSV2dhfr (Subrama ni, S. et al. (1981) Mol. Cell.
- the molecular chaperone protein can be obtained, for example, by transforming a desired host cell with the expression vector and expressing the polypeptide in the cell.
- a desired molecular chaperone protein can be produced in large quantities in bacterial cells by incorporating the desired polynucleotide into a vector expression vector.
- a large amount of molecular chaperone protein can be produced using yeast or insect cells.
- a desired molecular chaperone protein can be produced in a test tube by a known technique using the above-mentioned polynucleotide bound downstream of a predetermined promoter. More specifically, using the TNT system (Promega), a desired molecular chaperone protein can be obtained in vitro by a transcription / translation reaction in a test tube in which the above-mentioned polynucleotide bound downstream of the above promoter is in the shape of a cage. Can be produced.
- the molecular chaperone protein produced in the cells can be detected, quantified, and further purified.
- the protein can be detected and purified by Western plotting using an antibody that binds to a molecular chaperone protein, or by immunoprecipitation.
- the protein may be daltathione mono S-transferase (GS T), protein A, ⁇ -galatatosidase, maltose-binding protein (MBP), etc. and expressed as a fusion protein with an appropriate tag protein, Western plotting using antibodies specific for these tag proteins,
- the protein can be detected by immunoprecipitation.
- the protein can be purified using these tag proteins. More specifically, the protein can be purified using the tag protein as follows.
- a molecular chaperone protein for example, SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27
- a vector in which a polynucleotide encoding them is added to a protein of interest in which a GST tag or His tag is expressed, More specifically, for example, it is incorporated into PGEX-6P1 (Amersham) described in Example 1 or commercially available pET-28a (Novagen), etc., and introduced into bacteria.
- the former is a GST fusion protein, and in the latter case A molecular chaperone protein can be expressed as a His fusion protein.
- the fusion protein can be purified from the bacterial cell-derived protein extract in which they are expressed using the properties of the GST or His tag.
- the chaperone expression plasmid prepared using pGEX-6P1 is designed to add a V, GST tag to the N-terminus of the molecular chaperone protein!
- the protein can be purified from cells expressing the desired molecular chaperone protein using these GST tags.
- a molecular chaperone fused with a GST tag from the disrupted cell extract according to the well-known GST-pull down method Example 1
- a molecular chaperone fused with a GST tag from the disrupted cell extract according to the well-known GST-pull down method (Experimental Engineering, Voll 3, No. 6, pp. 528, Matsuchichigosan et al., 1994).
- Proteins can be isolated by centrifugation after binding to dartathione Sepharose beads (Glutathione Sepharose 4B; Amersham Fanolemacia).
- dartathione Sepharose 4B Glutathione Sepharose 4B
- His tag it is also known to use the His tag to purify the protein from cells expressing the desired molecular chaperone protein (Experimental Medicine Separate Volume, Protein Molecular Interaction Experiments, 1996, p. 32, Nakahara et al. ).
- the molecular chaperone protein fused with the His tag can be bound to Ni 2+ -NTA-A garose (Funakoshi) from the disrupted cell extract and isolated by centrifugation.
- a method that does not use a tag protein such as a molecular chaperone protein
- a method that does not use a tag protein can also be purified by various separation operations utilizing its physical and chemical properties. Specifically, use of ultrafiltration, centrifugation, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography can be exemplified.
- the test agent is not particularly limited, and examples thereof include commercially available compounds (including peptides), various known compounds (including peptides) registered in chemical files, combinatorial chemistry technology ( N. Terrett et al., Drug Discov. Today, 4 (1): 41, 1999), compound cultures, microorganism culture supernatants, natural components derived from plants and marine organisms, animal tissue extraction Products (including polynucleotides and polypeptides) or compounds obtained by chemically or biologically modifying them, and those having a clear pharmacological action.
- the pharmacological action includes not only desirable actions in medicine but also harmful effects on the living body.
- the use of the method of the present invention is considered to be a compound that is considered to be more useful than existing methods: (1) a compound that is difficult to modify in terms of chemical structure; Compounds whose action is lost or predicted to be lost, (3) compounds whose pharmacological activity is undetermined by decomposing or mixing metabolites and other congeners, and (4) synthesis and purification Or compounds whose natural materials are difficult to obtain due to the difficulty in obtaining raw materials (including natural products), etc.
- a solution obtained by extracting a protein from a primary target tissue for inducing pharmacological activity possessed by a test drug or cultured cells that maintain many of the properties of the tissue can be used.
- a preparation method according to the purpose it is preferable to use a preparation method according to the purpose. Specifically, according to known protein extraction methods, SDS, Triton X-100, or various surfactants such as CHAPS and CHAPSO are selected to suit the purpose and a buffer solution containing these at a concentration suitable for the purpose.
- the above-mentioned cells are crushed using, and after centrifugation, the supernatant is separated and collected, and then used as the cell extract in the method of the present invention. More specifically, in the buffer used for crushing, It is preferable to contain inhibitors of various proteolytic enzymes such as PMSF (phenylmethyl sulfonylfluoride), EGTA (Ethylene glycol—bis (j8-aminoethylether) —N, N, N ′, N′-tetraacetic Acid), etc. Until it is applied to the method of the present invention, it is preferably frozen and stored at a temperature of 80 ° C or lower so that the protein is stably maintained.
- PMSF phenylmethyl sulfonylfluoride
- EGTA Ethylene glycol—bis (j8-aminoethylether) —N, N, N ′, N′-tetraacetic Acid
- Molecular chaperone proteins are all thought to respond to many substrate proteins per molecule. However, it is considered that the substrate specificity differs depending on the type. Therefore, in order to truly and comprehensively search for targets of compounds having various structures, it is preferable to prepare molecular chaperone proteins belonging to many different families and simultaneously use them as probes. More preferably, as shown in Examples 2 (3), 3, 4 and 8 of the present invention, it is desirable to prepare a plurality of molecular chaperone proteins derived from different chaperone families and use them simultaneously.
- the protein to be used as a probe in the present invention, molecular chaperone protein
- the substrate specificity of the probe It can be expected that even a protein other than the original substrate will be recognized by the probe if it is a molecule similar to the substrate. Therefore, as in the in vitro pull-down method shown in Example 2 (2) of the present invention, the use of a system capable of reacting a large amount of probe protein compared to the cell-derived protein serving as a substrate is an exhaustive list of compounds. It is more preferable to enable target search.
- molecular chaperone proteins have been reported to act by forming multimers in vivo. Therefore, in carrying out the method of the present invention, as shown in Examples 2 (2) and 4, derived from a biological sample in which an endogenous chaperone capable of forming a complex with a molecular chaperone protein (probe) is mixed. It is more preferable to use cell extract and protein separation conditions. More specifically, a molecular chaperone protein (probe protein) is applied to a cell-derived cell extract containing an endogenous chaperone as in the in vitro pull-down method shown in Example 2 (2) of the present invention. Use of a reaction system is preferred.
- the molecular chaperone protein obtained above and a concentrated solution of the binding protein are separated by a known protein separation method to detect a protein that binds to the molecular chaperone protein.
- a known protein separation method to detect a protein that binds to the molecular chaperone protein.
- it can be separated by polyacrylamide gel electrophoresis to detect various known proteins such as silver staining, Coomassie Priant Blue staining, or existing gel staining such as negative gel staining (Wako Pure Chemical).
- the method (“Protein structure analysis for gene cloning" Hisashi Hirano, Tokyo Chemical Dojin, 1993, p37-p40) can detect proteins derived from sample cells bound to molecular chaperone proteins and molecular chaperone proteins. it can.
- the method used in the process of the present invention is not limited to the above method as long as it can detect a protein.
- the proteins that bind to the molecular chaperone protein with and without the test drug added are compared. By comparing the two, it is possible to identify a sample cell-derived protein whose binding to the molecular chaperone protein has changed when the test drug is added or not (ie, the target protein of the test drug).
- the well-known SDS polyacrylamide electrophoresis method can be mentioned. In that case, more accurate comparison is possible by developing with two-dimensional electrophoresis.
- the protein detected and selected by the above method is identified.
- the amino acid sequence present in the molecule is determined and the amino acid sequence information is used.
- the target protein of the test drug can be recovered from the gel and purified, and then its amino acid sequence can be determined by mass spectrometry or a known technique to identify the protein.
- the target protein separated by SDS polyacrylamide electrophoresis gel was fragmented using tribsin or the like, the resulting peptide mixture was recovered from the gel, and the protein was identified by mass spectral analysis. (Schevchenko et al., Analytical Chemistry, 68, 850-858, 1996).
- the target protein separated by SDS polyacrylamide electrophoresis gel was fragmented using trypsin or the like, and the resulting peptide mixture was gelled.
- the protein can be recovered and identified by mass spectrum analysis.
- the target protein identified by the method of the present invention is tested using a known gene function analysis technique. It is possible to confirm that it is a true target protein that brings about the pharmacological action of the test drug
- the presence or absence of direct binding between the test drug and the obtained target protein molecule can be examined by the method shown below.
- a part or full length region of the polypeptide to be examined for the ability to bind or a part or full length region of the polypeptide to be examined fused with a tag such as GST, Flag, or His is expressed in the cell.
- the target polypeptide expressed from the cells by affinity purification method using affinity with tags such as GST, Flag, His, etc., or immunoprecipitation method using antibodies responding to the tag, etc. Isolate 'purify. Subsequently, the purified polypeptide and the test drug are mixed, and the complex formed by binding of the test drug and the polypeptide is isolated.
- the complex is denatured by acid, heat, or other stimulus, the compound is separated again to remove only the protein, and then analyzed by mass spectrometry using mass spectrometry, and the test drug is contained in the sample.
- the test drug is contained in the sample.
- the target polymorphs can be examined by methods such as the known ELISA method, far western method, and binding assay method. It can be confirmed whether or not the peptide and the test drug are capable of binding.
- a labeled compound can be prepared by replacing an element in the molecule of the test drug with a radioisotope.
- the binding between the polypeptide and the test drug can be confirmed by an ELISA method in which the polypeptide to be examined purified by the above method is immobilized.
- the polypeptide to be studied is separated by a known SDS acrylamide gel electrophoresis method, transferred onto a nitrocellulose membrane, and then the polypeptide and the polypeptide are also subjected to the far western method using the labeled test agent as a probe. The binding of the test drug can be confirmed.
- a known protein three-dimensional structure prediction method (J Med Chem. 2004 Dec 30; 47 (27): 6804-11) becomes a keyhole where the test compound can bind to the target protein identified by the method of the present invention. You can check if a structure exists.
- gene knockdown experiments at the cellular level using the known RNA interference technology (Tuschl T. et al., Nat Biotechnol. 2002, 20 (5): p446-448.), Also known genes at the cellular level. Increase or decrease the expression level of the target protein by various biochemical and Z or genetic engineering experimental techniques such as overexpression experiments, and further, gene knockout animal production, gene overexpression animal production, etc.
- the identification method of the present invention can identify the target protein of the test drug that brings about the pharmacological action of the test drug.
- the identification method of the present invention is not limited to the desired pharmacological action (main action) among the pharmacological actions of the test drug. ) Is more suitable as a method for identifying a target protein that brings about. ⁇ The screening tool of the present invention and use for screening>
- the screening tool of the present invention comprises the following (1) to (3).
- Human ATP5B protein (polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2), A TP5B functional equivalent variant (amino acid sequence represented by SEQ ID NO: 2 or amino acid sequence represented by SEQ ID NO: 2) 1 to 10 (preferably 1 to 7, more preferably 1 to 5 and more preferably 1 to 3) amino acid sequences deleted, substituted, and Z or inserted amino acid sequences, And a polypeptide that binds biguanide and inhibits AMPK activation by biguanide by Z or overexpression), and ATP5B homologous polypeptide (identity of amino acid sequence represented by SEQ ID NO: 2 is 90% or more ( Preferably 95% or more, more preferably 98% or more) and binds to biguanide and inhibits AMPK activity by biguanide by Z or overexpression. ) (Hereinafter, consists of a polypeptide) tools, diabetes drug screening tool to share biguanide and medicinal target (hereinafter, referred as the polypeptide-type screening
- a screening pool for a therapeutic agent for diabetes that shares the drug target with biguanide (hereinafter referred to as the polynucleotide-type screening tool of the present invention), which also has the power of a polynucleotide encoding a polypeptide for tool (hereinafter referred to as a polynucleotide for tool).
- the polynucleotide-type screening tool of the present invention which also has the power of a polynucleotide encoding a polypeptide for tool (hereinafter referred to as a polynucleotide for tool).
- Screening tool for anti-diabetic drugs that share the drug target with biguanide which consists of cells (hereinafter referred to as tool cells) transformed with a vector containing the tool polynucleotide and expressing the tool polypeptide (hereinafter referred to as tool cells). This is called the cell type screening tool of the present invention).
- the use of the present invention for screening for a therapeutic drug for diabetes that shares a drug target with biguanide is also included in the present invention.
- screening tool refers to what is used for screening (specifically, a polypeptide, polynucleotide or cell used for screening).
- the “diabetic drug screening tool sharing a drug target with biguanide” refers to a test compound contacted in the screening method of the present invention in order to screen a diabetes drug sharing a drug target with biguanide.
- ATP5B functional equivalent variants and ATP5B homologous polypeptides are not limited to humans. As long as one of the polypeptide for tool is applicable, it includes those derived from all living organisms from vertebrates to bacteria as well as human variants of the amino acid sequence represented by SEQ ID NO: 2.
- the polypeptide is not limited to a natural polypeptide, and includes a polypeptide artificially modified by genetic engineering based on the amino acid sequence represented by SEQ ID NO: 2.
- ATP5B functional equivalent variant or ATP5B Polypeptides containing the marker sequences described below in homologous polypeptides are also included in the tool polypeptides.
- amino acid residues involved in the peripheral structure of site 20, particularly Glul75 and Asp 295 in the case of human ATP5B, are important for the binding of ATP5B and biguanide. There was a certain power. Therefore, when a mutation is introduced, amino acid residues involved in the surrounding structure of Site 20, especially Glul75 and Asp295 in the case of human ATP5B, are conserved, and by binding mutations to other parts, binding to biguanide A modified polypeptide that maintains its activity can be easily produced.
- the human ATP5B polypeptide strength of the tool polypeptide is used.
- a polypeptide encoding a human ATP5B polypeptide of the tool polynucleotide is used.
- Nucleotide (particularly preferably, the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1) is used as a cell type screening tool of the present invention in a vector containing a polynucleotide encoding human ATP5B among the tool cells. More preferred are cells that have been transformed and express human ATP5B.
- the tool polynucleotide can be easily produced and obtained by a general genetic engineering technique.
- a technique as described in the above ⁇ Method for producing molecular chaperone protein>, a known operation “Molecular Cloning” [Sambrook, J et al., Cold Spring Harbor Laboratory Press, 1989, etc.], for example, ( 1) a method using PCR, (2) a method using a conventional genetic engineering method (that is, a method for selecting a transformant containing a desired polypeptide, a transformant transformed with a cDNA library), or (3) List chemical synthesis methods.
- Each production method can be carried out in the same manner as described in WO01Z34785, as described in “Method for producing molecular chaperone protein” above.
- mRNA is extracted from human skeletal muscle, heart and / or tissue, and the same as described in ⁇ Method for producing molecular chaperone protein> above.
- a polynucleotide for a tool or a part thereof can be obtained by PCR using a primer. More specifically, for example, a polynucleotide for a tool can be produced by the method described in Example 5 (1).
- a method using a chemical synthesis method can produce a polynucleotide for a tool in the same manner as described in ⁇ Method for producing molecular chaperone protein> above.
- the tool polynucleotide obtained as described above may be prepared by a known method (for example, “Molecular Cloning, Sambrook, J. et al., Cold Spring” in the same manner as described in the above-described method for producing a molecular chaperone protein>.
- the polypeptide for tool can be expressed in a test tube or in a test cell by ligation downstream of an appropriate promoter by the method described in Harbor Laboratory Press, 1989 ”.
- the expression vector (tool expression vector) used for the production of the tool cell is not particularly limited as long as it contains the tool polynucleotide.
- an expression vector obtained by inserting a tool polynucleotide into a known expression vector appropriately selected according to the host cell to be used can be mentioned.
- the tool polypeptide can be obtained, for example, by transforming a desired host cell with a tool expression vector and expressing the tool polypeptide in the cell. More specifically, by incorporating the tool polynucleotide into a bacterial expression vector, the tool polypeptide can be produced in large quantities in the nocteria cells.
- a tool polypeptide can be produced in a test tube in a known manner. More specifically, as described in Example 5 (4), using the TNT system (Promega), transcription in vitro using the tool polynucleotide bound downstream of the promoter as described above.
- TNT system Promega
- the tool cell is a cell in which the tool polynucleotide is integrated into the chromosome of the host cell, as long as it is transformed with the tool expression vector and contains the tool polynucleotide.
- the cells may be contained in the form of an expression vector containing the tool polynucleotide. Used for transformation As cells, cells that respond to biguanides are preferred. More specifically, HeLaS3 cells, liver-derived cells, or skeletal muscle-derived cells are preferred. As long as it corresponds to the tool polynucleotide, the tool cell may contain a polynucleotide containing a marker sequence described later.
- the cell type screening tool herein is preferably a cell transformed with a tool expression vector.
- the desired transformed cells obtained above can be cultured according to a conventional method, and the polypeptide for tool is produced by the culture.
- the medium used for the culture various commonly used media can be appropriately selected according to the adopted host cells.
- serum components such as fetal bovine serum (FBS) are added.
- Medium such as Dulbecco's Modified Eagle Minimum Essential Medium (DMEM) can be used.
- the polypeptide for tool produced as described above can be separated or purified by various known separation procedures utilizing physical properties, biochemical properties, etc. of the polypeptide as necessary. I can do it.
- the marker polypeptide tag protein
- the expression of the polypeptide can be confirmed and purified using the tag protein.
- Marker sequences include, for example, FLAG epitope, hexahistidine tag, Hemagglutinin tag, myc epitope, glutathione S-transferase (GST), protein ⁇ , ⁇ -galactosidase, maltose-binding protein ( ⁇ ).
- fusion polypeptide in which a specific amino acid sequence recognized by a protease such as enterokinase, factor Xa, or thrombin is inserted between the marker sequence and the tool polypeptide is expressed and purified using a tag protein. It is also possible to cleave and remove the sequence portion with these proteases to obtain a tool polypeptide.
- a protease such as enterokinase, factor Xa, or thrombin
- the polypeptide for tool is, for example, a vector in which a GST tag or His tag is added to the target protein, more specifically, for example, pGEX-6P1 (Amersham) or pET— Using 28a (Novagen) etc., the former can be expressed as a GST fusion protein, and the latter can be expressed as a His fusion protein.
- the fusion protein is the same as described in ⁇ Method for producing molecular chaperone protein> from the protein extract derived from the bacterial cell in which they are expressed, using the properties of the GST or His tag. Further, it can be purified.
- polypeptide that binds biguanide means a polypeptide that binds to a low molecular compound (methformin, phenformin, pformin, etc.) classified as a biguanide, and the polypeptide is a biguanide. Whether or not to “combine” can be confirmed by the following method.
- the polypeptide to be examined for binding is isolated and purified.
- the above methods can be used for the expression, isolation and purification of the polypeptide.
- the ability of binding to phenformin is confirmed by the method of Example 5 (4).
- the concentration of phenformin to be added is preferably 10 ⁇ or less, more preferably 1.0 ⁇ or less, and even more preferably 0. It is determined that the polypeptide binds to biguanide.
- “Inhibiting biguanide activity by overexpression” means that a certain peptide was stimulated by biguanide under the condition that cells were present in excess of normal levels. This means that the activity of sputum in the cells, ie, the degree of phosphorylation, is reduced compared to normal cells. Whether or not “inhibit the activity” is confirmed by the method of Example 5 (7).
- Example 5 Under the conditions of (7), phosphatidylphosphatase stimulated with phenformin in cells overexpressing the polypeptide of interest compared to the control (cells overexpressing the polypeptide of interest). If the increase in wrinkles is reduced by 50%, preferably 70%, more preferably 90%, the polypeptide to be examined is determined to be a polypeptide that inhibits the biguanide wrinkle activity due to overexpression.
- ATP5B is a true target protein that binds to biguanide and contributes to the drug efficacy (main action) of the compound.
- the present inventors screened for a low molecular weight compound that binds to a polypeptide for a tool and activates AMPK, thereby obtaining a compound that can exhibit the same therapeutic effect as that of biguanide. It was clarified that it could be newly acquired, and a screening method for antidiabetic drugs was completed.
- the screening method of the present invention includes the following methods.
- a method for screening an antidiabetic agent comprising:
- step [1] is a step of contacting in the presence of a biguanide.
- Screening in the present specification is a substance in which a substance having a target activity is screened out from a large number of test substances, and a substance has a target property with respect to a certain test substance. It includes both detecting force or not.
- the purified tool polypeptide and the test substance are mixed and brought into contact (contacting), and the test is performed.
- the substance that binds and acts directly on the polypeptide i.e., diabetes
- mass spectrometry A method of analyzing (therapeutic agent) (the step of analyzing binding) and selecting a substance that binds to the polypeptide for the tool (ie, a therapeutic agent for diabetes).
- affinity selection mass spectrometry AS-MS method; Pharmacia (Japan Pharmaceutical Association) Vol. 41 No.
- the tool polypeptide is expressed in cells. Affinity purification method using affinity with tags such as GST, Flag, and His from the cells, or immunoprecipitation method using an antibody (for example, anti-ATP5B antibody, tag antibody) that responds to tool polypeptide
- an antibody for example, anti-ATP5B antibody, tag antibody
- the substance that does not bind to the polypeptide is removed with a resin or the like that adsorbs the low molecular weight compound.
- the complex is denatured by acid, heat, or other stimulus to separate low molecular weight substances in the complex, and only the remaining protein is removed. Analyze by mass spectrometry using mass spectrometry to identify substances contained in the sample, and select substances that bind to the tool polypeptide.
- the control does not go through the process of adsorbing and removing the test substance under the condition that the concentration of the test substance to be added is 1 ⁇ .
- the total amount of the test substance recovered in comparison with the above is preferably 5% or more, more preferably 10% or more, and still more preferably 50% or more as a substance that binds to the tool polypeptide.
- a test substance that binds to a tool polypeptide is prepared by applying a specific label to a test substance group and using a known ELISA method, first western method, binding assay method, or the like. A method of selection is also included. A specific example is the method of Example 5 (4). In these methods, a test substance that can detect binding to the polypeptide for tooling at a concentration of preferably 10 ⁇ m or less, more preferably 1 ⁇ m or less, and even more preferably 0.1 ⁇ m or less is used for tools. Select as a substance that binds to the polypeptide.
- the screening method of the present invention comprising the step of bringing a polypeptide for tool and a test substance into contact with each other in the presence of a biguanide, the labeled method or the modified biguanide and the tool polypeptide.
- the step of detecting the binding between the polypeptide and biguanide by ELISA, far western method or binding assay using tide the change in binding that occurs when the test substance is present at the same time is detected. This can be achieved by measuring. Specifically, various experimental methods exemplified below are used.
- the tool polypeptide is expressed in cells.
- Tools expressed by affinity purification methods using affinity with the cell force tag, or immunoprecipitation methods using antibodies that respond to tool polypeptides Isolate 'purify the polypeptide for use. Subsequently, the purified polypeptide, test substance and biguanide are mixed and the formed complex is isolated. The complex is then denatured by acid, heat, or other stimulus to separate the test substance and biguanide again to remove only the protein, and then analyzed by mass spectrometry using mass spectrometry. To determine whether the binding of the tool polypeptide to the biguanide is inhibited by the test substance.
- a method such as a known ELISA method, far western method, or binding assay method using a labeled biguanide prepared by modifying biguanides and labeling a part of their molecular structure as a probe.
- a labeled compound is prepared by substituting an element in a biguanide molecule with a radioisotope.
- the binding between the polypeptide and the biguanide is confirmed by ELISA using a purified tool polypeptide immobilized thereon.
- the polypeptide for the tool is separated by a known SDS acrylamide gel electrophoresis method, transferred onto a nitrocellulose membrane, and then the polypeptide is separated from the polypeptide by a far western method using the above-mentioned labeled biguanide as a probe.
- Check biguanide binding Alternatively, the labeled biguanide and the purified polypeptide for the tool are mixed, trapped on a filter and washed, and then the radiation dose derived from the labeled probe is measured to determine the compound-peptide complex formed. The binding of the tool polypeptide to the biguanide is confirmed by V, the so-called binding assay, which detects the total body mass.
- the label of biguanide is not limited to a radioisotope, and it does not affect its pharmacological activity. Can be achieved.
- labeling can be performed by modifying a part of the molecular structure of phenformin, metformin, or buformin and subjecting it to piotin.
- the binding between the polypeptide for tool and biguanide is confirmed by ELISA or far western method as described above by utilizing the binding of piotin and avidin and using a labeled avidin antibody or the like.
- the binding between the tool polypeptide and the biguanide is confirmed by the method of Example 5 (4).
- the step of confirming the binding between the tool polypeptide and the biguanide it is examined whether or not the binding of the polypeptide and the biguanide is inhibited by the presence of the test substance simultaneously with the biguanide.
- the biguanide concentration is 10 M
- a substance having an IC50 of preferably 10 M or less, more preferably a substance of 1 M or less, and further preferably a test substance of 0.1 M or less is selected as a therapeutic agent for diabetes. If the presence of a specific test substance prevents the binding of the tool polypeptide to the biguanide, the phenomenon is caused by the binding of the test substance to the biguanide binding site in the molecule of the tool polypeptide.
- the binding to the polypeptide is the result of competitive inhibition.
- the confirmation of competitive binding inhibition by the test substance is obtained by gradually inhibiting the biguanide binding to the polypeptide by gradually changing the ratio of the concentration of biguanide and test substance simultaneously present. It can be confirmed by examining what is done.
- a substance that is, a therapeutic agent for diabetes
- a substance that binds to the tool polypeptide can be selected by the method shown in Example 6 described later.
- the screening method of the present invention comprising the step of bringing a tool cell and a test substance into contact with each other.
- the tool cell and the test substance are mixed and brought into contact with each other (step of contacting), and the test substance and the polypeptide bind to each other.
- the isolated compound is then denatured and then separated and identified by mass spectrometry to analyze the substance that binds directly to the polypeptide (that is, a therapeutic agent for diabetes) (analyzes the binding).
- Step) a method of selecting a substance that binds to the tool polypeptide (ie, a therapeutic agent for diabetes). How to analyze the binding
- the AS-MS method described above can be used. Specifically, for example, it can be carried out as follows.
- the tool cell in which the tool polypeptide is expressed is brought into contact with the test substance.
- Affinity purification method using affinity with tags such as GST, Flag, His, etc., or immunoprecipitation method using antibodies that respond to tool polypeptides (eg anti-ATP5B antibody, tag antibody) Concentrate the complex of tool polypeptide and test substance binding to it. In this concentration process, it is desirable to include the same test substance obtained by treating the cells in the reaction solution. After removing the substance that does not bind to the polypeptide with a resin that adsorbs low molecular weight compounds, the next complex of the tool polypeptide and the test substance that binds to the polypeptide is acid, heat, etc. The low molecular weight substance in the complex is separated by denaturing with the stimulation of, and only the remaining protein is removed.
- the substances that bind to the polypeptide for the tool are selected from the test substances.
- the total amount of the test substance recovered is preferably 5% or more compared to a control that does not undergo the process of adsorbing and removing the test substance under the condition that the concentration of the test substance to be added is 1 ⁇ . More preferably, 10% or more, more preferably 50% or more of the substance is selected as a substance that binds to the tool polypeptide.
- the binding between the tool polypeptide and the biguanide is competitively inhibited by the test substance.
- cell force is obtained by the same process as the above-described binding analysis screening method using tool cells>, and the complex of biguanide bound to the polypeptide of the present invention is bound thereto. Concentrate the body. Subsequently, the binding between the tool polypeptide and the test substance is analyzed. The analysis of the binding can be carried out by the same method as described in “Competitive inhibition screening method using tool polypeptide” above.
- a modified biguanide such as a label
- a known binding assay method is used. This can be performed by quantifying the binding between the polypeptide for tool and the biguanide. If the presence of the test substance hinders the binding of the tool polypeptide to the biguanide, the test substance competitively inhibits the binding of the biguanide to the tool polypeptide, and the tool polypeptide and the test substance are bound. I can judge.
- the screening method of the present invention after praying for the binding between the tool polypeptide and the test substance, and selecting a substance that binds to the tool polypeptide, the selected test substance activates AMPK.
- the method further includes a step of confirming the above and a step of confirming that the compound has Z or diabetes therapeutic activity.
- the step of confirming that the selected substance activates AMPK can be performed, for example, as follows. Add test substance or solvent control to cells expressing tool polypeptide (eg HeLaS3 cells) and incubate. Using cell lysate prepared by lysing cultured cells, using known SDS electrophoresis and anti-phosphorylated AMPK antibody (eg Phospho-AMPK-a (Thr 172) Antibody, Daiichi Kagaku) Intracellular AMPK phosphorylation level (ie, activation level) is detected by Western blotting. Preferably, it can be confirmed by the method of Example 5 (7) that the selected substance activates AMPK.
- a substance that enhances AMPK phosphorylation is selected as a substance that activates AMPK compared to the control.
- a substance that enhances AMPK phosphorylation a substance that enhances AM PK phosphorylation by 50%, preferably 70%, more preferably 90% or more is selected as compared with the control.
- the step of confirming that the selected substance has diabetes treatment activity includes a step of performing a known evaluation method, for example, a method of analyzing the effect of treating the selected substance for diabetes as described below. Can be mentioned.
- the compound selected by the screening method of the present invention is continuously administered to a diabetic model animal, and the blood glucose lowering action after the oral glucose tolerance test is confirmed by confirming the blood sugar lowering action as needed according to a conventional method. Based on this, the presence or absence of diabetes treatment effect is determined.
- measure insulin resistance in humans and analyze the therapeutic effects of type 2 diabetes using the improvement in the value as an index. Insulin resistance is measured in two main ways in humans. One is to measure blood glucose level and insulin concentration after fasting, and the other is glucose tolerance test. This is a test method in which a glucose solution is orally administered and the clearance rate from the blood circulation is known. In addition, more accurate tests include the euglycemic 'hyperinsulinemia clamp method.
- This test is based on the principle that insulin and glucose in the blood are maintained at a certain concentration, and the total amount of glucose solution administered over time and the insulin concentration used for metabolism are determined. It is to be measured (“Diabetes mellitus”) Nakagawa Osamu 1999, 42 (2): p. Preferably, by the method described in Example 7, it can be confirmed that the selected substance has diabetes therapeutic activity.
- test substance used in the screening method of the present invention is not particularly limited.
- commercially available compounds including peptides and various known compounds (including peptides) registered in the chemical file.
- Combinatorial chemistry technology N. Terr ett et al., Drug Discov. Today, 4 (1): 41, 1999
- compound mixtures microbial culture supernatants, plants and marine organisms
- Examples include natural components derived from animals, animal tissue extracts, and compounds (including peptides) obtained by chemically or biologically modifying compounds (including peptides) selected by the screening method of the present invention. .
- a pharmaceutical composition for treating diabetes comprising a substance [for example, DNA, protein (including antibody or antibody fragment), peptide, or other compound] obtained by the screening method of the present invention as an active ingredient.
- the present invention also includes a method for treating diabetes, comprising administering an effective amount of the substance obtained by the screening method of the present invention to a subject in need of diabetes treatment.
- the present invention includes the use of the substance obtained by the screening method of the present invention for producing a pharmaceutical composition for treating diabetes.
- the active ingredient in the pharmaceutical composition of the present invention can be selected by the screening method of the present invention.
- Examples of compounds selected by the screening method of the present invention include 2-((E) — (1H-1,2,4 triazole-3-irimino) methyl described in Examples 6 (2) and 7 described later. And [Lu] phenol and 6-black 9H purine 1-2 amine.
- confirmation of the effectiveness of treating diabetes can be achieved by methods known to those skilled in the art or It can be carried out by using an improved method (see the above “step for confirming that the selected substance has anti-diabetic activity”).
- the preparation containing a substance [eg, DNA, protein (including antibody or antibody fragment), peptide, or other compound] obtained by the screening method of the present invention as an active ingredient depends on the type of the active ingredient.
- a substance eg, DNA, protein (including antibody or antibody fragment), peptide, or other compound
- it can be prepared as a pharmaceutical composition using pharmacologically acceptable carriers, excipients, and Z or other additives that are usually used for their formulation.
- oral administration such as tablets, pills, capsules, granules, fine granules, powders, or oral liquids, or injections such as intravenous, intramuscular or joint injections, suppositories And parenteral administration by transdermal administration agent or transmucosal administration agent.
- parenteral administration such as intravenous injection is preferred.
- one or more active substances and at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, It can be mixed with starch, polyvinylpyrrolidone, magnesium metasilicate, or the like.
- the composition may contain an additive other than an inert diluent, for example, a lubricant, a disintegrant, a stabilizer, or a solubilizer or solubilizer according to a conventional method.
- Tablets or pills can be coated with a sugar coating or a film such as a gastric or enteric substance, if necessary.
- Liquid compositions for oral use can include, for example, emulsions, solutions, suspensions, syrups, or elixirs, and are commonly used inert diluents such as purified water or Ethanol can be included.
- the composition may contain additive agents other than inert diluents, such as wetting agents, suspending agents, sweeteners, fragrances, or preservatives.
- Aseptic aqueous or non-aqueous solutions, suspensions, or emulsions may be included.
- the water-soluble solution or suspension may contain, for example, distilled water for injection or physiological saline as a diluent.
- diluents for water-insoluble solutions or suspensions include propylene glycol, polyethylene glycol, vegetable oil (eg, olive oil), alcohols (eg, ethanol), or polysodium. Rubate 80 etc. can be included.
- the composition may further contain a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilizing aid, or a preservative.
- the composition can be sterilized by, for example, filtration through a bacteria-retaining filter, formulation of a bactericide, or irradiation.
- a sterile solid composition can be produced, and can be used by dissolving in sterile water or other sterile injectable medium at the time of use.
- the dosage can be appropriately determined in consideration of the active ingredient, that is, the strength of the activity of the substance obtained by the screening method of the present invention, the symptoms, the age of the administration subject, or the sex.
- the dose in the case of oral administration, is usually about 0.1 to 1: LOOmg, preferably 0.1 to 50 mg per day for an adult (with a body weight of 60 kg).
- LOOmg preferably 0.1 to 50 mg per day for an adult (with a body weight of 60 kg).
- parenteral administration in the form of an injection, it is 0.01 to 50 mg per day, preferably ⁇ or 0.01 to LOmg.
- SEQ ID NO: 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, Or 25 chaperone proteins represented by the amino acid sequence of 27 (human HSPA1A, human HSPH1, human HSPCA, human HSPD1, human DNAJA1, human HS PB1, human HSPE1, human HSPA4, human HSP90B1, human CCT6B, human TCP1, Human HSPA 14, human HSPA9B, human STCH, human HYOU1, human HSPB5, human HSPB2, human DNAJA2, human DNAJB1, human DNAJB2, human HCG3, human DNAJB11, human DNAJC11, human DN AJC7, human DNAJC6)
- the oligonucleotide sequence shown in SEQ ID NO: 28-77 (even number 5 'side, odd number 3' side) is used as a primer (for example,
- the primer sets for human HSPA1A are SEQ ID NO: 28 and SEQ ID NO: 29.
- HSPH1, HSPE1, HSP90B1, HSPA9B and DNAJC11 are human liver-derived cDNA libraries (Clontech)
- HSPCA, HSPA1A and HSPD1 are human brain cDNA cDNA libraries (Clontech)
- DNAJA1 and HSPA4 are HeLa cells CDNA library (Clontech)
- HSPB1 is a human mammary gland-derived cDNA library (Clontech)
- TCP1, HSPA14, HYOU1 and HSPB2 are human skeletal muscle-derived cDNA libraries (Clontech)
- CCT6B, STCH, HSPB5, DNAJA2 DNAJB1, DNAJB2, HCG3, DNAJB11, human DNAJC7 and human DNAJC6 are commercially available cDNAs (Ultimate TM ORF Clones, Invitrogen), respectively, using DNA polymerase (Pyrobest DNA Polymerase, Takara Shuzo) at 95 ° C3 After a heat denaturation reaction
- a nucleotide sequence was determined using a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer Applied Systems) using the oligonucleotide shown in SEQ ID NO: 86 as a primer. !
- Each nucleotide sequence (RefSeq accession number NM—005345, NM—006644, NM—00 5348, NM—199440, NM—001539, NM—001540, NM—002157, NM—002154, NM—003299, NM—006584 , NM- 030752, NM- 016299, NM- 005347, NM- 006948, NM- 006389, NM- 001885, NM- 001541, NM- 005880, NM- 006145, NM- 006736, NM- 001001394, NM- 0 16306, NM-018198, NM-003315
- the GST fusion expression plasmid group obtained by cloning the 25 types of molecular chaperones obtained in (1) above was introduced into Escherichia coli BL 21 (Takara Bio Inc.) by transformation using the heat shock method. 2. After shaking overnight in 4 mL of culture medium, transfer the entire volume to 400 mL culture medium, shake culture at 37 ° C for 3 hours, and then IPTG (Sigma) to a final concentration of 2.5 mM.
- GST-HSPA1A (about 96 kDa), GST- HSPH1 (about 123 kDa), GST- HSPCA (about ll lkDa), GST- HSPD1 ( 87kD a), GST-DNAJA1 (approximately 71 kDa), GST—HSPBl (approximately 59 kDa), GST—HSPEl (approximately 37 kDa), GST—HSPA4 (approximately 120 kDa), GST—HSP90B1 (approximately 118 kDa), GS T—HSPA9B (approximately 100 kDa) ), GST—DNAJCl l (approximately 89 kDa), GST—TCPl (approximately 86 kDa), GST—HSPA14 (approximately 81 kDa), GST—HYOU
- GST protein a protein having only the GST tag portion
- GST protein expected molecular weight of about 26 kDa
- separation by SDS polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were performed, and it was confirmed that proteins of each expected molecular weight were purified.
- ER a A gene cDNA encoding the full-length region of estrogen receptor ⁇ (hereinafter referred to as ER a), which is a kind of human steroid hormone receptor shown in a known database, is obtained according to the sequence shown in RefSeq accession number NM-000125. Cloning was performed using the two designed DNA primers (SEQ ID NO: 78 and SEQ ID NO: 79). Specifically, using the primer set of SEQ ID NO: 78 and SEQ ID NO: 79, a PCR reaction was carried out using a HeLa cell-derived cDNA library (Clontech) as a saddle, and about 1. A 78 kbp DNA fragment was amplified.
- PCR reaction uses DNA polymerase (Pyrobest DN A Polymerase; Takara Shuzo), 98 ° C (1 minute), 98 ° C (5 seconds), 55 ° C (30 seconds), 72 ° C (5 minutes) This cycle was repeated 35 times.
- the obtained DNA fragment was expressed as an expression vector (pc DNA3.1 / ⁇ 5—13 ⁇ 45—Cing 0? 0; Invitrogen) was subcloned using the Cing 0–0 TA Cloning system (Invitrogen).
- the pcDNA-ER was transiently introduced into COS-7 cells (ATCC) cultured in a 70% confluent state on a 10 cm petri dish using Lipofectamine 2000 reagent (Invitrogen). After culturing for 30 hours, the medium was removed, and the cells were washed with ice-cold PBS. Then, 1.0 ml of buffer A (50 mM Tris-HCl (pH 7.5), 10% glycerol, 12 OmM NaCl, ImM EDTA, 0. ImM EGTA, 0.5 mM PMSF, 0.5% NP-40) was added and dissolved.
- buffer A 50 mM Tris-HCl (pH 7.5), 10% glycerol, 12 OmM NaCl, ImM EDTA, 0. ImM EGTA, 0.5 mM PMSF, 0.5% NP-40
- the cell extract was centrifuged at 1500 rpm for 5 minutes to remove the precipitate, and the supernatant soluble fraction (hereinafter referred to as ER receptor-expressing cell extract) was collected.
- the soluble fraction of this cell extract is known to be a target protein that exhibits the efficacy of 17—8 estradiol (hereinafter abbreviated as E2) (Green S. & Chambon P. Trends Genet 1988 Nov; 4 (ll): p309— 314.) ER a force is included.
- E2 17—8 estradiol
- control COS-7 cell extract hereinafter referred to as control COS-7 cell extract
- the target protein ER is actually detected by the identification method of the present invention using the chaperone protein HSPA4 protein. I checked if it was possible.
- the known GST-pull-down method first, 1 ⁇ g of GST-HSPA4 protein or GST protein (prepared in Example 1 (2) above) purified on dartathione sepharose beads and the above-mentioned ER receptor-expressing cell extract or control COS — Each of the 7 cell extracts was mixed and shaken at 4 ° C for 1 hour with or without 10 M E2. Thereafter, the protein that binds to GST-HSPA4 or GST protein on the beads was coprecipitated by centrifugation.
- Example 2 (2) Detection of 17- ⁇ estradiol target protein by the identification method of the present invention
- a protein that binds to a molecular chaperone protein is added to the molecular chaperone protein, or a test drug is added. It was proved that the target protein of the test drug can be detected by comparison when the sample is not added.
- the identification method of the present invention was useful even when molecular chaperones other than those used in Example 2 (2) were used.
- Example (3) using the 25 chaperone proteins of Example 1 (2), the target protein ER was converted to the identification method of the present invention as in Example 2 (2). To see if it could actually be detected.
- HSPA4 protein as a chaperone protein
- the above 25 types of GST fusion proteins were divided into 8 groups or 9 groups of 3 groups according to molecular weight, and 0.2 g each was mixed.
- the breakdown of each 8 or 9 types of mixed GST-fused chaperone proteins is a group of relatively high molecular weight chaperones: GST—HSPH1, GST—HSPA4, GST—HSPCA, GST—HYOUl, GST-DNAJC6, GST—HSP90B1 GST-HSPA9B ⁇ and GST-DNAJCl l, medium molecular weight chaperones, GST-HSPA1A, GST- HSPD1, GST—DNAJA1, GST—TCP1, GST—CCT6B, GST—HSPA14, GST — GJ—HSPB1, GST—HSPE1, GST—DNAJA2, GST—DNAJB1 1, GST—DNAJB1, GST—DNAJB2, GST—HSPB2 A group consisting of a mixture of DNAJC7 and GST—STCH and relatively low molecular weight chaperones , GST-HSPB5, and GST-HCG3.
- ER (L), ER (M), and ER (H) in the table are the above-mentioned relatively low molecular weight chaperone group, medium molecular weight chaperone group, and relatively high molecular weight chaperone group, respectively. Show the results.
- Example 3 Example 4, and Example 8, it is shown that target proteins of various drugs can be identified by the identification method of the present invention using various molecular chaperones.
- the experiment was performed in accordance with the above-described Example 2.
- the signal intensity of the band obtained by Western blotting was quantified as a measured value of density per unit area using VersaDoc Imaging System (Bio-Rad).
- VersaDoc Imaging System Bio-Rad
- FKBP12 is known to be one of the target proteins of FK506 and FK1706 (J Biol Chem. 1993 Nov 5; 268 (31): 22992-22999, Eur J Pharmaco 1. 2005 Feb 10; 509 (1) : 11—19).
- GR Darcocorticoid receptor
- DHFR Human hydrofolate reductase
- MTX methotrexate
- LNCaP cells In the soluble fraction of human prostate-derived cells, LNCaP cells (ATCC), it is known to be a target protein of 5a-dihydrotestosterone (hereinafter DHT) (J Steroid Biochem Mol Biol. 1993 Dec; 46 (6): 699-711) Androgen receptor (AR) is included.
- DHT 5a-dihydrotestosterone
- AR Androgen receptor
- HUVEC HUVEC
- MR Mineralcorticoid receptor
- the following cell extract-soluble fraction is prepared, and the identification method of the present invention using each chaperone protein group as the target protein of each drug present in the cell extract-soluble fraction.
- We investigated whether it could be detected by Of the drugs FK506 (Japanese Patent Publication No. 03-038276) and F K1706 (European Patent No. 346427) were synthesized, and the other drugs were purchased from Sigma.
- the FKBP12, GR, and DHFR bands are all Western plots using an antibody that recognizes the V5 tag added to each target protein, as in Example 2 (3) above.
- the AR and MR bands are commercially available antibodies (N -20 and C-19, Santa Cruz)).
- HUVEC soluble fraction Z chaperone protein group (e) HUVEC soluble fraction Z chaperone protein group; GST—HSPA1A, GST—HSPH1, GST—HSPCA, GST—HSPA4 mixed protein group
- control HeLa cell extract Cell extract extract soluble fraction of HeLa cells (hereinafter referred to as control HeLa cell extract; control (1) to (c) above)
- L6 cells which are rat skeletal muscle-derived cells, are suspended in collagen medium-coated 15 cm diameter plates containing 10% urinary fetal serum (FCS) (X minimum essential medium (a MEM, Invitrogene)). The cells were cultured until confluent on Asahi Techno Glass Co., Ltd. The cells were washed twice with 15 ml of ice-cold phosphate buffer (PBS), and then lysed with 2 ml of the above-mentioned buffer A.
- FCS urinary fetal serum
- PBS ice-cold phosphate buffer
- the cell extract was collected using a scraper, centrifuged at 1500 rpm for 5 minutes to remove the precipitate, and the soluble fraction of the supernatant was collected.
- a type of biguanide that has been shown to have a hypoglycemic effect in the clinical setting (UK Prospective Diabetes Study (UKPDS) Group: Lancet, 352, 854 (1998)) Implemented above with or without added 2 Pulled down 1 ⁇ g of GST-HSPA4 protein purified on dartathione sepharose beads in the same manner as in (2), followed by a pull-down experiment at 4 ° C for 1 hour, followed by centrifugation for GST on the beads.
- the protein that binds to HSPA4 was co-precipitated, but here, in order to prevent the molecular chaperone protein from losing force on the beads, the molecular chaperone protein and beads were reinforced by a known method. Used in a cross-linked state. Specifically, the GST-HSPA4 protein purified on dartathione sepharose beads was washed with 0.2 M sodium borate solution, suspended in 20 mM DMP, and treated at 4 ° C for 45 minutes. This was washed with a 0.2M monoethanolamine solution to stop the reaction, and then washed with a 20 mM dartathione solution, and the one excluding the uncrosslinked molecular chaperone protein was used as a probe.
- This protein is considered to be one of the target proteins of phenformin, which is brought into a three-dimensional structure change by adding phenformin. Therefore, this band was cut out and the protein was fragmented using trypsin V, and the resulting peptide mixture was recovered from the gel, and the known method (Schevchenko et al., Analytical Chemistry, 68th, 850-858). (1996), the protein was identified by mass spectral analysis. As a result, it was revealed that the protein in the band was ATP5B (RefSeq accession number NP-599191).
- PCR reaction uses DNA polymerase (TAKARA LA Taq; Takara Shuzo), 94 ° C (3 minutes), 94 ° C (30 seconds), 58 ° C (1.5 minutes), 72 ° C (4 minutes) ) Was repeated 35 times, and the PCR product was made into a saddle and PCR was performed under the same conditions. PCR products were separated by agarose gel electrophoresis, resulting in amplification of a DNA fragment of approximately 1600 base pairs. I confirmed it. Therefore, the DNA fragment in the reaction solution was cloned into an expression vector (pcDNA3.1 / V5-His-TOPO; Invitrogen) using the TOPO TA Cloning system (Invitrogen).
- pcDNA3.1 / V5-His-TOPO Invitrogen
- the primer shown in SEQ ID NO: 81 used at this time was V3 epitope derived from the vector on the 3rd side after cloning (derived from V protein of paramyxovirus SV5, Southern JA, J. Gen. Virol. 72, 1551— 1557, 1991) and And the His6 tag (Lindner P BioTechniques 22, 140-149, 1997) were designed to remove the stop codon sequence of human ATP5B so that it would continue in the same frame as the triplet of the human ATP5B gene.
- ATP5 B which was found by the identification method of the present invention and considered to be a biguanide target protein, was a true target protein that brought about the pharmacological action (main action) of biguanide.
- all of the conventional methods for searching for a target protein of a compound use only direct binding between the compound and the protein as an index, a large number of proteins that bind to the compound can be obtained. The probability of being found was generally low.
- the method of the present invention seeks to find a target of a compound by using a change in the three-dimensional structure of the protein to which the compound is bound as an index, and the found protein is inevitably a simple binding protein of the compound. Therefore, it can be expected to be a protein molecule whose function can be greatly changed by the compound.
- the obtained cDNA fragment of about 560 base pairs was cloned into pcDNA3.1 / V5-His-TOPO, and then the restriction enzyme sites BamHI and Xhol were used to attach the DHFR cDNA fragment to both ends of the above primers.
- the betater power was also cut out.
- pGEX-6P-1 (Amersham), which is a GST fusion protein expression vector, was cleaved with restriction enzymes BamHI and Xhol, respectively, to make it linear.
- DNA ligase solution DNA ligation kit II; Takara Shuzo Co., Ltd.
- pGEX-DHFR a plasmid inserted into the multicloning site of pGEX-6P-1) (Hereinafter abbreviated as pGEX-DHFR).
- pGEX-DHFR a plasmid inserted into the multicloning site of pGEX-6P-1)
- the pGEX-DHFR prepared in Example 5 (2) was introduced into E. coli BL21 by transformation using the heat shock method. 2. After overnight shaking culture in 4 mL of culture solution, transfer the entire volume to 400 mL culture solution, shake culture at 37 ° C for 3 hours, and then add IPTG (Sigma) to a final concentration of 2.5 mM. ) And further cultured with shaking for 3 hours to induce the expression of GST-fused DHFR protein (hereinafter abbreviated as GST-DHFR). The bacterial cells were collected, and GST-DHFR was purified on glutathione sepharose beads according to a known GST-pull-down method.
- GST protein a protein having only a GST tag portion
- E. coli BL21 transformed with pGEX-6P-1 a protein having only a GST tag portion
- Separation by SDS polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were performed according to known methods, and it was confirmed that the expected molecular weight protein (GST-DHFR; 45 kDa, GST protein; 26 kDa) was purified. .
- DHFR methotrexate (2S) — 5— [(3 ⁇ [ ⁇ [Amino (2S) — 5— [(3 ⁇ [ ⁇ (Amino ( Imino) methyl] amino ⁇ (imino) methyl] amino ⁇ propyl) amino] 2— ( ⁇ 4— [[(2, 4 diaminopteridine— 6-yl) methyl] (methyl) amino] benzoyl ⁇ amino ) -5-oxopentanoic acid dihydrochloride (hereinafter abbreviated as MTX-phenformin), this compound was fixed on GST-DHFR purified on glutathione sepharose beads, fixed at the MTX portion, and protruded The presence or absence of binding to ATP5B protein on the phenformin side was confirmed by the GST pull-down method.
- MTX phenformin was prepared using a well-known organic synthesis technique according to the following reaction formula.
- Crystal structure data was acquired from RCSB Protein Data Bank with ID 1BMF .
- 1BMF includes 7-mer molecule (3 ⁇ subunits (A, B, C chain), 3 j8 subunits (D, E, F) of bovine mitochondrial Fl -ATPase Chain) and one ⁇ subunit (G chain)).
- ATP5B is the ⁇ subunit of F1-ATP synthase, the extracellular region of FIFO—ATP synthase (Nature. 1997; 386: 299-302, Nature. 1994; 370 (6491): 621—628)
- Ushi ATP5 B retains 99% amino acid identity with human ATP5B (Human ATP5B has 529 residues and Ushi ATP5B has 482 residues.
- Human ATP5B is N-terminal. 46 residues long and 1 residue longer at the C-terminus).
- D-chain which is one of ⁇ subunits of ushi-ATP5B, and the adjacent ex-subunits, ⁇ and C-chain Only (hereinafter referred to as complex ⁇ a) was used.
- Ph4Dock a function provided in Chemical Computing Group Inc. (DMOE (Molecular Operating Environment) software), which is a combination of ligands and receptors. This is a function that automatically finds a stable complex structure including the bond position by using a computer just by giving a three-dimensional structure (J Med Chem.
- Table 2 shows the sites where metformin and phenformin are bound, and the energy value (U_ele + U_vdw) for the binding at that time!
- the sites in the table represent binding sites, and for convenience, the larger and more hydrophobic binding sites are numbered sequentially.
- U—ele represents electrostatic interaction energy
- U—vdw represents van der Waals energy
- U—ele + U—vdw is small! / .
- the force field used was mmff94s.
- site 20 and site 29 bind to metformin and phenformin in common.
- a mixture of equal amounts of the two PCR products obtained above is converted into a saddle shape, and this time a PCR reaction is performed using the primers shown in SEQ ID NO: 80 and SEQ ID NO: 81.
- a full-length cDNA encoding ATB5B was obtained.
- the nucleotide sequence inserted into the vector was identified using the synthetic DNA primer shown in SEQ ID NO: 90, and the Glul75Val mutation in ATB5B It was confirmed that this occurred.
- the obtained expression plasmid is hereinafter abbreviated as pcDNA-ATP5B (E175V).
- AMPK AMP-activated kinase
- ATP5B found by the identification method of the present invention is a true target molecule responsible for the biguanide's efficacy (main action)
- the ATP5B protein is upstream of AMPK activation in the intracellular signal transduction system operated by biguanide. Should be located at. Therefore, in order to verify that the ATP5B molecular force that binds to biguanide is actually upstream of AMPK activity in experiments, the effect of AMPK activity by biguanide has an effect in the state where ATP5B is overexpressed in cells. I examined whether to receive.
- the PCR reaction was performed under the same conditions as in Example 5 (1) above, and the amplified DNA fragment of about 1300 base pairs was inserted into the expression vector pcDNA3.1 / V5-His-TOPO.
- the nucleotide sequence of the inserted DNA fragment of the completed plasmid it was confirmed that it was a clone consisting of the full-length cDNA sequence of human LKB1 shown in RefSeq accession number NM-000455.
- this expression plasmid is abbreviated as pcDNA-LKB1.
- HeLaS3 cells This expression plasmid pcDNA-LKB1, pcDNA-ATP5B prepared in Example 5 (1) above, or an empty vector (pcDNA3.1) (Invitrogen) was introduced into HeLaS3 cells.
- HeLaS3 cells (ATCC) consist of 70% confluent in 6well culture plate (well diameter 35mm) culture dish containing 2ml of 10% fetal calf serum (Sigma) and 2% of each well. It was cultured until it reached a state.
- phosphate buffer hereinafter abbreviated as PBS
- 0.15 ml of the above buffer A per well (however, the NaCl concentration was 150 mM and various phosphatase inhibitors; 2 mM Na VO , 10mM NaF, 25mM ⁇ -glycose phosphate, 0.2
- AMPK activity in the cells was detected as the phosphorylation level of the molecule by Western blotting using electrophoresis and anti-phosphorylated ⁇ antibody (Phospho- ⁇ -a (Thrl 72) Ant ibody, Daiichi Kagaku) .
- a Western plot using an anti- ⁇ antibody was simultaneously used. went.
- Figure 4 As shown in Fig.
- both the cells introduced with the above-mentioned empty vector and the cells into which pcDNA-LKBl was introduced to express LKB1 were treated with phenformin as compared with the case of treatment with solvent. It was observed that the level of phosphate was significantly increased and activated! However, in cells in which pcDNA—ATP5B was introduced and human ATP5B was expressed, the activity of AMPK by phenformin treatment was not observed.
- ATP5B protein in the cell changes the activity of AMPK, which contributes to the efficacy of biguanides such as phenformin and metformin, and the intracellular signal of biguanide ATP5B is clearly located upstream of AMPK.
- ATP5B is a true target that binds biguanide and contributes to the medicinal properties of the compound. You can conclude that it is a protein.
- the phenomenon in which the activation of AMPK by biguanide, ie, the enhancement of phosphorylation, was prevented by overexpression of ATP5B is explained by one of the following theories.
- ATP5B originally has the effect of suppressing AMPK phosphate, and it is possible that biguanide binds to ATP5B and interferes with its AMPK inhibitory action to activate AMPK.
- the ATP5B molecule acting by complexing with an endogenous molecule alone was overexpressed, causing the biguanide to be purged (removed) by binding to excess single ATP5B.
- the biguanide is no longer able to see the enhancement of AMPK phosphate that occurs via a complex molecule containing ATP5B.
- ATP5B is a target protein that contributes to the efficacy of biguanides.
- the substance that binds to ATP5B has the same medicinal effect (main action) as biguanide.
- the target protein of the test drug contributing to the main action of the compound can be identified by the identification method of the present invention.
- test substance that binds to the polypeptide ie, antidiabetic agent
- the test substance that binds to the polypeptide is selected using the change in binding of the polypeptide and biguanide as an index. Screening methods that can be used.
- the binding between ATP5B and biguanide can be detected.
- the concentration of MTX-phenformin present 1.0, 10, 100 i u M
- the binding to the ATP5B protein becomes clearer. Detected.
- free phenformin (10 M) was added as a test substance to this system, it was shown that the binding between the protein and MTX-phenformin was hindered as shown in FIG.
- the phenformin added here was evaluated, and by replacing it with a test substance, it was possible to examine whether the test substance has the ability to change the binding between ATP5B protein and MTX-phenformin (biguanide).
- the binding to the tool polypeptide can be competitively inhibited, and a substance that binds to the tool polypeptide, that is, a therapeutic agent for diabetes can be screened.
- a substance that binds to the tool polypeptide that is, a therapeutic agent for diabetes.
- each of these compounds showed significant activity activity in the detection experiment of activity activity shown in Example 5 (7) described above. 5 As in the case of phenformin and metformin shown in (7), it disappeared due to overexpression of ⁇ 5 ⁇ .
- phenformin and metformin shown in (7) it disappeared due to overexpression of ⁇ 5 ⁇ .
- both of the above-mentioned compound ⁇ and ⁇ compound ⁇ interact directly with the biguanide binding site of ⁇ 5 and activate intracellular AMPK.
- the compound having the same AMPK activity ability as that of biguanide can be selected by the method for screening a therapeutic agent for diabetes using the polypeptide of the present specification shown in Example 6 (1).
- Example 7 Measurement of blood glucose lowering effect of hit compound and effect on blood lactic acid level
- Compound A and Compound B found by the screening method of the present invention Has AMPK activity.
- the effects of the above-mentioned two compounds were also investigated on the increase in blood lactate, which is a side effect of biguanide.
- Diabetes model mice, dbZdb mice (BKS. Cg— + Leprdb / + Leprdb / Jcl; Claire, Japan), 15 11-week-old males were divided into 3 groups of 5 each.
- Metformin (Sigma) and the above compound A were dissolved in a solvent (5% Cremophor, 0.2% methylcellulose: MC) at a concentration of 30 mgZml or lOmgZml, respectively.
- a solvent 5% Cremophor, 0.2% methylcellulose: MC
- metformin 300 mgZkg per body weight and compound A lOOmgZkg per body weight were administered intraperitoneally, and compared with the group administered only the same volume of solvent (5% cremophor, 0.2% MC). .
- the animals were fasted at the same time as the administration, and blood was collected from the tail at 0 minutes, 90 minutes, and 180 minutes after the start, and blood glucose and blood lactate levels were measured.
- the blood glucose level is measured by a simple blood glucose meter (Accu-Checker Lactic acid levels in blood were measured using a simple lactic acid analyzer (Latatate Pro; Arkray Marketing Co., Ltd.)
- 12 18-week-old males from dbZdb mice 4 were divided into 3 groups: metformin (Sigma) and the above-mentioned compound B were dissolved in physiological saline at a concentration of 30 mgZml or 9 mgZml, respectively.
- metformin showed a significant hypoglycemic effect at 90 and 180 minutes in all experiments compared to the solvent-administered group. Both Compound A and Compound B exhibited a hypoglycemic effect with a significant difference at 90 minutes and 180 minutes (FIGS. 8A and C).
- the metformin-treated group caused a significant increase in blood lactate after 90 minutes in either experiment, but neither Compound A nor Compound B caused an increase in blood lactate ( Figures 8B and D). ). From these results, it was confirmed that a novel antidiabetic agent having a significant hypoglycemic action similar to biguanide can be found by the screening method of the present invention.
- the screening method of the present invention enables the main action (pharmacological action of biguanide; It was confirmed that a new antidiabetic drug could be screened without side effects (ie, increased blood lactate level).
- THP-1 a human blood cell culture cell
- FCS urinary fetal serum
- FCS urinary fetal serum
- the cells were collected by centrifugation at 1200 rpm for 3 minutes, washed once with 10 ml of ice-cold PBS, lysed with the above-mentioned Noffer A 2. Oml, and the cell extract was collected. 15 cell extracts The precipitate was removed by centrifugation at OOrpm for 5 minutes, and the soluble fraction of the supernatant was collected. Thalidomide ((-)-thalidomide, Sigma) was added to a final concentration of 100 M, or purified under the conditions of the unexcluded potassium extract on the soluble fraction of this cell extract on Dartathione Sepharose beads.
- GST-fused chaperone proteins (GST—HSPA1A, GST—HSPH1, GST—HSPCA, GST—HSPD1, GST—DNAJA1, GST—HSPB1, GST—HSPE1, GST—HSPA4; 0.5 ⁇ g of each protein Were mixed) and a pull-down experiment was conducted.
- the pull-down experiment process and conditions were the same as in Example 2 (2). That is, after shaking at 4 ° C for 1 hour, the protein binding to the GST fusion chaperone protein mixture on the beads was coprecipitated by centrifugation.
- each chaperone protein and beads were used after being chemically cross-linked by a known method.
- thalidomide having a final concentration of 100 M was added or suspended in 5 ml of the above-mentioned buffer A′O. Without addition, and coprecipitated again by centrifugation. After this operation was repeated four times, the protein in the precipitate was separated by SDS polyacrylamide gel electrophoresis according to a known method, and the protein was detected by negative staining (Wako Pure Chemical Industries). As a result, the presence of multiple protein bands that were present only when thalidomide was added was detected.
- proteins are a protein group in which the binding between the protein and the molecular chaperone protein mixture is changed by the addition of thalidomide, that is, a protein group in which the three-dimensional structure is changed by the addition of thalidomide.
- TARDBP is a transcription factor that has a binding activity to nucleic acids such as DNA and RNA, and is known to have an inhibitory action on HIV (Ou SH et al., Virol. 1995 Jun; 69 (6): 35 84— 3596 ). Thalidomide is known to have a significant anti-HIV effect (Franks ME et al., Lancet. 2004; 363 (9423): 1802-1811;), and TARDBP is a target protein of thalidomide. There is no contradiction as white matter.
- TARDBP of thalidomide found by the method of the present invention in Example 8 described above is used as an index to determine whether it is a true target molecule responsible for the main action of thalidomide or not. We tried to verify it by experiment.
- the PCR reaction was performed using DNA polymerase (TAKARA LA Taq; Takara Shuzo), 94 ° C (3 minutes), 94 ° C (30 seconds) ⁇ 58 ° C (1.5 minutes) '72 ° C (4 Min) was repeated 35 times.
- the stop codon sequence of the gene was removed so that the tag (Lindner P BioTechniques 22, 140-149, 1997) followed in the same frame as the triplet of the TARDBP gene.
- the primer (TOPO TA Cloning kitZ Invitrogen; SEQ ID NO: 89) that binds the nucleotide sequence of the inserted DNA fragment in the resulting plasmid to the T7 promoter motor region on the vector and the sequencing kit (Applied system) ) And a sequencer (ABI 3700 DNA sequencer Applied Biosystems).
- pcDNA—TARDBP or empty vector (pcDNA3.1) was introduced into HeLaS 3 cells. Specifically, the HeLaS3 cells are first prepared in a 12-well culture plate in a culture dish of 70% confluent with the minimum essential medium DMEM (GIBCO) containing 10% fetal calf serum (Sigma) in each well. Incubated until. Replace 0.5 ml of serum-free medium OPTI MEM 1 (Invitrogen) per well, and use 0.8 ⁇ g / well of pcDNA—TARDBP or pcDNA3.1 each using Lipofectamine 2000 (Invitrogen). Introduced transiently.
- DMEM minimum essential medium DMEM
- OPTI MEM 1 containing 10% fetal calf serum
- okadaic acid (Wako Pure Chemical Industries) was further cultured for 16 hours under the condition of 5 OnM final concentration or not added. At this time, some cells were added with 100 M thalidomide simultaneously with okadaic acid treatment. After removing the medium and washing the cells twice with ice-cold PBS, these cells were frozen and stored at 80 ° C.
- RNA was prepared from each cell force frozen in Example 9 (2) above using an RNA extraction reagent (Isogen; Nitsubon Gene) according to the instructions. Each prepared total RNA was then treated with deoxyribonuclease (Nitsubon Gene), treated with phenol Z chloroform, ethanol precipitated and dissolved in sterile water. Using 1 ⁇ g of this total RNA, reverse transcription to single-stranded cDNA was performed in a 20 ⁇ 1 system using an inverse transcription reaction kit (Advantage RT-for-PCR Kit; Clontech).
- Isogen deoxyribonuclease
- phenol Z chloroform phenol Z chloroform
- SEQ ID NO: 97 SEQ ID NO: 102 Six oligonucleotides (SEQ ID NO: 97 SEQ ID NO: 102) were designed and synthesized as PCR primers for gene expression level measurement. SEQ ID NO: 97 and SEQ ID NO: 98 for human j8-actin gene, SEQ ID NO: 99 and SEQ ID NO: 100 for human TNF- ⁇ gene, SEQ ID NO: 101 and sequence for human TARDBP gene Each was used in combination with number 102.
- [TNF-a or TARDBP corrected expression level] [TNF- a or TARDBP gene expression level (raw data)] / [j8 actin gene expression level (raw data)]
- TARDBP TARDBP highly expressing cells
- TNF- ⁇ expression from control cells was significantly increased ( ⁇ 80 times) by okadaic acid treatment. It was confirmed that thalidomide supplementation suppressed the increase in TNF- ⁇ expression by okadaic acid to nearly 50%, and that the action of thalidomide could be detected in the cells.
- TARDBP is known to be a transcription factor that has binding activity with nucleic acids such as DNA and RNA.
- TARDBP is a molecule in a functional complex consisting of multiple transcription factors essential for inducing TNF-a expression.
- TNF-a Even if it overexpresses itself alone, it does not enhance the induction of TNF-a expression. However, if thalidomide inhibits the function of the molecule, transcription is essential for inducing TNF-a expression. As a result of the loss of function of the complex, the production of TNF-a is thought to be suppressed.
- the protein TARDBP which is found by the identification method of the present invention and binds to a molecular chaperone only when thalidomide is added, is involved in the regulation of TNFa expression, which is considered to be one of the molecular mechanisms responsible for the drug efficacy of thalidomide. It was considered a molecule.
- the identification method of the present invention can be used for identification of a target protein without modifying the test drug. It was done.
- the screening method of the present invention can be applied to the use of screening for a therapeutic drug for diabetes.
- the screening tool of the present invention can be used for the screening.
- the identification method of the present invention is useful as a method for identifying a target protein useful for improvement studies of existing drugs.
- the numerical heading 223> in the following sequence table describes “Artificial Sequence. Specifically, it is represented by the sequence numbers 28 to 77, 84 to 89, 91 and 92 in the sequence table. Each base sequence is an artificially synthesized primer sequence.
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE602006010522T DE602006010522D1 (de) | 2005-08-12 | 2006-08-09 | Verfahren zur identifizierung des zielproteins eines wirkstoffs und verfahren zum screening eines therapeutischen wirkstoffs gegen diabetes mittels des zielproteins |
JP2007530962A JP4992716B2 (ja) | 2005-08-12 | 2006-08-09 | 薬剤の標的蛋白質を同定する方法及び標的蛋白質を用いた糖尿病治療薬のスクリーニング方法 |
US11/909,031 US8003331B2 (en) | 2005-08-12 | 2006-08-09 | Method for identifying target protein of drug and method for screening therapeutic agent for diabetes using the target protein |
EP06782561A EP1860438B1 (en) | 2005-08-12 | 2006-08-09 | Method for identifying target protein of agent and method for screening therapeutic agent for diabetes using target protein |
CA002601869A CA2601869A1 (en) | 2005-08-12 | 2006-08-09 | Method for identifying target protein of agent and method for screening therapeutic agent for diabetes using target protein |
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JP2005234673 | 2005-08-12 | ||
JP2005-234673 | 2005-08-12 | ||
JP2005279582 | 2005-09-27 | ||
JP2005-279582 | 2005-09-27 |
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WO2007020853A1 true WO2007020853A1 (ja) | 2007-02-22 |
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PCT/JP2006/315745 WO2007020853A1 (ja) | 2005-08-12 | 2006-08-09 | 薬剤の標的蛋白質を同定する方法及び標的蛋白質を用いた糖尿病治療薬のスクリーニング方法 |
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US (1) | US8003331B2 (ja) |
EP (2) | EP2124062A1 (ja) |
JP (1) | JP4992716B2 (ja) |
CA (1) | CA2601869A1 (ja) |
DE (1) | DE602006010522D1 (ja) |
ES (1) | ES2336605T3 (ja) |
WO (1) | WO2007020853A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008126920A1 (ja) * | 2007-04-11 | 2008-10-23 | Ajinomoto Co., Inc. | 糖尿病治療薬 |
JP2009058500A (ja) * | 2007-08-06 | 2009-03-19 | Genome Soyaku Kenkyusho:Kk | 血糖値を降下させる物質の評価方法、スクリーニング方法及び製造方法 |
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US8579964B2 (en) | 2010-05-05 | 2013-11-12 | Neovasc Inc. | Transcatheter mitral valve prosthesis |
EP2688907B1 (en) * | 2011-03-24 | 2016-04-27 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Dominant negative hsp110 mutant and its use in prognosing and treating cancers |
US9308087B2 (en) | 2011-04-28 | 2016-04-12 | Neovasc Tiara Inc. | Sequentially deployed transcatheter mitral valve prosthesis |
US9554897B2 (en) | 2011-04-28 | 2017-01-31 | Neovasc Tiara Inc. | Methods and apparatus for engaging a valve prosthesis with tissue |
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Cited By (6)
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---|---|---|---|---|
WO2008126920A1 (ja) * | 2007-04-11 | 2008-10-23 | Ajinomoto Co., Inc. | 糖尿病治療薬 |
US8357680B2 (en) | 2007-04-11 | 2013-01-22 | Ajinomoto Co., Inc. | Remedy for diabetes |
US8772281B2 (en) | 2007-04-11 | 2014-07-08 | Ajinomoto Co., Inc. | Remedy for diabetes |
JP5672699B2 (ja) * | 2007-04-11 | 2015-02-18 | 味の素株式会社 | 血糖降下作用化合物のスクリーニング法 |
US9440980B2 (en) | 2007-04-11 | 2016-09-13 | Ea Pharma Co., Ltd. | Remedy for diabetes |
JP2009058500A (ja) * | 2007-08-06 | 2009-03-19 | Genome Soyaku Kenkyusho:Kk | 血糖値を降下させる物質の評価方法、スクリーニング方法及び製造方法 |
Also Published As
Publication number | Publication date |
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JP4992716B2 (ja) | 2012-08-08 |
ES2336605T3 (es) | 2010-04-14 |
US8003331B2 (en) | 2011-08-23 |
JPWO2007020853A1 (ja) | 2009-02-26 |
EP1860438B1 (en) | 2009-11-18 |
EP1860438A1 (en) | 2007-11-28 |
EP2124062A1 (en) | 2009-11-25 |
EP1860438A4 (en) | 2008-11-12 |
DE602006010522D1 (de) | 2009-12-31 |
CA2601869A1 (en) | 2007-02-22 |
US20090041754A1 (en) | 2009-02-12 |
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