Patent Grant
8003331
This application is a 371 National Stage Entry of International Application No. PCT/JP2006/315745 filed on Aug. 9, 2006 and claims the benefit of Japanese Patent Application Nos. P. 2005-234673 filed on Aug. 12, 2005, and P. 2005-279582 filed Sep. 27, 2005 in the Japanese Intellectual Property Office, the disclosures of each of which are incorporated herein in their entirety by reference.
The present invention relates to a method for identifying the target protein of a compound having a pharmacological action by detecting a tertiary structural change of the target protein by binding a compound having a pharmacological action to said target protein, with the use of a chaperone protein having a characteristic of binding to the protein by recognizing a tertiary structural change of the protein.
The present invention also relate to a method for screening therapeutic agent for diabetes using a target protein found by the identification method of the present invention.
A large number of low molecular compounds used as pharmaceutical agents are still unclear in regard to their mechanism of action despite their distinct pharmacological actions. In general, most of the pharmaceutical agents act upon specific proteins in vivo and alter functions of the proteins, and induce pharmacological actions as a result. In the agents whose mechanisms of action are unclear, proteins as their targets have not been identified. In recent years, as a result of advances in the elucidation of signal transduction system in vivo at the molecular level, a large number of specific protein molecules necessary for inducing specific pharmacological actions have been identified. As a result, development of the agents so-called molecule targeting agents which target at such specific protein molecules is in progress recently, and their ratio is rapidly increasing. In the case of a compound whose target protein is evident, its in vivo functional mechanism is clear, and structure of the compound can be modified with the use of its strength to bind to said protein, or a change in the enzyme activity possessed by the protein, as the index. Thus, it is easy to carry out studies with the aim of improving pharmacokinetics including absorption and degradation, as well as pharmacological activity, so that it is markedly advantageous in developing agents. In the case of a compound whose target protein is unclear to the contrary, it is not easy to attempt improvement of its chemical structure for the purpose of improving the activity, even when distinct pharmacological action is found (cf. Non-patent Reference 1).
In addition, though there are differences in degree, pharmaceutical agents generally have both desirable pharmacological actions (principal effects) and undesirable pharmacological actions (adverse side effects). Even in the case of molecule targeting agents whose target proteins carry the principal effects and are already put on the market, there are many cases in which information on the target proteins concerned in the adverse side effects is scanty, and this causes a problem requiring time and cost for studying improvement for avoidance of the adverse side effects (cf. Non-patent Reference 1).
In fact, there are a large number of pharmaceutical agents whose significant pharmacological actions are known but their target proteins are unclear. As representative examples, biguanide which has been used for a long time as an agent for treating diabetes (cf. Non-patent Reference 2) and thalidomide in which its presence has been reconsidered in view of its drastic therapeutic effect on multiple myeloma may be cited (cf. Non-patent Reference 3). Though biguanide has a significant hypoglycemic action, and thalidomide a significant angiogenesis inhibitory action, direct target protein for each of these agents in vivo has not been identified. Thus, in spite of the useful pharmacological actions possessed by these agents, it was difficult to carry out improvement studies for enhancing the effects. In addition to this, serious adverse side effects such as lactic acidosis by biguanide (cf. Non-patent Reference 4) and teratogenicity by thalidomide (cf. Non-patent Reference 3) are known, but studies for avoiding these problems have not been advanced, because their targets are unclear. Thus, identification of the target proteins of these agents is in demand.
Conventionally, a method in which a protein which directly binds to a low molecular compound is detected and separated by physical and/or chemical means was a general means for identifying a target protein upon which said compound acts. For example, a method is known in which a part of the structure of a compound is modified and bind to high molecular weight affinity beads, and a target protein bound to the compound is separated and purified by gravity or the like physical force. Also, a method is carried out in which a tag to be used as a label is attached to a part of the structure of a compound and the target protein bound to said compound is chemically detected (cf. Non-patent Reference 5). In recent years, attempts have also been made to screen and identify, from a cDNA library, a gene fragment coding for a protein which binds to the compound of interest, by a yeast two hybrid method (cf. Non-patent Reference 6), a phage display method (cf. Non-patent Reference 7) and the like molecular biological techniques.
However, in spite of the aforementioned attempts by various methods, a case in which a target protein of an agent was actually identified from the studies in this field so far is not many. The reasons for the low frequency of success include that it is necessary to modify a part of the structure of a compound because beads or a tag is bound to the compound to be used as the probe in every case of the aforementioned methods, so that it is unavoidable to screen for a protein which binds to an artificial structure different from the original compound (cf. Non-patent References 1 and 5). That is, this becomes a reason of mistakenly identifying, as the target protein, a nonspecific protein which binds to a tag, beads, a complex thereof with a compound, or the like artificial substance which is different from the agent having original pharmacological activity. In addition, though it is essential, for the purpose of finding the true target protein, to apply modification of the structure of a compound to a region which does not exert influence upon the pharmacological action of said compound, agents and compounds having unclear targets are generally poor in information on the correlation between their structures and pharmacological activities, so that there are many cases in which compounds modified at optional regions have to be used. Because of this, there is a high frequency of selecting a compound which lost its original pharmacological activity as the probe. Essentially, it is desirable to verify firstly that said compound to which a tag or bead is added by modification is still keeping its original pharmacological activity and then use it as the probe, but since cell membrane permeability, stability and the like various parameters exert influences, it is not easy to judge the presence or absence of the pharmacological activity. Also, since modification of the structure of compounds requires time, cost and special techniques, these cause the aforementioned methods to hardly become a general purpose studying means.
On the other hand, it is possible to verify binding of a specified protein to a compound labeled by replacing an element in the molecule of a compound with a radioisotope (its structure is the same as the before labeling), but since it is not a fixable modification, it is not easy to screen the target protein from a large number of proteins. In addition, this method has a disadvantage in that the compound becomes unstable by the labeling and the cost runs up.
As another reason of the low success ratio of compound target screening by the conventional methods, a point can be exemplified that since each of the aforementioned methods carries out detection and separation of a target making use of the direct binding of a compound with a protein as the index, it is difficult to achieve target finding when the binding affinity between the compound and the target protein is low. Actually, in each of the only few cases of succeeding in finding a target by the aforementioned methods, the binding affinity between the compound and the protein is high (cf. Non-patent Reference 4). However, the degree of pharmacological activity of a compound and its binding affinity for a target protein do not always have a correlation based on the knowledge so far obtained. Rather, it is considered that strong binding of a compound to target protein may not be necessary for the induction of pharmacological action excluding irreversible inhibition (cf. Non-patent Reference 8). Based on the above problems, concern has been directed toward a method for identifying target proteins of agents, which were not able to be found by the conventional methods.
Molecular chaperone is a group of proteins which assist structure formation of protein, such as folding or denaturation (unfolding) of a protein molecule, multimer formation and the like (cf. Non-patent Reference 9). It is known now that a large number of molecules generally referred to as heat shock protein, in which its expression is accelerated by heat stimulation, act as chaperone. Among the molecular chaperones, a group of molecules generally referred to as Hsp60 family are particularly called “chaperonin” as a typical molecular chaperone.
These molecular chaperones represented by the heat shock protein interact with unstable proteins before completion of their tertiary structures in their translation process and keep them stably, and also have the action to maintain and control the protein structure such that influences upon the function of intracellular protein are not caused accompanied by an environmental change and to accelerate ubiquitination and subsequent degradation of substrate which became an abnormal state (cf. Non-patent Reference 10).
Thus, the chaperone has a property as a functional molecule which recognizes non-natural structure of a protein molecule as the substrate.
On the other hand, a screening method for identifying the ligand of already known target protein has been reported, which uses molecular chaperon for the determination of the degree of folded state and unfolded state of the target protein in the presence or absence of a ligand candidate (cf. Non-patent References 1 to 6).
Insulin is secreted from the 13 cell of pancreatic islets of Langerhans and reduces blood sugar level by acting mainly upon muscles, the liver and fat to store and consume blood sugar through its intake into cells. Diabetes is induced by the insufficient action of this insulin, and there are two types in its patients, namely, type I having a disorder in the production or secretion of insulin, and type II in which acceleration of glucose metabolism by insulin becomes difficult to occur. Though the blood sugar level becomes higher than that of healthy people in both of these patients, blood insulin becomes absolutely scarce in type I, while insulin resistance in which intake or consumption of blood sugar by cells is not accelerated in spite of the presence of insulin is generated in type II. The type II diabetes is a so-called life style-related disease which is induced by overeating, less exercise, stress and the like causes in addition to hereditary basic factor. These days, this type II patient is rapidly increasing in advanced nations accompanied by the increase of caloric intake, and it occupies 95% of diabetes patients in Japan. Thus, the necessity of not only a simple hypoglycemic agent but also treatment of type II diabetes for accelerating glucose metabolism through the improvement of insulin resistance is increasing as agents for treating diabetes.
Currently, insulin injections are prescribed for the treatment of type I diabetes patients. On the other hand, as the hypoglycemic agent prescribed for type II diabetes patients, a sulfonylurea system hypoglycemic agent (SU agent) which accelerates secretion of insulin by acting upon β cells of the pancreas and an α-glucosidase inhibitor which delays digestion absorption of glucose are known, in addition to the insulin injections. Though these improve insulin resistance indirectly, a thiazolidine derivative has been used in recent years as an agent which more directly improves insulin resistance. Its action is to accelerate intake of glucose into cells and use of glucose in the cells. It has been shown that this thiazolidine derivative acts as an agonist of peroxisome proliferator activated receptor gamma (PPAR γ) (cf. Non-patent Reference 11). However, it is known that the thiazolidine derivative not only improves insulin resistance but also has adverse side effects of inducing fat accumulation and edema (cf. Non-patent Reference 12). Since this induction of edema is a serious adverse side effect which results in cardiac hypertrophy, more useful new drug target molecule instead of PPAR γ is in demand for the improvement of insulin resistance. As a leading agent which produces a glucose metabolism improving action other than these, a hypoglycemia agent biguanide which has been used for a long time is known (cf. Non-patent Reference 13). The biguanide agent has been reported to have actions to enhance glucose metabolism by anaerobic glycolytic action, suppression of gluconeogenesis, suppression of appetite and suppression of intestinal absorption of glucose, and as a result, biguanide improves insulin sensitivity in the liver and muscles. Since biguanide does not act upon the pancreas and does not increase secretion of insulin, it has a characteristic in that it does not cause obesity and hardly cause hypoglycemia. The action of biguanide does not include undesirable actions possessed by the aforementioned thiazolidine derivative and insulin preparations, and there are many cases in which it is prescribed in combination with the aforementioned other hypoglycemic agents in reality. Combined with the recent year's reconsideration on its strong pharmacological action, the biguanide agent now holds its position net to the thiazolidine derivative as insulin resistance improving agent. But on the other hand, it is known that biguanide agent has an adverse side effect of causing lactic acidosis by increasing accumulation of lactic acid (cf. Non-patent Reference 14). In spite of the very old history of biguanide as an agent, a distinct target protein, like the case of PPARγ of the thiazolidine derivative, has not been identified yet. Since information on the structural activity correlation regarding biguanide agents and the target protein has not been obtained, not only a dissociate study on adverse side effect such as improvement of lactic acidosis but also an improvement study aimed at increasing hypoglycemia as the principal effect has been difficult to carry out up to the present. ATP5B protein is the β subunit of F1F0-ATP synthase, which is encoded on the genome and perform its action after transferred to mitochondria (cf. Non-patent References 15 and 16). Also, regarding the existing amounts of ATP5B, it has been reported that both of the amounts of its gene expression and protein amount are lowered in muscles of type II diabetes patients in comparison with those of healthy people (cf. Non-patent References 17 and 18 and Patent Reference 7). In addition, it has been reported that phosphorylation level of ATP5B in muscles of diabetes patients and fasting blood sugar level take inverse correlation (cf. Non-patent Reference 18 and Patent Reference 7), and those (e.g., a nucleic acid fragment) which control expression of ATP5B, a polypeptide, an antibody, a polynucleotide or a compound which binds to a polypeptide, and the like can be agents for treating diabetes-associated diseases (cf. Patent Reference 7). There is a report which discloses various polypeptides (3025 substances) included in human heart mitochondrial proteome including ATP5B, and describes that these are related to the screening for an agent for treating diseases (including diabetes) associated with mitochondrial functions (cf. Patent Reference 8). However, there are no reports stating that ATP5B protein binds to biguanide.
The present invention aims at providing a method for identifying a target protein of a low molecular compound, with the use of not the binding of said compound with a protein but a change in the tertiary structure of protein responding to the compound as the index, and without requiring structural modification of the aforementioned compound.
Also, the present invention aims at providing a method for screening for a novel agent for treating diabetes.
When a compound acts and thereby exerts influence upon the function of a target protein, it is expected that tertiary structure of said protein undergoes a change by its interaction with the compound. Thus, it is considered that the factor necessary for the pharmacological action produced by the compound is not a simple binding between the compound and the target but a change in the tertiary structure of the target protein by the action of the compound. Accordingly, the present inventors have considered that when it is possible to screen a protein making use of said change as the index, a true target protein which carried pharmacological action of a compound can be screened with a probability of higher than conventional compound target screening means. In addition, by developing a method for picking up and detecting the aforementioned change not from the compound side but from the side of a target protein in response to the compound, identification of the target protein of said compound was realized without requiring compound structure modification which was unavoidable by the conventional methods.
That is, the inventors have found that a molecular chaperone protein known as a functional molecule which recognizes non-natural structure of a protein as the substrate recognizes a change of the tertiary structure of protein by a compound (an agent whose target protein is unknown), and thereby have constructed a method for detecting and identifying a target protein of a compound (an agent whose target protein is unknown) making use, as the index, of a change in the binding of an intracellular protein with a molecular chaperone protein. Illustratively, the inventors have succeeded in detecting estrogen receptor as the target protein of a low molecular compound 17β-estradiol (Example 2), and also have succeeded in detecting FKBP12 as the target protein of FK506 and FK1706, detecting glucocorticoid receptor as the target protein of dexamethasone, detecting androgen receptor as the target protein of dihydrotestosterone, detecting mineralcorticoid receptor as the target protein of androsterone and detecting hydrofolate reductase as the target protein of methotrexate (Example 3). Also, we have succeeded in detecting and identifying the target protein of an agent for treating diabetes, biguanide, whose target protein was unclear in the past, and have fount that this is ATP5B (Example 4). In addition, by the method of the present invention, we have succeeded in finding TARDBP as the target protein of thalidomide whose target protein was unclear up to this time (Example 8).
Also, we have revealed that, when the aforementioned ATP5B protein as the β subunit of F1F0-ATP synthase existing on the mitochondria membrane, whose function to bind to a diabetes-treating agent biguanide has been found by the inventors, is excessively expressed in a cell, activation of intracellular AMP kinase (to be referred to as AMPK hereinafter) by biguanide is obstructed (Example 5). Based on these findings, the inventors have revealed that the ATP5B protein is the target protein concerned in the pharmacological action (principal effect) of biguanide, and thereby constructed a new screening method of an agent for treating diabetes, which uses said protein. By finding that a substance obtained by the screening method of the present invention certainly has the diabetes-treating effect and does not have adverse side effects, new screening tool and screening method of an agent for treating diabetes and a pharmaceutical composition for diabetes treatment were provided.
That is, the present invention relates to:
<1> a method for screening for an agent for treating diabetes, which comprises
[1] a step of allowing (1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:2, (2) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO:2 wherein 1 to 10 amino acids thereof are deleted, substituted and/or inserted, and which also binds to biguanide and/or inhibits activation of AMPK by biguanide due to over-expression, (3) a polypeptide which comprises an amino acid sequence having 90% or more of homology with the amino acid sequence represented by SEQ ID NO:2 and which also binds to biguanide and/or inhibits activation of AMPK by biguanide due to over-expression, or (4) a cell transformed with a vector which comprises a polynucleotide coding for the polypeptide described in (1) to (3), to be in contact with a substance to be tested, and
[2] a step of analyzing binding of said polypeptide with the substance to be tested,
<2> the screening method described in <1>, wherein the step of [1] is a contacting step in the coexistence of biguanide,
<3> the screening method described in <1> or <2>, which further comprises a step of confirming that it activates AMPK activity and/or has a therapeutic activity for diabetes,
<4> a screening tool for an agent for treating diabetes having the same medicinal target with biguanide, which consists of (1) the polypeptide described in <1>, (2) a polynucleotide coding for the polypeptide described in <1> or (3) the transformed cell described in <1>,
<5> use of (1) the polypeptide described in <1>, (2) a polynucleotide coding for the polypeptide described in <1> or (3) the transformed cell described in <1>, for the screening of an agent for treating diabetes having the same medicinal target with biguanide,
<6> a pharmaceutical composition for treating diabetes, which comprises a substance obtained by the method described in <1> to <3>,
<7> a method for treating diabetes, which comprises administering an effective amount of a substance obtained by the method described in <1> to <3> to a subject in need of diabetes treatment,
<8> use of a substance obtained by the method described in <1> to <3> for the manufacture of a pharmaceutical composition for treating diabetes,
<9> a method for identifying a target protein of an agent to be tested, which comprises
[1] (1) a step of allowing an agent to be tested, a molecular chaperone protein and a sample cell protein to be in contact with each other, and
(2) a step of detecting a protein which binds to the molecular chaperone protein, [2] (3) a step of allowing a molecular chaperone protein to be in contact with a sample cell protein, and
(4) a step of detecting a protein which binds to the molecular chaperone protein, and
[3] a step of comparing the protein detected by (2) with the protein detected by (4),
<10> the identification method described in <9>, wherein the molecular chaperone protein is a protein consisting of a polypeptide which comprises an amino acid sequence of 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 wherein 1 to 10 amino acids thereof are deleted, substituted and/or inserted and also binds to a protein by recognizing a change in the tertiary structure of the protein, or consisting of a polypeptide which comprises an amino acid sequence having 90% or more of identity with the amino acid sequence represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 and also binds to a protein by recognizing a change in the tertiary structure of the protein, and
<11> the identification method described in <9>, wherein the molecular chaperone protein is a protein consisting of the amino acid sequence represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27.
On the priority date of this application, properties of molecular chaperone as a functional molecule which recognizes a non-natural structure of a protein molecule that becomes the substrate (Non-patent Reference 9 and Non-patent Reference 10) and a method for identifying a ligand of an already known protein using molecular chaperone were known (Patent References 1 to 6), but a method for identifying a target protein of a low molecular compound using molecular chaperone was not known.
On the priority date, reduction of the existing amount of ATP5B in muscles of type II diabetes patients (cf. Non-patent References 17 and 18 and Patent Reference 7) and inverse correlation between phosphorylation level of ATP5B in muscles of diabetes patients and fasting blood glucose level (cf. Non-patent Reference 18 and Patent Reference 7) were known, but the Patent Reference 7 describes that the ATP5B itself and antibodies become agents for treating diseases associated with diabetes, but it was unclear about the way of regulating expression of ATP for generating therapeutic effect of diabetes. There is a report which discloses various polypeptides (3025 substances) included in human heart mitochondrial proteome including ATP5B, and describes that these are related to the screening of a therapeutic agent for diseases including diabetes related to a large number of mitochondrial functions (cf. Patent Reference 8), but there is no basis that ATP5B is related to the screening of an agent for treating diabetes. In addition, in a reference opened to the public after the priority date of this application (International Publication No. 2005/090992), polypeptides including ATP5B as two or more modifiers of the PTEN pathway are disclosed, and a system for detecting binding of these modifiers with a candidate compound is disclosed, but a relationship between an agent specifically binding to the modifier and diabetes is not described or suggested. Since there are no reports in these references stating that ATP5B protein and biguanide bind together, binding of ATP5B protein and biguanide is the knowledge found for the first time by the present inventors, and the diabetes treating agent-screening method which uses ATP5B and has the principal effect similar to that of biguanide (particularly a diabetes treating agent-screening method which uses ATP5B and is carried out in the coexistence of biguanide) is an invention carried out for the first time by the present inventors.
The method of the present invention for identifying a target protein which responds to a compound with the use of a change in the tertiary structure of the target protein as the index, without requiring structural modification of the compound and without using binding strength of said compound and target protein as the index, is useful as an identification method of target proteins useful in studying improvement of already existing agents, and is a novel means which sweeps away various problems present in the conventional screening methods of compound target proteins.
By the screening method of the present invention which uses the screening tool of the present invention (e.g., ATP5B which is the target protein of biguanide), a compound which becomes an agent for treating diabetes and has a new structurally-non-analogous mother compound can be obtained, in addition to a structurally-analogous compound o biguanide. This structure-non-analogous compound can become a new diabetes-treating agent which does not cause obesity as a characteristic of biguanide and also has an effect of hardly causing hypoglycemia. In addition to this, by the use of the binding with ATP5B protein as the index, it becomes possible to modify molecular structure of the obtained compound while keeping its principal effect, so that it becomes possible to develop an agent for treating diabetes having more high principal effect and more reduced adverse side effect in comparison with the conventional biguanide agents.
The following describes the present invention in detail. The gene manipulation techniques in this description can be carried out in accordance with the conventionally known techniques of “Molecular Cloning” Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, and the like unless otherwise noted, and the protein manipulation techniques can be carried out in accordance with the conventionally known techniques of “Tanpaku Jikken Protocol (Protein experiment Protocol)” (Shujun-sha, 1997) and the like unless otherwise noted.
One of the present invention is a method for identifying a protein (target protein) whose tertiary structure was changed in response to a compound with the use of a molecular chaperone protein which is a protein having a function to recognize a change in the tertiary structure of a protein in vivo, by inclusively examining difference in the change of binding of molecular chaperone with an endogenous protein (target protein) at the time of adding or not adding a specific compound (an agent whose target protein is unclear), thereby selecting a protein whose binding amount is increased only when an agent to be tested is added or whose binding amount is reduced only when the agent to be tested is added.
The identification method of the present invention is a method for identifying a target protein of an agent to be tested, which comprises
[1] (1) a step of allowing an agent to be tested, a molecular chaperone protein and a sample cell protein to be in contact with each other, and
(2) a step of detecting a protein which binds to the molecular chaperone protein,
[2] (3) a step of allowing a molecular chaperone protein to be in contact with a sample cell protein, and
(4) a step of detecting a protein which binds to the molecular chaperone protein, and
[3] a step of comparing the protein detected by (2) with the protein detected by (4)
The sample cell protein according to this description means a group of proteins contained (expressed) in a cell considered to be containing the target protein to be screened (to be referred to as “sample cell” hereinafter). According to the identification method of the present invention, it is not limited by the contacting order, condition of the molecular chaperone protein (whether it is isolated, expressed in a cell or contained in a cell extract) and condition of the sample cell protein (whether it is expressed in an intact cell or contained in a cell extract), with the proviso that the agent to be tested, the molecular chaperone protein and the sample cell protein are contacted with one another. That is, the identification method of the present invention includes a method which uses an isolated and purified molecular chaperone protein and a sample cell protein contained in a sample cell extract (the first identification method), a method which uses a molecular chaperone protein expressed in a sample cell transformed with a vector comprising a partial or whole length region of a polynucleotide coding for the molecular chaperone protein and a sample cell protein expressed in the transformed sample cell (intact cell) (the second identification method), and a method which uses a molecular chaperone protein under a condition of being contained in the aforementioned extract of transformed cell and a sample cell protein under a condition of being contained in the same extract (the third identification method).
In the first identification method of the present invention, the molecular chaperone molecule is isolated. For example, it is produced in a large amount by expressing a partial or whole length region of the molecular chaperone molecule or a partial or whole length region of the molecular chaperone molecule fused with GST, Flag, His or the like tag in Escherichia coli or the like bacterium, yeast an insect cell or the like, or by a chemical synthesis method, and then it can be purified using an antibody of the molecular chaperone protein, antibodies of various tags fused to the molecular chaperone protein or affinity beads or affinity column having high affinity for the tag. Alternatively, it is also possible to produce and purify the molecular chaperone protein by effecting transcription and translation of a DNA fragment of the molecular chaperone gene in vitro. In the first identification method of the present invention, the purified molecular chaperone protein is mixed and contacted with a protein mixed liquid extracted from a sample cell (namely a liquid containing a sample cell protein) in vitro under a condition of adding or not adding an agent to be tested, and then both the molecular chaperone protein and proteins binding thereto are concentrated in accordance with the method described above. Preferably, a protein derived from a sample cell which binds to the molecular chaperone protein only when an agent to be tested is not added, or a protein derived from a sample cell which binds to the molecular chaperone protein only when an agent to be tested is added, can be detected by the methods described in Example 2(2) (3), 3, 4 or 8.
The second identification method of the present invention is a method for identifying a target protein of an agent to be tested, which comprises
[1] (1) a step of allowing an agent to be tested, a molecular chaperone protein expressed in a sample cell transformed with a vector comprising a polynucleotide coding for the molecular chaperone protein, and a sample cell protein expressed in the aforementioned transformed cell to be in contact with each other, and
(2) a step of detecting a protein which binds to the molecular chaperone protein,
[2] (3) a step of allowing a molecular chaperone protein expressed in a sample cell transformed with a vector comprising a polynucleotide coding for molecular chaperone protein to be in contact with a sample cell protein expressed in the aforementioned transformed cell, and
(4) a step of detecting a protein which binds to the molecular chaperone protein, and
[3] a step of comparing the protein detected by (2) with the protein detected by (4).
The third identification method of the present invention is a method for identifying a target protein of an agent to be tested, which comprises
[1] (1) a step of allowing an agent to be tested, a molecular chaperone protein under a condition of being contained a cell extract of a sample cell transformed with a vector comprising a polynucleotide coding for the molecular chaperone protein and a sample cell protein under a condition of being contained in the aforementioned extract to be in contact with each other, and
(2) a step of detecting a protein which binds to the molecular chaperone protein,
[2] (3) a step of allowing a molecular chaperone protein under a condition of being contained a cell extract of a sample cell transformed with a vector comprising a polynucleotide coding for the molecular chaperone protein to be in contact with a sample cell protein under a condition of being contained in the aforementioned extract, and
(4) a step of detecting a protein which binds to the molecular chaperone protein, and
[3] a step of comparing the protein detected by (2) with the protein detected by (4).
The second identification method and the third identification method of the present invention include a step of transforming a cell considered to be containing the target protein to be screened with a vector comprising a partial or whole length region of a polynucleotide coding for the molecular chaperone protein, and expressing a partial or whole length region of a polypeptide as the molecular chaperone protein, or a partial or whole length region of said polypeptide to which GST, Flag, His or the like tag is fused, in said cell. In the second identification method, a compound desired to screen its target protein (to be referred to as agent to be tested hereinafter) is added (contacted) or not added (un-added) to the aforementioned transformed cell of a living state. By this, the molecular chaperone protein expressing in the aforementioned transformed sample cell, a sample cell protein and a sample agent, or the molecular chaperone protein expressing in the aforementioned transformed sample cell and the sample cell protein, can be contacted. In the third identification method, an agent to be tested is added (contacted) or not added (un-added) to a protein mixed liquid extracted from the aforementioned transformed cell (namely a sample cell extract containing a molecular chaperone protein and a sample cell protein). By this, the molecular chaperone protein under a state of being contained in an extract of the aforementioned transformed sample cell, the sample cell protein under a state of being contained in the same extract and the sample agent, or the molecular chaperone protein under a state of being contained in an extract of the aforementioned transformed sample cell and the sample cell protein under a state of being contained in the same extract, can be contacted.
In the second identification method and the third identification method of the present invention, the protein binding to the molecular chaperone protein is concentrated in accordance with the same method of the first identification method.
<Molecular Chaperone Protein>
As the molecular chaperone protein which can be used in the identification method of the present invention, any conventionally known molecular chaperone protein can be used. Illustratively, typical proteins belonging to the respective families of Hsp90 (HtpG; the parenthesized part shows name of Escherichia coli), Hsp70 (DnaJ), Hsp60 (GroEL), Hsp40 (DnaJ), Hsp27 (IbpAB), Hsp104 (C1pB) and GRP78 (DnaK) can be exemplified (A. Sreedhar et al., Pharmacology & Therapeutics, 2004, vol. 101, no. 3, p. 227-257; D. S. Latchman et al., Cardiovascular Research, 2001, vol. 51, p. 637-646). In addition, FKBP56 and Hsp32 known as heme oxygenase-1, low molecular sHSPs (small heat shock proteins) and the like can also be used as chaperones (P. Laksanalamai, Extremphiles, 2004, vol. 8, no. 1, p. 1-11).
As the molecular chaperone protein which can be used in the identification method of the present invention, a conventionally known molecular chaperone or a polypeptide which comprises an amino acid sequence representing a conventionally known molecular chaperone protein wherein 1 to 10 (preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3) amino acids thereof are deleted, substituted and/or inserted, and also binds to a protein by recognizing a change in tertiary structure of said protein (to be referred to as “functionally equivalent variant” hereinafter) is included. Also, a polypeptide which comprises an amino acid sequence having 90% or more (preferably 95% or more, more preferably 98% or more) of identity with the amino acid sequence representing the above-described known molecular chaperone protein, also binds to a protein by recognizing a change in tertiary structure of said protein (to be referred to as “homologous polypeptide” hereinafter) is included.
Also, origins of the functionally equivalent variant and homologous polypeptide are not limited to specific organism species. In addition, they are not limited to natural polypeptides, with the proviso that they come under either the functionally equivalent variant or the homologous polypeptide, and a polypeptide artificially modified by means of genetic engineering based on an amino acid sequence representing a conventionally known molecular chaperone protein is also included therein.
In this connection, the aforementioned “identity” in this description means the value Identity obtained using the parameters arranged as default by NEEDLE program (J. Mol. Biol., 1970; 48: 443-453) retrieval. The aforementioned parameters are as follows.
Gap penalty=10
Extend penalty=0.5
Matrix=EDNAFULL
Preferred as the molecular chaperone protein are proteins represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 (human HSPA1A; RefSeq accession number NP—005336, human HSPH1; RefSeq accession number NP—006635, human HSPCA; RefSeq accession number NP—005339, human HSPD1; RefSeq accession number NP—955472, human DNAJA1; RefSeq accession number NP—001530, human HSPB1; RefSeq accession number NP—001531, human HSPE1; RefSeq accession number NP—002148, human HSPA4; RefSeq accession number NP—002145, human HSP90B1; RefSeq accession number NP—003290, human CCT6B; RefSeq accession number NP—006575, human TCP1; RefSeq accession number NP—110379, human HSPA14; RefSeq accession number NP—057383, human HSPA9B; RefSeq accession number NP—005338, human STCH; RefSeq accession number NP—008879, human HYOU1; RefSeq accession number NP—006380, human HSPB5; RefSeq accession number NP—001876, human HSPB2; RefSeq accession number NP—001532, human DNAJA2; RefSeq accession number NP—005871, human DNAJB1; RefSeq accession number NP—006136, human DNAJB2; RefSeq accession number NP—006727, human HCG3; RefSeq accession number NP—001001394, human DNAJB11; RefSeq accession number NP—057390, human DNAJC11; RefSeq accession number NP—060668, human DNAJC7; RefSeq accession number NP—003306, human DNAJC6; RefSeq accession number NP—055602), and a polypeptide which comprises an amino acid sequence of 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 wherein 1 to 10 (preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3) amino acids thereof are deleted, substituted and/or inserted and also binds to a protein by recognizing a change in the tertiary structure of the protein, or a polypeptide which comprises an amino acid sequence having 90% or more (preferably 95% or more, more preferably 98% or more) of identity with the amino acid sequence represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 and also binds to a protein by recognizing a change in the tertiary structure of the protein.
The term “binds to a protein by recognizing a change in the tertiary structure of the protein” means that the molecular chaperone binds to a protein by responding to a change in the tertiary structure of the protein caused by its binding with an agent to be tested, or the molecular chaperone once bound to a protein of unchanged state is separated by responding to a change in the tertiary structure of the protein caused by its binding with an agent to be tested. Whether or not the molecular chaperone “binds” by responding to a change in the tertiary structure of the protein can be verified in the same manner as the method for “detecting a protein which binds to molecular chaperone protein” of the identification method of the present invention. Particularly, regarding the functionally equivalent variant and homologous polypeptide of a polypeptide consisting of an amino acid sequence represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27, to “bind to a protein by recognizing a change in the tertiary structure of the protein” is verified, by responding to a change in the tertiary structure of the ATP5B protein caused by its binding to biguanide (agent to be tested), based on the separation of the molecular chaperone protein once bound to the ATP5B protein of unchanged state by responding to a change in the tertiary structure of the ATP5B protein caused by its binding to biguanide. Alternatively, it is verified by the binding of the molecular chaperone to a protein by responding to a change in the tertiary structure of the TARDBP protein caused by its binding to thalidomide (agent to be tested). These verifications are carried out under the conditions of Example 4 or Example 8, using a functionally equivalent variant or homologous polypeptide to be examined instead of the molecular chaperone protein used in Example 4 or Example 8.
In the identification method of the present invention, among the molecular chaperone proteins, use of the proteins represented by 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, 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, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and/or SEQ ID NO:27 is particularly desirable. Since these proteins belong to the aforementioned respective families of different chaperones (Hsp90, Hsp70, Hsp60, Hsp40, Hsp27, Hsp104 and GRP78), it is expected that each of them has a property characteristic to each family of the molecular chaperone protein.
<Production Method of Molecular Chaperone Protein>
In the identification method of the present invention, a molecular chaperone protein can be easily produced and obtained by a general genetic engineering and/or biochemical means using a polynucleotide coding for the molecular chaperone protein. Said polynucleotide can be easily produced and obtained by a general genetic engineering technique based on the sequence information disclosed in this description or conventionally known sequence information.
For example, it can be obtained in the following manner, but it can be obtained not only by this method but also by conventionally known operations (“Molecular Cloning” [Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, and the like]). For example, (1) a method which uses PCR, (2) a method which uses a usual genetic engineering technique (namely a method in which a transformant containing the desired polypeptide is selected from the transformants transformed with a cDNA library), or (3) a chemical synthesis method can be cited. Respective production methods can be carried out in the same manner as described in WO 01/34785.
In the method which uses PCR, a polynucleotide coding for a molecular chaperone protein can be produced, for example, by the procedure described in the “Mode for Carrying Out the Invention” 1) Production method of protein gene, a) First production method, of the aforementioned patent reference. For example, mRNA is extracted from human liver, brain, mammary gland or the like tissue. Next, a first strand cDNA is synthesized by carrying out a reverse transferase reaction of this mRNA in the presence of random primers or oligo(dT) primers. A polynucleotide coding for the molecular chaperone protein or a part thereof can be obtained by subjecting the thus obtained first strand cDNA to a polymerase chain reaction (PCR) using two primers interposing a partial region of the gene of interest. More illustratively, a polynucleotide coding for a molecular chaperone protein can be produced by the method described in Example 1.
In the method which uses a usual genetic engineering technique, a polynucleotide coding for a molecular chaperone protein can be produced, for example, by the procedure described in the “Mode for Carrying Out the Invention” 1) Production method of protein gene, b) Second production method, of the aforementioned patent reference.
In the method which uses a chemical synthesis method, a polynucleotide coding for a molecular chaperone protein can be produced, for example, by the procedure described in the “Mode for Carrying Out the Invention” 1) Production method of protein gene, c) Third production method, d) Fourth production method, of the aforementioned patent reference. Illustratively, it can be produced by liquid phase and solid phase peptide synthesis methods. The synthesis may be carried out by successively binding amino acids one by one, or by synthesizing a polypeptide fragment consisting of several amino acids and then binding it. The polypeptide of the present invention obtained by these means can be purified in accordance with various conventionally known methods.
Mutation of the sequence sometimes occurs by a natural mutation, but can also be prepare by carrying out artificial modification. The present invention does not care about the cause and means of the mutation. Regarding the artificial means for preparing the aforementioned mutants, in addition to the gene engineering techniques such as the base-specific substitution method (Methods in Enzymology, (1987) 154, 350, 367-382) of a polynucleotide coding for the aforementioned polypeptide, phosphotriester method, phospho-amidide method and the like chemical synthesis means (Science, 150, 178, 1968) can for example be cited. It is possible to obtain a polynucleotide which accompanies the desired base substitution by their combination. Alternatively, it is possible to generate substitution of a non-specific base in the polynucleotide molecule by repeating the operation of PCR or adding manganese ion or the like to its reaction liquid.
The molecular chaperone protein can be expressed in vitro or in a cell to be tested, by connecting the molecular chaperone protein-encoding polynucleotide obtained as described in the above to the downstream of an appropriate promoter by the method described in “Molecular Cloning, Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989” or the like.
Illustratively, by adding a polynucleotide containing a specific promoter sequence to the upstream of the initiation codon of the polynucleotide obtained as described in the above, and using this as the template, expression of the molecular chaperone protein by transcription and translation of the gene in a cell-free system can be carried out. Alternatively, when the molecular chaperone protein-encoding polynucleotide is integrated into an appropriate vector plasmid and transform a host cell by the plasmid, expression of said polypeptide becomes possible. Still alternatively, a cell in which such a construction is integrated into chromosomal DNA may be prepared and used. More illustratively, when a fragment containing the isolated polynucleotide is again integrated into an appropriate vector plasmid, it can transform host cells of eukaryote and prokaryote. In addition, when an appropriate promoter and a sequence concerned in the gene expression are transferred into these vectors, it becomes possible to effect expression of the molecular chaperone protein in respective host cells. The host cell is not particularly limited, and it may be any cell which can realize expression of the molecular chaperone protein in an amount sufficient for the purpose of applying to the method of the present invention. As the host cell, for example, a monkey cell COS cell (Gluzman, Y. (1981) Cell, 23, 175-182), a dehydrofolate reductase deficient strain of Chinese hamster ovary cell (CHO) (Urlaub, G. and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. USA, 77, 4216-4220), human fetal kidney derived HEK293 cell and 293-EBNA cell in which EBNA-1 gene of Epstein Barr virus is transferred into the same cell (mfd. by Invitrogen), and the like can be cited.
The method for expressing a gene by transforming a host cell can be carried out, for example, by the method described in the recombinant protein production method of the “Mode for Carrying Out the Invention” 2) of the aforementioned patent reference. The expression vector to be used in the production of a molecular chaperone expression cell (expression vector for molecular chaperone expression) is not particularly limited, with the proviso that it contains the desired polynucleotide. For example, an expression vector obtained by inserting the polynucleotide of interest into a conventionally known expression vector optionally selected in response to the host cell to be used can be cited. As the conventionally known expression vector, for example, pSV2dhfr having SV40 early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol., 1, 854-864), pEF-BOS having human elongation factor promoter (Mizushima, S, and Nagata, S. (1990) Nucleic Acids Res., 18, 5322), pCEP4 having cytomegalovirus promoter (Invitrogen), pME18S (Maruyama, K. and Takebe, Y. (1990) Med. Immunol., 20, 27-32), pCDM8 (Seed, B. (1987) Nature, 329, 840-842) and the like can be cited. The molecular chaperone protein can be obtained, for example, by transforming a desired host cell with the aforementioned expression vector, and effecting expression of the aforementioned polypeptide in said cell. More illustratively, by integrating a desired polynucleotide into a bacterial expression vector, a desired molecular chaperone protein can be produced in bacterial cells in a large amount. Also, the molecular chaperone protein can also be produced in a large amount using yeast, an insect cell or the like. In addition, a desired molecular chaperone protein can be produced in vitro by a conventionally known method using the aforementioned polynucleotide linked to the downstream of a certain promoter. More illustratively, a desired molecular chaperone protein can be produced in vitro by carrying out transcription and translation reactions in vitro using, as the template, the aforementioned polynucleotide linked to the downstream of the aforementioned promoter.
By culturing the aforementioned cell, the molecular chaperone protein produced in the cells can be detected, determined and further purified. For example, it is possible to detect and purify said protein by western blotting or immunoprecipitation using an antibody which binds to the molecular chaperone protein. Alternatively, by expressing said protein as a fusion protein with an appropriate tag protein such as glutathione-S-transferase (GST), protein A, β-galactosidase, maltose-binding protein (MBP) or the like, said protein can be detected by western blotting or immunoprecipitation using an antibody specific for such a tag protein. In addition, the aforementioned protein can be purified making use of these tag proteins. More illustratively, the aforementioned protein can be purified making use of a tag protein in the following manner.
In the method of the present invention, a polynucleotide coding for a molecular chaperone protein (e.g., a polypeptide represented by 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 27) is integrated for example into a vector by which GST tag or His tag is added to the expressed protein of interest, more illustratively the pGEX-6P1 (mfd. by Amersham) described in Example 1 or a commercially available pET-28a (Novagen) for example, and transferred into a bacterium, and the molecular chaperone protein can thereby expressed as a GST fusion type protein in the case of the former, and as a His fusion type protein in the case of the latter. Said fusion type protein can be purified from a protein extract derived from the bacterial cell expressing the protein, making use of the properties of the GST or His tag. For example, each of the chaperone expression plasmids prepared in Example 1 using pGEX-6P1 is designed in such a manner that the GST tag is added to the N-terminal of the molecular chaperone protein. By this, the desired molecular chaperone protein can be purified from a cell in which said protein was expressed, making use of the GST tag. More illustratively, the molecular chaperone protein fused with GST tag can be isolated from a disrupted cell extract by binding to glutathione Sepharose beads (Glutathione Sepharose 4B; Amersham) and centrifuging it, in accordance with the conventionally known GST pull-down method (N. Matsu et al., Jikken Kogaku (Experimental Engineering), Vol. 13, No. 6, page 528, 1994). On the other hand, regarding the purification of a desired molecular chaperone protein from a cell expressing said protein making use of His tag, the molecular chaperone protein fused with His tag can be isolated from a disrupted cell extract by binding to Ni2+-NTA-Agarose (mfd. by Funakoshi) and centrifuging it, in accordance with the conventionally known method (Nakahara et al., Jikken Igaku Bessatsu Tanpakushitsu no bunshikan sogo sayo jikken-ho (Experimental Medicine, supplement, Experimental methods of the intermolecular interaction of protein), page 32, 1996).
Alternatively, as occasion demands, the molecular chaperone protein can also be purified by a method which does not use a tag protein, for example by various separation operations making use of its physical properties and chemical properties. Illustratively, application of ultrafiltration, centrifugation, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography can be exemplified.
<Agent to be Tested>
Though not particularly limited, examples of the agent to be tested include commercially available compounds (including peptides), various conventionally known compounds (including peptides) registered in chemical files, a group of compounds obtained by the combinatorial chemistry techniques (N. Terrett et al., Drug Discov. Today, 4(1): 41, 1999), microbial culture supernatants, natural components derived from plants and marine organisms, animal tissue extracts (including polynucleotides and peptides), or those which are chemically or biologically modified compounds thereof and have distinct pharmacological actions. Not only the actions desirable for medical treatments but also the actions toxic for the living bodies are included in the pharmacological actions. As compounds considered to be useful in the application of the method of the present invention in comparison with the conventional methods, (1) compounds to which modifications can hardly be applied due to their chemical structures, (2) compounds in which the aforementioned pharmacological actions are lost, or considered to be lost, by modifications, (3) compounds having unidentified chemical structures which produce pharmacological actions by their mixing with degradation or metabolic products or other contaminants, (4) compounds (including natural substances) whose available amounts are limited due to difficulties in performing synthesis and purification or obtaining their raw materials, and the like can be exemplified.
<Cell Extract>
A liquid obtained by extracting protein from a primary target tissue for use in inducing pharmacological action possessed by the agent to be tested, or from a cultured cell keeping most properties of said tissue, can be used as the cell extract to be used in the method of the present invention. Regarding the method for extracting protein from a cell, it is desirable to use a preparation method in response to the purpose. Illustratively, in accordance with conventionally known protein extraction methods, a substance fitted to the purpose is selected from SDS, Triton-X, or CHAPS, CHAPSO and the like various surfactants, the aforementioned cell is disrupted and centrifuged using a buffer containing the substance, and then the supernatant is separated and recovered to be used as the cell extract of the method of the present invention. More illustratively, it is desirable that inhibitors of various living body-derived proteolytic enzymes, such as PMSF (phenylmethyl sulfonylfluoride), EGTA (ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid) and the like, are contained in the buffer to be used in the disruption, and it is desirable to store it under a condition of −80° C. or lower which can stably maintain the frozen protein until its application to the method of the present invention.
<Step of Detecting a Protein which Binds to Molecular Chaperone Protein>
It is considered that each molecular chaperone protein responds to a large number of substrate proteins per molecule. However, it is considered that the substrate specificity varies depending on their kinds. Thus, in order to truly inclusively screen targets of compounds having various structures, it is desirable to arrange molecular chaperone proteins belonging to a large variety of different families and use simultaneously as the probe. More preferably, as shown in Example 2(3), 3, 4 and 8 of the present invention, it is desirable to arrange two or more molecular chaperone proteins derived from different chaperone families and use them simultaneously.
Also, in the case of a biochemical test system, when the protein to be used as the probe (molecular chaperone protein in the case of this present invention) is present in large excess in comparison with the protein to be used as the substrate, substrate specificity of the probe is thinned, so that it can be expected that even a protein other than the original protein is recognized by the prove when it is a molecule analogous to said substrate. Accordingly, like the case of the in vitro pull-down method shown in Example 2(2) of the present invention, use of a system in which a large amount of the probe protein can be used in the reaction, in comparison with the cell-derived protein to be used as the substrate, is more desirable in enabling inclusive target screening of compounds.
In addition, it has been reported that many molecular chaperone proteins undergo their actions by forming a polymer in vivo. Accordingly, in carrying out the method of the present invention, it is more desirable to use such a living body sample-derived cell extract and separation conditions that an endogenous chaperone capable of forming a complex with the molecular chaperone protein (probe) is present therein as a mixture, as shown in Examples 2(2) and 4. More illustratively, it is desirable to use a system in which a molecular chaperone protein (probe protein) is allowed to react with a cell extract derived from a cell which contains an endogenous chaperone, like the in vitro pull-down method shown in Example 2(2) of the present invention.
In order to “detect a protein which binds to a molecular chaperone protein”, the following operation is carried out. A molecular chaperone protein and a protein which binds to the molecular chaperone protein can be concentrated from a sample cell extract extracted from a cell to which an agent to be tested was added or not added, or a sample cell extract to which an agent to be tested was added or not added, by the conventionally known immunoprecipitation method using an antibody of the molecular chaperone protein or an antibody of a tag fused to the molecular chaperone protein. Alternatively, the molecular chaperone protein and a protein which binds to the molecular chaperone protein can also be concentrated by a conventionally known method which uses affinity beads or affinity column having affinity for the aforementioned tag. As an illustrative method, a GST pull-down method which uses the peptide purified by attaching GST or the like tag can be exemplified.
The protein binding to the molecular chaperone protein is detected by separating the concentrated liquid of molecular chaperone protein and its binding protein obtained in the above by a conventionally known protein separation method. For example, after separating them by polyacrylamide gel electrophoresis, the molecular chaperone protein and the sample cell-derived protein linked to the molecular chaperone protein can be detected by various conventional methods for detecting proteins including already existing protein staining methods such as silver staining, Coomassie Brilliant Blue staining or Negative Gel Staining (Wako Pure Chemical Industries) (“Idensgi Cloning no Tame no Tanpakushitsu Kozo Kaiseki (Protein Structure Analysis for Gene Cloning)” Hisashi Hirano Tokyo Kagaku Dojin 1993 p. 37-p. 40). The method to be used in this step of the present invention is not limited to the above method with the proviso that it can detect the protein.
Regarding the proteins detected by the above method, the proteins which bind to the molecular chaperone protein in the case of adding and not adding the agent to be tested are compared. By comparing both cases, a sample cell-derived protein whose binding to the molecular chaperone protein was changed at the time of adding or not adding the agent to be tested (namely, the target protein of the agent to be tested) can be identified. As the method for detecting and comparing a group of sample cell-derived proteins at the time of adding or not adding the agent to be tested, the conventionally known SDS polyacrylamide gel electrophoresis can be exemplified. In that case, more precise comparison can be made when developed by a two dimensional electrophoresis. By comparing the bands developed by the electrophoresis based on the results of the case of the addition of the agent to be tested and the results of the case of the un-addition of the agent to be tested (namely, by comparing whether there is a case in which the quantity of a band increases only at the time of the addition of the agent to be tested or a case in which the quantity of a band increases only at the time of the un-addition of the agent to be tested), a protein whose binding quantity increases only at the time of the addition of the agent to be tested, or whose binding quantity decreases only at the time of the addition of the agent to be tested, can be selected. As the protein whose binding quantity increases only at the time of the addition of the agent to be tested, or whose binding quantity decreases only at the time of the addition of the agent to be tested, it is desirable to select a protein which binds only at the time of the addition of the agent to be tested or does not bind only at the time of the addition of the agent to be tested.
Subsequently, the proteins detected and selected by the above method are identified. The amino acid sequences presenting in their molecules are determined by conventionally known protein purification methods and protein identification methods (Schevchenko et al., Analytical Chemistry, vol. 68, p. 850-p. 858, 1996), and based on this amino acid sequence information, a protein whose binding with the molecular chaperone protein changes at the time of the addition or un-addition of the agent to be added (namely a target protein of the agent to be tested) can be identified. Illustratively, the target protein of the agent to be tested can be identified by recovering and purifying the protein from the gel and then determining its amino acid sequence by a mass spectrum method or a conventionally known method. More illustratively, identification of the protein can be carried out by the mass spectrum analysis after digesting the protein of interest separated by the SDS polyacrylamide gel electrophoresis into fragments using trypsin or the like and recovering the thus formed peptide mixture from the gel (Schevchenko et al., Analytical Chemistry, vol. 68, p. 850-p. 858, 1996). Alternatively, after eluting the protein of interest from the gel by an electric elution method or the like, or after blotting the protein of interest on the gel on a film of PVDF (polyvinylidene fluoride) or the like, it is made into fragments by an enzyme digestion or a chemical digestion as occasion demands, and, as occasion further demands, the thus obtained peptide fragments are separated by a liquid chromatography, a capillary electrophoresis or the like, and then identification of the protein can be carried out by a mass spectrum analysis or an N-terminal or C-terminal amino acid sequence analysis (H. Hirano, Proteome Kaiseki—Rixon to Hoho— (Proteome Analysis—Theory and Method) Tokyo Kagaku Dojin, 2001). In this connection, the identification method to be used in the method of the present invention is not limited to the method described in the above, with the proviso that identification of a group of purified said target proteins can be carried out.
More illustratively, as described in Example 4 and Example 8, identification of the target protein separated by the SDS polyacrylamide gel electrophoresis can be carried out by digesting the protein into fragments using trypsin or the like, recovering the thus formed peptide mixture from the gel and then carrying out a mass spectrum analysis.
It is possible to verify by the conventionally known gene function analyzing techniques that the target protein identified by the method of the present invention is a true target protein which produces pharmacological actions of the agent to be tested. Firstly, illustratively, the presence or absence of direct binding between the agent to be tested and the thus obtained target protein molecule can be examined by the method shown below.
A part or the whole length region of a polypeptide to be examined for whether or not it binds thereto or a part or the whole length region of a polypeptide to be examined to which GST, Flag, His or the like tag was fused is expressed in a cell. The expressed polypeptide to be examined is isolated and purified from said cell by an affinity purification method making use of the affinity for the GST, Flag, His or the like tag, or by an immunoprecipitation method making use of an antibody which responds to said tag. Subsequently, the thus purified a polypeptide is mixed with an agent to be tested, and the complex formed by the binding of said agent to be tested and the polypeptide is isolated. Next, by examining whether or not said agent to be tested is contained in the sample by carrying out a mass spectrometry analysis using a mass spectrometer, after re-separating the compounds by denaturing said complex by an acid, heat or other stimulation and thereby removing the protein alone, the binding of the peptide to be examined with said agent to be tested can be verified. In addition, as another method, whether or not the polypeptide to be examined and said agent to be tested bind to each other can be verified by conventionally known ELISA, western blotting, binding assay and the like methods using, as the probe, a labeled agent to be tested prepared by labeling a part of the molecular structure of the agent to be tested. It is desirable that a radioisotope which does not exert influence upon the binding with the target protein is used as the label of the agent to be tested. Illustratively, for example, a labeled compound can be prepared by substituting an element in the molecule of the agent to be tested with a radioisotope. By using said labeled agent to be tested as the probe, binding of a polypeptide with said agent to be tested can be verified by an ELISA method in which the polypeptide to be examined purified by the aforementioned method is immobilized. Alternatively, after separating the polypeptide to be examined by a conventionally known SDS acrylamide gel electrophoresis and transferring it on a nitrocellulose film, binding of said polypeptide with said agent to be tested can also be verified by a far western method using the aforementioned labeled agent to be tested as the probe. Also after mixing the labeled agent to be tested with the polypeptide to be examined purified by the aforementioned method and washing them by trapping on a filter, binding of said polypeptide with said agent to be tested can be verified by a so-called binding assay in which total amount of the thus formed complex of the compound and peptide is detected by measuring the radiation dose derived from the labeled probe.
Also, whether or not a keyhole structure is present in the target protein identified by the method of the present invention, to which said compound to be tested can bind, by a conventionally known protein tertiary structure predicting technique (J. Med. Chem., Dec. 30, 2004; 47 (27): 6804-11). In addition, expression quantity of the aforementioned target protein can be increased or decreased by various biochemical and/or genetic engineering test methods, such as a conventionally known gene knockout test at the cell level using an RNA interference technique (Tuschl T. et al., Nat. Biotechnol., 2002, 20(5): p. 446-448) and a conventionally known gene over-expression test also at the cell level, as well as the preparation of a gene knockout animal or preparation of a gene over-expressing animal, and since the effect to accelerate or suppress the principal effect or an adverse side effect is found when the principal effect or an adverse side effect is tested under these conditions, it can be confirmed that a target protein encoded by a gene whose expression was changed is the true target protein.
Though the identification method of the present invention can identify the target protein of an agent to be tested which produces pharmacological action of the agent to be tested, the identification method of the present invention is more suited as a method for identifying a target protein which produces a desired pharmacological action (principal effect) among the pharmacological actions of the agent to be tested.
<Screening Tool of the Present Invention and its Use for Screening>
The screening tool of the present invention consists of the following (1) to (3).
(1) A diabetes-treating agent screening tool which has the same medicinal target with biguanide (to be referred to as polypeptide type screening tool of the present invention hereinafter), consisting of a human ATP5B protein (a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:2), an ATP5B functionally equivalent variant (a polypeptide which comprises the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence of the amino acid sequence represented by SEQ ID NO:2 wherein 1 to 10 (preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3) amino acids thereof are deleted, substituted and/or inserted, and also binds to biguanide and/or inhibits AMPK activation of biguanide by its over-expression), and an ATP5B homologous protein (a polypeptide which comprises an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more) of identity with the amino acid sequence represented by SEQ ID NO:2, and also binds to biguanide and/or inhibits AMPK activation of biguanide by its over-expression) (to be referred to as polypeptide for tool use hereinafter),
(2) a diabetes-treating agent screening tool which has the same medicinal target with biguanide (to be referred to as polynucleotide type screening tool of the present invention hereinafter), consisting of a polynucleotide coding for the polypeptide for tool use (to be referred to as polynucleotide for tool use hereinafter), or
(3) a diabetes-treating agent screening tool which has the same medicinal target with biguanide (to be referred to as cell type screening tool of the present invention hereinafter), consisting of a cell which is transformed with the polynucleotide for tool use and thereby expressing the polypeptides for tool use (to be referred to as cell for tool use hereinafter).
In addition, use of
(1) polypeptides for tool use,
(2) polynucleotide for tool use, or
(3) cell for tool use
for the screening of a diabetes-treating agent which has the same medicinal target with biguanide is also included in the present invention.
According to this description, the “screening tool” means a substance for use in the screening (illustratively, the polypeptide, polynucleotide or cell to be used in the screening). The “screening tool of a diabetes-treating agent, which has the same medicinal target with biguanide” is a cell or polypeptide which becomes the object to which a compound to be tested is contacted, or a polynucleotide for obtaining or expressing a polypeptide which becomes the object to which a compound to be tested is contacted, for use in the screening of a diabetes-treating agent which has the same medicinal target with biguanide by the screening method of the present invention.
Origins of the ATP5B functionally equivalent variant and ATP5B homologous polypeptide are not limited to human. With the proviso that it comes under any one of the polypeptides for tool use, not only the mutants of the amino acid sequence represented by SEQ ID NO:2 in human but those derived from all kinds of organisms ranging from a vertebrate to a bacterium are also included therein, and not only natural polypeptides but also polypeptides artificially modified by genetic engineering techniques based on the amino acid sequence represented by SEQ ID NO:2 are included therein. In addition, with the proviso that it binds to biguanide and/or inhibits AMPK activation of biguanide by its over-expression, a polypeptide in which a marker sequence which is described later is contained in the ATP5B functionally equivalent variant or ATP5B homologous polypeptide is also included in the polypeptide for tool use.
In the Example 5(5) and (6) which are described later, it was revealed that amino acid residues concerned in the peripheral structure of site 20, particularly Glu 175 and Asp 295 in the case of human ATP5B, are important for the binding of ATP5B with biguanide. Accordingly, when a mutation is introduced, a modified polypeptide which keeps its activity to bind with biguanide can be easily produced by preserving the amino acid residues concerned in the peripheral structure of site 20, particularly Glu 175 and Asp 295 in the case of human ATP5B, and introducing a mutation into a part other than this.
Human ATP5B polypeptide is more desirable among the polypeptides for tool use, as the polypeptide type screening tool of the present invention, and a polynucleotide coding for human ATP5B polypeptide (particularly preferably the polynucleotide represented by the nucleotide sequence of SEQ ID NO:1) among the polynucleotides for tool use, as the polynucleotide type screening tool of the present invention, and a cell which is transformed with a vector comprising a polynucleotide coding for human ATP5B and is thereby expressing human ATP5B, among the cells for tool use, as the polynucleotide type screening tool of the present invention.
The polynucleotide for tool use can be easily produced and obtained by general genetic engineering techniques based on the sequence information disclosed in this description or conventionally known gene sequence information. As such techniques, as described in the aforementioned <Production method of molecular chaperone protein>, conventionally known operations “Molecular Cloning” [Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, and the like], for example, (1) a method which uses PCR, (2) a method which uses a usual genetic engineering technique (namely a method in which a transformant containing the desired polypeptide is selected from the transformants transformed with a cDNA library), or (3) a chemical synthesis method can be cited. Respective production methods can be carried out in the same manner as described in WO 01/34785.
In the method which uses PCR, a polynucleotide for tool use or a part thereof can be obtained, for example, by extracting mRNA from human skeletal muscle, brain or the like tissue, and using a first strand cDNA in the same manner as described in the aforementioned <Production method of molecular chaperone protein>, by subjecting it to PCR using two primers interposing a partial region of the polynucleotide for tool use. More illustratively, the polynucleotide for tool use can be produced, for example, by the method described in Example 5(1).
Also by the method which uses a usual genetic engineering technique or the method which uses a chemical synthesis method, the polynucleotide for tool use can be produced in the same manner as described in the aforementioned <Production method of molecular chaperone protein>.
In the same manner as described in the aforementioned <Production method of molecular chaperone protein>, the polynucleotide for tool use obtained in the above manner can be expressed in vitro or in a cell to be tested by connecting the polynucleotide for tool use to the downstream of an appropriate promoter by a conventionally known method (e.g., the method described in “Molecular Cloning, Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989” or the like).
The expression vector to be used in the production of the cell for tool use (expression vector for tool use) is not particularly limited, with the proviso that it comprises the polynucleotide for tool use. For example, an expression vector prepared by inserting the polynucleotide for tool use into a conventionally known expression vector optionally selected in response to the host cell to be used can be cited. The polypeptide for tool use can be obtained, for example, by transforming a desired host cell with the expression vector for tool use, and effecting expression of the polypeptide for tool use in said cell. More illustratively, the polypeptide for tool use can be produced in a large amount in a bacterial cell by integrating the polynucleotide for tool use into a bacterial expression vector. In addition, the polypeptide for tool use can be produced in vitro by a conventionally known method using the polynucleotide for tool use linked to the downstream of a predetermined promoter. More illustratively, as described in Example 5(4), the polypeptide for tool use can be produced in vitro by carrying out transcription and translation reactions in vitro using, as the template, the aforementioned polynucleotide for tool use linked to the downstream of a promoter and using the TNT system (mfd. by Promega).
The cell for tool use is not particularly limited with the proviso that it is transformed with the expression vector for tool use and comprises the polynucleotide for tool use, so that it may be a cell in which the polynucleotide for tool use is integrated into the chromosome of a host cell, or a cell which comprises the polynucleotide for tool use in the form of an expression vector. As the cell to be used in the transformation, a cell which responds to biguanide is desirable, and more illustratively, HeLa S3 cell, a liver-derived cell or a skeletal muscle-derived cell is desirable. As the cell for tool use, it may contain a polynucleotide comprising a marker sequence which is described later, with the proviso that it comes under the polynucleotide for tool use. As the cell type screening tool according to this description, a cell transformed with the expression vector for tool use is desirable.
The desired transformant cell obtained in the above can be cultured in accordance with a usual method, and the polypeptide for tool use is produced by said culturing. As the medium to be used in said culturing, generally used various media can be optionally selected in response to the employed host cell, and in the case of the aforementioned HeLa S3 cell for example, Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS) or the like serum component, and the like can be used.
As occasion demands, the polypeptide for tool use produced by the above manner can be separated or purified by various conventionally known separation operations making use of the physiological properties, biochemical properties and the like of said polypeptide. Also, when a marker sequence (tag protein) is contained in the polypeptide for tool use, it is possible to carry out expression confirmation, purification and the like of said polypeptide making use of the tag protein. Examples of the marker sequence include FLAG epitope, Hexa-Histidine tag, Hemagglutinin tag, myc epitope, glutathione-S-transferase (GST), protein A, β-galactosidase, maltose-binding protein (MBP) and the like.
In addition, it is possible also to obtain the polypeptide for tool use by effecting expression of a fusion polypeptide in which a specific amino acid sequence which can be recognized by enterokinase, factor Xa, thrombin and the like proteases is inserted between a marker sequence and the polypeptide for tool use, purifying it making use of the tag protein, and then digesting and removing the marker sequence moiety by these proteases.
More illustratively, the polypeptide for tool use can be expressed, for example, using a vector by which the GST tag or His tag is added to the protein of interest, more illustratively a commercially available pGEX-6P1 (Amersham) or pET-28a (Novagen) for example, as a GST fusion type protein in the former case or a His fusion type protein in the latter case. Said fusion type proteins can be purified from the protein extracts derived from the bacterial cells which expressed them, in the same manner as described in the aforementioned <Production method of molecular chaperone protein> making use of the properties of the GST tag or His tag.
The “polypeptide which binds to biguanide” according to this description means a polypeptide which binds to a low molecular compound classified into biguanide (metformin, phenformin, buformin or the like), and whether or not the polypeptide “binds” to biguanide can be verified by the following method.
The polypeptides to be examined on whether or not they bind thereto are isolated and purified. Expression of the polypeptides and their isolation and purification can be carried out making use of the aforementioned methods. Subsequently, whether or not they bind to phenformin is verified by the method of Example 5(4). A polypeptide whose binding to phenformin at its adding concentration of preferably 10 μM or less, more preferably 1.0 μM or less, further preferably 0.1 μM or less, can be confirmed under the conditions of said Example is regarded as a polypeptide which binds to biguanide.
In addition, the term “inhibits AMPK activation of biguanide by its over-expression” means that activation of AMPK in a cell when stimulated with biguanide under such a condition that a certain polypeptide is present in the cell in an excess amount than usual, namely degree of phosphorylation, is reduced in comparison with the cells of usual state. Whether or not it “inhibits AMPK activation” can be verified by the method of Example 5(7). When increase in the phosphorylation of AMPK by phenformin stimulation in a cell over-expressing the polypeptide to be examined is reduced by a factor of 50%, preferably 79%, further preferably 90%, in comparison with the control (a cell not over-expressing the polypeptide to be examined) under the conditions of Example 5(7), it is judged that the polypeptide to be examined is a polypeptide which inhibits AMPK activation of biguanide by its over-expression.
<Screening Method of the Present Invention>
The inventors have found that when human ATP5B (SEQ ID NO:2) as one of the polypeptides for tool use is over-expressed in HeLa S3 cell, AMPK activation in said cell by treatment with phenformin, metformin or the like biguanide is inhibited (Example 5(7)). This means that activation ability of AMPK contributing to the drug effect of biguanide changes by the change of the amount of ATP5B protein which exists in the cell, and shows that ATP5B is evidently positioned at the upstream of AMPK in terms of the intracellular signal of biguanide. In addition, the inventors have found that ATP5B biochemically binds to phenformin as a species of biguanide (Example 5(4)). Based on these findings, it can be concluded that ATP5B is a true target protein which binds to biguanide and contributes to the drug effect (principal effect) of said compound. In accordance with said findings, the inventors have revealed that a compound capable of showing a drug effect similar to the diabetes-treating effect of biguanide can be newly obtained by carrying out screening of a low molecular compound which activates AMPK, and thereby have accomplished a screening method of an agent for treating diabetes.
The following methods are included in the screening method of the present invention.
<I> Binding Assay Screening Method
A method for screening for an agent for treating diabetes, which comprises
[1] a step of allowing a polypeptide for tool use or a cell for tool use and a substance to be tested to be in contact with each other, and
[2] a step of analyzing binding of said polypeptide with the substance to be tested.
<II> Competitive Inhibition Screening Method
The screening method described in <I>, wherein the step [1] is a step of allowing them to be in contact with each other in the coexistence of biguanide.
The “screening” according to this description both of the screening of a substance having the activity of interest from a large number of substances to be tested and the detection of whether or not the substance is a substance having the property of interest.
<Binding Assay Screening Method which Uses a Polypeptide for Tool Use>
Included in the screening method of the present invention, which comprises a step of allowing a polypeptide for tool use and a substance to be tested to be in contact with each other, is a method in which a purified polypeptide for tool use and a substance to be tested are allowed to be in contact with each other by mixing them (a step of contacting), a substance which directly binds to said polypeptide and acts thereon (namely an agent for treating diabetes) is analyzed by identifying, by a mass spectrometry, the compounds separated by denaturing a complex formed by the binding of said substance to be tested and polypeptide after its isolation (a step of analyzing the binding), and then a substance which binds to the polypeptide for tool use (namely an agent for treating diabetes) is selected. Such a method can be carried out by an affinity selection-mass spectrometry (AS-MS method; cf. Pharmacia (Pharmaceutical Society of Japan) Vol. 41 No. 6 p. 564 2005, J. Protein Chem. 1997, 16, 5, 505-511, J. Comb. Chem. 1999, 1, 82-90, Anal. Biochem. 324 (2004) 241-249, J. Biomol. Screening 9 (6); 2004 498-505). In the screening method of the present invention, these steps can be carried out illustratively, for example in the following manner. A polypeptide for tool use is expressed in a cell. The expressed said polypeptide is isolated and purified from said cell by an affinity purification method making use of its affinity for GST, Flag, His or the like tag, or an immunoprecipitation method or the like using an antibody which responds to the polypeptide for tool use (e.g., an anti-ATP5B antibody or a tag antibody). Subsequently, the purified said polypeptide are mixed to effect their contact, and then the substance which does not bind to said polypeptide is removed by a low molecular compound-adsorbing resin or the like. A complex of the remaining protein and substance to be tested is isolated, and then the low molecular substance in the complex is separated by denaturing said complex by an acid, heat or other stimulation, and the remained protein alone is removed. A substance which binds to the polypeptide for tool use is selected by identifying the substance contained in the same sample through the mass spectrometry analysis using a mass spectrometer. When concentration of the polypeptide for tool use is 1 μM or 10 μM in the method described in the above, a substance having preferably 5% or more, more preferably 10% or more, further preferably 50% or more, as the total amount of recovered said substances to be tested, under a condition of 1 μM as the adding concentration of the substances to be tested, in comparison with the control which does not go through the step of adhering and removing the substances to be tested, is selected as the substance which binds to the polypeptide for tool use.
In addition, a method in which a substance which binds to the polypeptide for tool use is selected making use of a conventionally known ELISA method, far western method, binding assay method or the like method, by applying a specified label to a group of substances to be tested is also included in the method of the present invention. As an illustrative example of such a case, the method of Example 5(4) can be cited. In such a method, a substance to be tested which can detect binding with the polypeptide for tool use at a concentration of preferably 10 μM or less, more preferably 1 μM or less, further preferably 0.1 μM or less, is selected as the substance which binds to polypeptide for tool use.
<Competitive Inhibition Screening Method which Uses a Polypeptide for Tool Use>
The screening method of the present invention which comprises a step of allowing a polypeptide for tool use and a substance to be tested to be in contact with each other in the coexistence of biguanide can be accomplished by measuring, in the step of detecting binding of the polypeptide for tool use and biguanide by an ELISA method, far western method, a binding assay or the like using a labeled or modified biguanide and said polypeptide, a change in said binding which occurs when a substance to be tested is allowed to be present at the same time.
Illustratively, various experimental means exemplified in the following are used. A polypeptide for tool use is expressed in a cell. The expressed polypeptide for tool use is isolated and purified from said cell by an affinity purification method making use of its affinity for a tag, or an immunoprecipitation method or the like using an antibody which responds to the polypeptide for tool use (e.g., an anti-ATP5B antibody or a tag antibody). Subsequently, the purified polypeptide is mixed a substance to be tested and biguanide, and the thus formed complex is isolated. Next, the substance to be tested and biguanide are again separated by denaturing said complex by an acid, heat or other stimulation, the remained protein alone is removed, and then whether or not the binding of the polypeptide for tool use and biguanide is inhibited by the substance to be tested is examined by examining whether or not a corresponding biguanide compound is contained in the same sample through the mass spectrometry analysis using a mass spectrometer. In addition, as another means, binding of the polypeptide for tool use and biguanide can be verified by a conventionally known ELISA method, far western method, binding assay or the like method using, as the probe, a labeled biguanide prepared by labeling a part of its molecular structure. Illustratively, for example, a labeled compound is prepared by substituting an element in the molecule of biguanide with a radioisotope. Making use of said labeled biguanide as the probe, binding of the purified polypeptide for tool use and biguanide is verified by a said polypeptide-immobilized ELISA method. Alternatively, polypeptides for tool use are separated by the conventionally known SDS acrylamide gel electrophoresis and transferred on a nitrocellulose film, and then binding of said polypeptide with biguanide is also verified by the far western method which uses the aforementioned labeled biguanide as the probe. Also alternatively, binding of a polypeptide for tool use and biguanide is verified by a so-called binding assay in which labeled biguanide and purified polypeptide for tool use are mixed and washed by trapping on a filter, and then total amount of the formed complex of the compound and peptide is detected by measuring the radiation dose derived from the labeled probe. The labeling of biguanide is not limited to a radioisotope, and it is possible to attain the purpose by modifying a part of its molecular structure within such a range that it does not exert influences upon its pharmacological activities. For example, it can be labeled by modifying through the biotinylation of a part of the molecular structure of phenformin, metformin or buformin. Also in this case, binding of the polypeptide for tool use and biguanide is verified by the ELISA or far western method in the same manner as described in the above making use of biotin and avidin and by the use of a labeled avidin antibody or the like. Preferably, binding of the polypeptide for tool use and biguanide is verified by the method of Example 5(4).
In the aforementioned step of verifying binding of the polypeptide for tool use and biguanide, whether or not the binding of said polypeptide and biguanide is inhibited is examined in the presence a substance to be tested together with biguanide. When the concentration of biguanide is 10 μM, a substance having an IC50 value of preferably 10 μM or less, more preferably a substance of 1 μM or less, further preferably a substance to be tested of 0.1 μM or less, is selected as an agent for treating diabetes. When binding of the polypeptide for tool use and biguanide is obstructed by the presence of a specific substance to be tested, it can be judged tat this phenomenon is a result of the competitive inhibition of the binding of biguanide and said polypeptide caused by the binding of said substance to be tested to the biguanide binding site in the molecule of the polypeptide for tool use. Regarding the verification that it is evidently competitive binding inhibition by the substance to be tested, it can be verified by examining stepwise obstruction of biguanide to be bound to said polypeptide, by carrying out stepwise changing of the ratio of concentrations of the biguanide and substance to be tested to be present at the same time. By the same method, a substance which binds to the polypeptide for tool use in competition with biguanide (namely an agent for treating diabetes) can be selected. More illustratively, a substance which binds to the polypeptide for tool use can be selected for example by the method shown in Example 6 which is described later.
<Binding Assay Screening Method which Uses a Cell for Tool Use>
Included in the screening method of the present invention, which comprises a step of allowing a cell for tool use and a substance to be tested to be in contact with each other, is a method in which a cell for tool use and a substance to be tested are allowed to be in contact with each other by mixing them (a step of contacting), a substance which directly binds to said polypeptide and acts thereon (namely an agent for treating diabetes) is analyzed by identifying, by a mass spectrometry, the compounds separated by denaturing a complex formed by the binding of said substance to be tested and polypeptide after its isolation (a step of analyzing the binding), and then a substance which binds to the polypeptide for tool use (namely an agent for treating diabetes) is selected. As the method for analyzing the binding, the aforementioned AS-MS method and the like can be used. Illustratively, it can be carried out for example in the following manner.
Firstly, a cell for tool use in which a polypeptide for tool use is expressed by transforming a cell with a vector comprising the polypeptide for tool use is allowed to be in contact with a substance to be tested. A complex of the polypeptide for tool use and the substance to be tested binding thereto is concentrated from the aforementioned cell by an affinity purification method making use of its affinity for GST, Flag, His or the like tag, or an immunoprecipitation method or the like using an antibody which responds to the polypeptide for tool use (e.g., an anti-ATP5B antibody or a tag antibody). It is desirable to contain the same substance to be tested used in the above for treating the cell, in the reaction liquid of this concentration step. Substances which do not bind to said polypeptide are removed by a low molecular compound-adsorbing resin or the like, and then the low molecular substance in the complex is separated by denaturing the thus obtained complex of the polypeptide for tool use and the substance to be tested binding thereto with an acid, heat or other stimulation, and the remained protein alone is removed. A substance which binds to the polypeptide for tool use is selected from substances to be tested by identifying the substances contained in the same sample through the mass spectrometry analysis using a mass spectrometer. Illustratively, under a condition of 1 μM in concentration of the test substance to be added, a substance having preferably 5% or more, more preferably 10% or more, further preferably 50% or more, as the total amount of recovered said substances to be tested, in comparison with the control which does not go through the step of adhering and removing the substances to be tested, is selected as the substance which binds to the polypeptide for tool use.
<Competitive Inhibition Screening Method which Uses a Cell for Tool Use>
By allowing a cell for tool use and a substance to be tested to be in contact with each other in the coexistence of biguanide, whether or not the binding of the polypeptide for tool use and biguanide is competitively inhibited by said substance to be tested and the substance to be tested and the polypeptide for tool use are bound with each other can be examined. Illustratively, a complex of the polypeptide of the present invention and the biguanide binding thereto is concentrated from a cell in the coexistence of biguanide, by the same process of the aforementioned <Binding assay screening method which uses a cell for tool use>, Subsequently, binding of the polypeptide for tool use and a substance to be tested is analyzed. Analysis of the binding can be carried out by the same method described in the aforementioned <Competitive inhibition screening method which uses a polypeptide for tool use>. For example, in the case of the use of biguanide modified with a label or the like, it can be carried out by determining binding of the polypeptide for tool use and biguanide using the conventionally known binding assay or the like method. When binding of the polypeptide for tool use and biguanide was obstructed by the presence of a substance to be tested, it can be judged that binding of biguanide and the polypeptide for tool use was competitively inhibited by the substance to be tested, and the polypeptide for tool use and the substance to be tested were bound to each other.
It is desirable that the screening method of the present invention further comprises a step, after analyzing binding of the polypeptide for tool use and a substance to be tested and selecting the substance which binds to the polypeptide for tool use, of verifying activation of AMPK by the selected substance to be tested, and/or a step of verifying its possession of diabetes-treating activity.
The step of verifying activation of AMPK by the selected substance can be carried out for example in the following manner. A cell expressing the polypeptide for tool use (e.g., HeLa S3 cell) is cultured after adding a substance to be tested or a vehicle control thereto. Using a cell lysate prepared by lysing the cultured cells, phosphorylation level (namely activation level) of intracellular AMPK is detected by conventionally known SDS electrophoresis and western blotting which uses an anti-phosphorylation AMPK antibody (e.g., Phospho-AMPK-α(Thr 172) Antibody, mfd. by Daiichi Pure Chemicals). Preferably, activation of AMPK by the selected substance can be verified by the method of Example 5(7). By comparing with the control, a substance which accelerates phosphorylation of AMPK is selected as a substance which activates AMPK. As the substance which accelerates phosphorylation of AMPK, a substance which accelerates phosphorylation of AMPK by a factor of 50%, preferably 70%, more preferably 90% or more, in comparison with the control is selected.
As the step of verifying possession of diabetes-treating activity by the selected substance, a step of carrying out a conventionally known evaluation method, for example a method for analyzing diabetes-treating effect of a selected substance as shown below, can be exemplified.
The presence or absence of the diabetes-treating effect is judged by continuously administering a compound selected by the screening method of the present invention to a diabetes model animal and verifying its hypoglycemic action on demand in accordance with the usual way, or by carrying out verification of hyperglycemia suppressing action after oral glucose tolerance test. Alternatively, its effect to treat type II diabetes is analyzed by measuring insulin resistance of human and using improvement of the value as the index. The insulin resistance in human is measured by mainly two methods. One is to measure blood glucose level and insulin concentration after fasting, and the other is a method so-called glucose tolerance test in which glucose liquid is orally administered and its clearance ratio from the blood circulation is checked. In addition, a euglycemic hyperinsulinemia clamp method can be exemplified as a more accurate method. This method is based on the theory that insulin and glucose in blood are maintained at certain concentrations, and periodically measures total amount of the administered glucose liquid and concentration of insulin used for its metabolism (“Tonyobyo (Diabetes)” O. Nakagawa 1999 vol. 42(2): pages 111-113). Possession of diabetes-treating activity by the selected substance can be verified preferably by the method described in Example 7.
Though not particularly limited, examples of the test substance to be used in the screening method of the present invention include commercially available compounds (including peptides), various conventionally known compounds (including peptides) registered in chemical files, a group of compounds obtained by the combinatorial chemistry techniques (N. Terrett et al., Drug Discov. Today, 4(1): 41, 1999), microbial culture supernatants, natural components derived from plants and marine organisms, animal tissue extracts, or compounds (including peptides) prepared by chemically or biologically modifying compounds (including peptides) selected by the screening method of the present invention.
<Pharmaceutical Composition for Diabetes Treatment Use, Method for Treating Diabetes and Use of a Substance for Producing the Pharmaceutical Composition for Diabetes Treatment Use>
A pharmaceutical composition for diabetes treatment use, which comprises a substance obtained by the screening method of the present invention [e.g., DNA, a protein (including antibody or antibody fragment), a peptide or a compound other than that] as the active ingredient, is included in the present invention. Also, a method for treating diabetes, which comprises administering a substance obtained by the screening method of the present invention at an effective dose to an object that requires treatment of diabetes, is included in the present invention. In addition, use of a substance obtained by the screening method of the present invention, for producing the pharmaceutical composition for diabetes treatment use, is included in the present invention.
The active ingredient in the pharmaceutical composition of the present invention can be selected by the screening method of the present invention. As the compound selected by the screening method of the present invention, the 2-[(E)-(1H-1,2,4-triazol-3-ylimino)methyl]phenol and 6-chloro-9H-purine-2-amine described in Examples 6(2) and 7 which are described later can be exemplified. In this connection, verification of the presence of diabetes-treating effect can be carried out by using a conventional method known to those skilled in the art or a modified method thereof (c.f., the aforementioned “step of verifying that the selected substance has diabetes-treating activity”).
Pharmaceutical preparations for diabetes treatment use, which comprise a substance obtained by the screening method of the present invention [e.g., DNA, a protein (including antibody or antibody fragment), a peptide or a compound other than that] as the active ingredient, can be prepared as pharmaceutical compositions in response to the type of the aforementioned active ingredient, using pharmacologically acceptable carriers, fillers and/or other additive agents which are generally used in preparing them.
As the administration, oral administration by tablets, pills, capsules, granules, fine subtilaes, powders, solutions for oral use or the like, or parenteral administration by injections for intravenous injection, intramuscular injection, intraarticular injection or the like, suppositories, percutaneous administration preparations, transmucosal administration preparations or the like can be exemplified. Particularly in the case of peptides which are digested in the stomach, intravenous injection or the like parenteral administration is desirable.
In the solid composition for use in the oral administration, one or more active substances can be mixed with at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinyl pyrrolidone, aluminum magnesium silicate or the like. In accordance with the usual way, the aforementioned composition may contain other additives than the inert diluent, such as a lubricant, a disintegrating agent, a stabilizing agent, a solubilizing or solubilization assisting agent or the like. As occasion demands, tablets or pills may be coated with a sugar coating or with a film of a gastric or enteric substance or the like.
The liquid composition for oral administration may include emulsions, solutions, suspensions, syrups, elixirs or the like and may contain a generally used inert diluent such as purified water or ethanol. In addition to the inert diluent, the aforementioned composition may contain other additive agent such as a moistening agent, a suspending agent, a sweetener, an aromatic or an antiseptic.
The injections for parenteral administration may include aseptic aqueous or non-aqueous solutions, suspensions or emulsions. The aqueous solutions or suspensions may include distilled water for injection, physiological saline or the like as a diluent. The non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, a plant oil (e.g., olive oil), an alcohol (e.g., ethanol), polysorbate 80 or the like as the diluent. The aforementioned composition may further contain a moistening agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilization assisting agent, an antiseptic or the like. The aforementioned compositions can be sterilized by filtration through a bacteria retaining filter, blending of a germicide or irradiation. Alternatively, they may be used by firstly producing sterile solid compositions and dissolving them in sterile water or other sterile medium for injection prior to their use.
The dose can be optionally decided by taking into consideration strength of the activity of the substance obtained by the screening method of the present invention, symptoms, age, sex and the like of each object to be administered.
For example, in the case of oral administration, the dose is generally from about 0.1 to 100 mg, preferably from 0.1 to 50 mg, per day per adult (as 60 kg in body weight). In the case of parenteral administration in the form of injections, it is from 0.01 to 50 mg, preferably from 0.01 to 10 mg.
The following describes the present invention in detail based on examples, but the present invention is not restricted by said examples. In this connection, unless otherwise noted, they can be carried out in accordance with the conventionally known methods (“Molecular Cloning” Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989, and the like). Also, when commercially available reagents and kits are used, they can be carried out in accordance with the instructions attached to the articles on the market.
(1) Cloning of Molecular Chaperone Gene and Preparation of GST Fusion Molecular Chaperone Protein Expression Plasmid
Using oligonucleotides represented by SEQ ID NOs:28 to 77 (even numbers for 5′ side, odd numbers for 3′ side) as primers (e.g., a primer set for human HSPA1A by SEQ ID NO:28 and SEQ ID NO:29, the same shall apply hereinafter), cDNA sequences coding for the complete length regions of 25 species of chaperone proteins (human HSPA1A, human HSPH1, human HSPCA, human HSPD1, human DNAJA1, human HSPB1, human HSPE1, human HSPA4, human HSP90B1, human CCT6B, human TCP1, human HSPA14, human HSPA9B, human STCH, human HYOU1, human HSPB5, human HSPB2, human DNAJA2, human DNAJB1, human DNAJB2, human HCG3, human DNAJB11, human DNAJC11, human DNAJC7, human DNAJC6) represented by the amino acid sequences of 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 27 were prepared by carrying out PCR using a DNA polymerase (Pyrobest DNA Polymerase, Takara Shuzo) under conditions of 3 minutes of thermal denaturation reaction at 95° C., subsequent 35 repetition of a cycle consisting of 10 seconds at 98° C., 30 seconds at 60° C. and 1 minute and 30 seconds at 74° C. and final heating at 74° C. for 7 minutes, using respective templates; a human liver-derived cDNA library (Clontech) for HSPH1, HSPE1, HSP90B1, HSPA9B and DNAJC11, a human grain-derived cDNA library (Clontech) for HSPCA, HSPA1A and HSPD1, a HeLa cell-derived cDNA library (Clontech) for DNAJA1 and HSPA4, a human mammary gland-derived cDNA library (Clontech) for HSPB1, a human skeletal muscle-derived cDNA library (Clontech) for TCP1, HSPA14, HYOU1 and HSPB2, and commercially available cDNA samples (Ultimate TM ORF Clones, Invitrogen) for CCT6B, STCH, HSPB5, DNAJA2, DNAJB1, DNAJB2, HCG3, DNAJB11, human DNAJC7 and human DNAJC6. The thereby formed DNA fragments of about 2.58 kbp (HSPH1), 2.2 kbp (HSPCA), 1.93 kbp (HSPA1A), 1.72 kbp (HSPD1), 1.19 kbp (DNAJA1), 0.62 kbp (HSPB1), 0.3 kbp (HSPE1), 2.52 kbp (HSPA4), 2.41 kbp (HSP90B1), 2.04 kbp (HSPA9B), 1.69 kbp (DNAJC11), 1.67 kbp (TCP1), 1.05 kbp (HSPA14), 3.0 kbp (HYOU1), 0.53 kbp (HSPB2), 1.59 kbp (CCT6B), 1.42 kbp (STCH), 0.53 kbp (HSPB5), 1.24 kbp (DNAJA2), 1.02 kbp (DNAJB1), 0.98 kbp (DNAJB2), 0.44 kbp (HCG3), 1.077 kbp (DNAJB11), 1.46 kbp (human DNAJC7) and 2.74 kbp (DNAJC6) were obtained. From the sequences contained in the primers, each of the cDNA samples was designed in such a manner that the following restriction enzyme sites are added to both termini thereof. BglII site and XhoI site are added in the case of the cDNA of HSPH1, and EcoRV site and NotI site in the case of the cDNA of HSPA4, EcoRI site and XhoI site in the case of the cDNA of STCH, HSPB5, DNAJC6 and DNAJC11, BglII site and NotI site in the case of the cDNA of TCP1, and BamHI site and NotI site in the case of the cDNA of DNAJA2. In the case of other cDNA molecules, they were designed such that BglII site and XhoI site are added. In order to insert these cDNA molecules into a GST fusion expression vector pGEX-6P-1 (Amersham Bioscience), each of the cDNA fragments obtained by the aforementioned PCR reaction was respectively digested at the respectively added restriction enzyme sites. 1) In the case of the cDNA digested with BamHI (or BglII) and XhoI, the vector was used by making it into a linear form through its digestion with the restriction enzymes BamHI and XhoI, and 2) in the case of the cDNA digested with restriction enzymes EcoRV and NotI, the vector was used by treating it with the restriction enzymes SmaI and NotI, 3) in the case of the cDNA fragment digested with restriction enzymes EcoRI and XhoI, the vector was used by treating it with the restriction enzymes EcoRI and XhoI, and 4) in the case of the cDNA fragment digested with restriction enzymes BamHI and NotI, the vector was used by treating it with the restriction enzymes BamHI and NotI, respectively. A mixture of each of the restriction enzyme-treated chaperone cDNA fragments and the vector was mixed with a DNA ligase liquid (DNA ligation kit II; Takara Shuzo) and treated at 16° C. for 3 hours, thereby preparing a plasmid in which each chaperone cDNA was inserted into the multi cloning site of pGEX-6P-1. By carrying out determination of nucleotide sequences using the oligonucleotide shown in SEQ ID NO:86 as the primer and using a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems), those which are the reported respective nucleotide sequences (RefSeq accession numbers NM—005345, NM—006644, NM—005348, 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—016306, NM—018198, NM—003315, NM—014787) and into which the coding region of each molecular chaperone cDNA and the GST tag translation frame of pGEX vector were simultaneously inserted were respectively selected.
(2) Purification of GST Fusion Molecular Chaperone Protein
A group of GST fusion expression plasmids obtained in the aforementioned (1) prepared by cloning 25 species of molecular chaperone were respectively transferred into Escherichia coli BL 21 (Takara Bio) through its transformation by a heat shock method. After overnight shaking culture using 2.4 ml of a culture liquid, its entire volume was transferred into 400 ml of the culture liquid and cultured at 37° C. for 3 hours on a shaker, and then IPTG (Sigma) was added thereto to a final concentration of 2.5 mM and the shaking culture was further carried out for 3 hours to induce respective GST fusion chaperone proteins (hereinafter, respectively referred to as GST-HSPA1A (ca. 96 kDa), GST-HSPH1 (ca. 123 kDa), GST-HSPCA (ca. 111 kDa), GST-HSPD1 (ca. 87 kDa), GST-DNAJA1 (ca. 71 kDa), GST-HSPB1 (ca. 59 kDa), GST-HSPE1 (ca. 37 kDa), GST-HSPA4 (ca. 120 kDa), GST-HSP90B1 (ca. 118 kDa), GST-HSPA9B (ca. 100 kDa), GST-DNAJC11 (ca. 89 kDa), GST-TCP1 (ca. 86 kDa), GST-HSPA14 (ca. 81 kDa), GST-HYOU1 (ca. 137 kDa), GST-HSPB2 (ca. 46 kDa), GST-CCT6B (ca. 84 kDa), GST-STCH (ca. 78 kDa), GST-HSPB5 (ca. 46 kDa), GST-DNAJA2 (ca. 72 kDa), GST-DNAJB1 (ca. 64 kDa), GST-DNAJB2 (ca. 62 kDa), GST-HCG3 (ca. 43 kDa), GST-DNAJB11 (ca. 67 kDa), GST-DNAJC7 (ca. 81 kDa) and GST-DNAJC6 (ca. 125 kDa)) (parenthesized numbers are respectively expected molecular weights). The cells were recovered, and each GST fusion molecular chaperone protein was purified on glutathione Sepharose beads in accordance with the conventionally known GST pull-down method. As the control, a protein of the GST tag moiety alone (to be referred to as GST protein hereinafter; expected molecular weight about 26 kDa) was expression-induced from the Escherichia coli BL 21 transformed with pGEX-6P-1 and purified, in the same manner as described in the above. By carrying out separation by SDS polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining in accordance with the conventionally known methods, it was confirmed that respective proteins having expected molecular weight were purified.
(1) Cloning of Estrogen Receptor Gene and Preparation of V5 Tag Fusion Estrogen Receptor Expression Plasmid
A gene cDNA coding for the complete length region of estrogen receptor α (to be referred to as ERα hereinafter) as a species of the steroid hormone receptors shown by a conventionally known data base was cloned using two species of DNA primers (SEQ ID NO:78 and SEQ ID NO:79) designed in accordance with the sequence shown by the RefSeq accession number NM—000125. Illustratively, a DNA fragment of about 1.78 kbp coding for the complete length region of ERα was amplified by carrying out PCR using a primer set of SEQ ID NO:78 and SEQ ID NO:79 and using a HeLa cell-derived cDNA library (Clontech) as the template. The PCR reaction was carried out at 98° C. (1 minute) and subsequent 35 repetition of a cycle consisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5 minutes), using a DNA polymerase (Pyrobest DNA Polymerase; Takara Shuzo). The thus obtained DNA fragment was sub-cloned into an expression vector (pcDNA3.1/V5-His-TOPO; Invitrogen) using TOPO TA Cloning System (Invitrogen). The primer represented by SEQ ID NO:79 was designed in such a manner that a vector-derived V5 epitope (derived from the V protein of paramyxovirus SV5, Southern J A, J. Gen. Virol. 72, 1551-1557, 1991) and a His 6 tag (lindner P, BioTechniques 22, 140-149, 1997), excluding the stop codon, are continued in the same frame of ERα gene triplet on the 3′ side after the cloning. Nucleotide sequence of the inserted DNA fragment in the thus obtained plasmid was determined using primers which bind to the T 7 promoter region on the vector (TOPO TA Cloning kit; Invitrogen; SEQ ID NO:89) and a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer; Applied Biosystems). As a result, it was confirmed that the cDNA coding for the human ERα shown by the RefSeq accession number NM—000125 is inserted the aforementioned expression vector pcDNA3.1N5-His-TOPO. Hereinafter, this expression plasmid is referred to as pcDNA-ER.
(2) Preparation of Human Estrogen Receptor Expression Cell and Detection of 17-β Estradiol Target Protein by the Identification Method of the Present Invention
Using Lipofect amine 2000 reagent (Invitrogen), the aforementioned pcDNA-ER was transiently transferred into COS-7 cell (ATCC) cultured on a 10 cm plate to a 70% confluent state. After 30 hours of culturing and subsequent removal of the medium, the cells were washed with ice-cooled PBS and then lysed by adding 1.0 ml of a buffer A (50 mM Tris-HCl (pH 7.5), 10% glycerol, 120 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 0.5% NP-40). This cell extract was centrifuged at 1500 rpm for 5 minutes to remove the precipitate, and soluble fraction of the supernatant (hereinafter, ER expression cell extract) was collected. ERα known as a target protein which shows the drug effect of 17-β estradiol (to be referred to as E2 hereinafter) (Green S. & Chambon P. Trends Genet. 1988 November; 4(11): p. 309-314) is contained in the soluble fraction of this cell extract. In addition, as a control, a cell extract soluble fraction of un-transferred COS-7 cell (hereinafter, control COS-7 cell extract) was prepared in the same manner. Whether or not a target protein, ER, can be actually detected by the identification method of the present invention using a chaperone protein, HSPA4 protein, was examined. A 1 μg portion of GST-HSPA4 protein or GST protein (prepared in the aforementioned Example 1(2)) purified on the glutathione Sepharose beads was mixed with the aforementioned ER expression cell extract or control COS-7 cell extract and shaken at 4° C. for 1 hour under a condition of adding or not adding 10 μM of E2. Thereafter, the protein binding to the GST-HSPA4 or GST protein on the beads was co-precipitated by centrifugation. This was suspended in the aforementioned buffer A′ (a buffer prepared by changing NaCl concentration in the buffer A to 100 mM) and again co-precipitated by centrifugation. After repeating this operation 4 times, proteins in the precipitate were separated by SDS polyacrylamide gel electrophoresis in accordance with a conventionally known method, and the amounts of ERα as the target protein of E2 were compared by western blotting using an anti-ERα antibody (MC-20; Santa Cruz). As a result (
(3) Detection of Target Protein of 17-β Estradiol by the Identification Method of the Present Invention
In the Example 2(2) described in the above, it was proved that detection of the target protein of an agent to be tested can be detected by comparing the protein which binds to a molecular chaperone protein at the time of adding or not adding said agent to be tested, using the molecular chaperone protein. In the following example, it was confirmed that the identification method of the present invention is useful even when molecular chaperones other than the one used in the Example 2(2) are used.
That is, in this Example (3), whether or not the target protein ER can be detected in reality by the identification method of the present invention in the same manner as in the Example 2(2) was examined using the 25 species of chaperone proteins of Example 1(2). The test was carried out in the same manner as in the Example 2(2), except that a mixture in which the aforementioned 25 species of GST fusion proteins (Example 1(2)) were divided into 3 groups 8 or 9 species in accordance with their molecular weight and mixed in 0.2 μg portions, instead of the use of HSPA4 protein as the chaperone protein, or 1.4 μg of GST protein was used, and that western blotting was carried out using an anti-V5 antibody (Invitrogen)which recognizes the V5 epitope fused to ER, and signal strengths of the thus obtained bands were converted into numerals as measured values of the density per unit area using VersaDoc Imaging System (Bio-Rad). Details of the mixed 8 or 9 species of GST fusion chaperone proteins are 3 groups; a group in which a group of chaperons having relatively high molecular weights, GST-HSPH1, GST-HSPA4, GST-HSPCA, GST-HYOU1, GST-DNAJC6, GST-HSP90B1, GST-HSPA9B and GST-DNAJC11, were mixed, a group in which a group of chaperons having medium degree of molecular weights, GST-HSPA1A, GST-HSPD1, GST-DNAJA1, GST-TCP1, GST-CCT6B, GST-HSPA14, GST-DNAJC7 and GST-STCH, were mixed, and a group in which a group of chaperons having relatively low molecular weights, GST-HSPB1, GST-HSPE1, GST-DNAJA2, GST-DNAJB11, GST-DNAJB1, GST-DNAJB2, GST-HSPB2, GST-HSPB5 and GST-HCG3, were mixed. As a result (Table 1), in each case of the use of the chaperon protein mixture groups, an ERα band of about 70 kDa, which is not detected when the control COS-7 cell extract is used, was detected from the ER expression cell extract, under the E2 un-addition condition, and the band of ERα was detected only slightly under the E2-added condition. The ER(L), ER(M) and ER(H) in the table show the results of using the aforementioned a group of chaperons having relatively low molecular weights, a group of chaperons having medium degree of molecular weights and a group of chaperons having relatively high molecular weights, respectively.
Based on these results, it was confirmed that detection of the target protein of an agent to be tested can be detected by comparing the protein at the time of adding or not adding said agent to be tested, even when a large variety of molecular chaperone proteins are simultaneously used.
The following shows that it was able to identify various target proteins of respective agents by the identification method of the present invention using various molecular chaperones, in Example 3, Example 4 and Example 8. Regarding those which were not described in detail in each Example, tests were carried out in accordance with the aforementioned Example 2. In this connection, signal strengths of the bands obtained by the western blotting were converted into numerals as measured values of the density per unit area using VersaDoc Imaging System (Bio-Rad).
It is known that FKBP 12 is one of the target proteins of FK 506 and FK 1706 (J. Biol. Chem. 1993 Nov. 5; 268(31): 22992-22999, Eur. J. Pharmacol. 2005 Feb. 10; 509(1): 11-19).
It is known that glucocorticoid receptor (hereinafter GR) as a species of human steroid hormone receptors is a target protein which shows the drug effect of dexamethasone (J. Clin. Invest. 1995 June; 95(6): 2435-2441).
It is known that human dehydrofolate reductase (to be referred to as DHFR hereinafter) is a target protein which shows the drug effect of methotrexate (hereinafter MTX) (J. Med. Chem. 2000 Oct. 19; 43(21): 3852-3861).
Androgen receptor (hereinafter AR) which is known as the target protein of 5a-dihydrotestosterone (hereinafter DHT) (J. Steroid Biochem. Mol. Biol. 1993 December; 46(6): 699-711) is contained in the soluble fraction of a human prostate-derived cell, LNCaP cell (ATCC).
Mineralcorticoid receptor (hereinafter MR) which is known as the target protein of aldosterone (Science 1987 Jul. 17; 237(4812): 268-275) is contained in the soluble fraction of a human blood vessel-derived cell, HUVEC (ATCC).
By preparing the following respective cell extract soluble fractions, whether or not the aforementioned target proteins of respective agents which are present in said cell extract soluble fractions can be detected by the identification method of the present invention using respective chaperone protein groups. In this connection, among the agents, FK 506 (JP-B-03-038276) and FK 1706 (European Patent No. 346427) were synthesized, and other agents were used by purchasing from Sigma. Each of the bands of FKBP 12, GR and DHFR was detected by the western blotting using an antibody which recognizes the V tag added to respective target proteins in the same manner as in the aforementioned Example 2(3), and the bands of AR and MR by the western blotting using commercially available antibodies (N-20 and C-19, Santa Cruz).
(a) Cell extract soluble fraction of HeLa S3 cell (ATCC) in which a gene cDNA coding for the complete length region of human FKBP 12 (RefSeq accession number NM—054014) was over-expressed (to be referred to as FKBP 12 expression cell extract hereinafter)/a mixed group of respective proteins of chaperone proteins; GST-HSPH1, GST-HSPA4, GST-HSPCA, GST-HYOU1, GST-DNAJC6, GST-HSP90B1, GST-HSPA9B, GST-HSPA1A and GST-DNAJC11
(b) Cell extract soluble fraction of HeLa S3 cell in which a gene cDNA coding for the complete length region of GR (RefSeq accession number NM—001024094) was over-expressed (to be referred to as GR expression cell extract hereinafter)/a mixed group of respective proteins of chaperone proteins; GST-HSPA1A, GST-HSPH1, GST-HSPCA and GST-HSPA4
(c) Cell extract soluble fraction of HeLa S3 cell in which a gene cDNA coding for the complete length region of DHFR (RefSeq accession number NM—000791) was over-expressed (to be referred to as DHFR expression cell extract hereinafter)/a mixed group of respective proteins of chaperone proteins; GST-HSPD1, GST-DNAJA1, GST-HSPB1 and GST-HSPE1
(d) LNCaP soluble fraction/a mixed group of respective proteins of chaperone proteins; GST-HSPA1A, GST-HSPH1, GST-HSPCA and GST-HSPA4
(e) HUVEC soluble fraction/a mixed group of respective proteins of chaperone proteins; GST-HSPA1A, GST-HSPH1, GST-HSPCA and GST-HSPA4
(f) Cell extract soluble fraction of HeLa cell (hereinafter, control HeLa cell extract; control of the aforementioned (a) to (c))
As a result (Table 1), when the FKBP 12 expression cell extract, GR expression cell extract and DHFR expression cell extract were used, the bands of FKBP 12, GR and DHFR which are not detected when the control HeLa cell extract is used was detected more distinctively under added condition of each agent, in comparison with the un-added condition. In addition, the band of AR was detected more distinctively under the un-added condition of DHT, and the band of MR was detected more distinctively under the added condition of aldosterone. Based on these results, it was confirmed similar to the case of Example 2 that target protein of agents to be tested can be detected by comparing proteins binding to molecular chaperone proteins at the time of adding or not adding said agents to be tested, by using various molecular chaperone proteins.
Using the aforementioned method shown in Example 2(2), attempt was made to screen the target protein of biguanide whose direct target protein has been unclear in spite of its significant drug effect as an agent for treating diabetes. Firstly, a rat skeletal muscle-derived cell, L 6 cell (ATCC), was suspended in a minimum essential medium (αMEM, Invitrogen) containing 10% fetal calf serum (FCS) and cultured on a collagen-coated plate of 15 cm in diameter (Asahi Techno Glass) until reaching the confluent state. The resulting cells were washed twice with 15 ml of ice-cooled phosphate buffer (PBS) and then lysed by adding 2.0 ml of the aforementioned buffer A, and the cell extract was collected using a scraper. This cell extract was centrifuged at 1500 rpm for 5 minutes to remove the precipitate, and the supernatant soluble fraction was collected. Phenformin (Sigma) which is a species of biguanide and whose hypoglycemic action has been clinically recognized (UK Prospective Diabetes Study (UKPDS) Group: Lancet, 352, 854 (1998)) was added to the soluble fraction of this cell extract to a final concentration of 50 nM, or not added thereto, and under such a condition, the pull-down test was carried out by adding 1 g of the GST-HSPA4 protein purified on the glutathione Sepharose beads in the same manner as in the aforementioned Example 2(2). After 1 hour of shaking at 4° C., the protein binding to the GST-HSPA4 on beads was co-precipitated by centrifugation. In this connection, in order to prevent drop out of the molecular chaperone protein from the beads, the molecular chaperone protein and beads were used by chemically crosslinking them in advance by a conventionally known method. Illustratively, the GST-HSPA4 protein purified on the glutathione Sepharose beads was washed with 0.2M sodium borate solution and then treated at 4° C. for 45 minutes by suspending in 20 mM DMP. After washing this with 0.2 M monoethanolamine solution to stop the reaction, this was washed by adding 20 mM glutathione solution to remove un-crosslinked molecular chaperone protein, and the resulting substance was used as the probe. After co-precipitating the protein binding to the GST-HSPA4, this was suspended in the aforementioned buffer A′ to which 50 nM in final concentration of phenformin was added or not added and again co-precipitated by centrifugation. After repeating this operation 4 times, proteins in the precipitate were separated by SDS polyacrylamide gel electrophoresis in accordance with a conventionally known method, and the proteins were detected by a conventionally known silver staining method. As a result, a large protein band of about 60 kDa, in which its amount to bind to the molecular chaperone protein is reduced at the time of adding phenformin in comparison with the case of no addition, was detected. It is considered that said protein is one of the target protein of phenformin, whose tertiary structure is changed by the addition of phenformin. Thus, this band was cut out, the protein was digested into fragments using trypsin, and the thus formed peptide mixture was recovered from the gel to carry out identification of the protein by mass spectrum analysis in accordance with the conventionally known method (Schevchenko et al., Analytical Chemistry, Vol. 68, pages 850 to 858, 1996). As a result, it was revealed that the protein in said band is ATP5B (RefSeq accession number NP 599191).
(1) Cloning of Human ATP5B Gene and Preparation of Human ATP5B Expression Plasmid
Firstly, cloning of human ATP5B gene was carried out. By synthesizing the primers represented by SEQ ID NO:80 and SEQ ID NO:81, and using said primers, an attempt was made to amplify complete length cDNA of human ATP5B from a human skeletal muscle-derived cDNA library (Clontech) by PCR. The PCR reaction was carried out using a DNA polymerase (TAKARA LA Taq; Takara Shuzo), by heating at 94° C. (3 minutes) and then repeating 35 times of a cycle consisting of 94° C. (30 seconds), 58° C. (1.5 minutes) and 72° C. (4 minutes), and PCR was again carried out using the PCR product as the template under the same conditions. As a result of separating the PCR product by an agarose gel electrophoresis, it was confirmed that a DNA fragment of about 1600 base pairs was amplified. Accordingly, the same DNA fragment in the reaction liquid was cloned into an expression vector (pcDNA3.1N5-His-TOPO; Invitrogen) using TOPO TA Cloning System (Invitrogen). The primer used in this case, represented by SEQ ID NO:81, was designed for excluding the stop codon sequence of human ATP5B so that a vector-derived V5 epitope (derived from the V protein of paramyxovirus SVS, Southern J A, J. Gen. Virol. 72, 1551-1557, 1991) is continued to the human ATPSB gene triplet by the same frame on the 3′ side after the cloning. Nucleotide sequence of the inserted DNA fragment in the thus obtained plasmid was determined using primers which bind to the T7 promoter region on the vector (TOPO TA Cloning kit; Invitrogen; SEQ ID NO:89) and a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer; Applied Biosystems). As a result, it was confirmed that it is a clone which comprises the complete length cDNA sequence for human ATPSB shown by SEQ ID NO:1. Hereinafter, this expression plasmid is referred to as pcDNA-ATPSB.
It was inspected by the following test that the ATP5B, which was found by the identification method of the present invention and considered to be a target protein of biguanide, is the true target protein which produces the pharmacological action (principal effect) of biguanide. In this connection, since all of the conventional methods for screening target proteins of compounds use only direct binding of a compound and a protein as the index, a large number of proteins capable of binding to said compound are obtained, but the probability of finding a protein actually concerned in the drug effect of the compound is low in general. Different from the conventional methods, the method of the present invention aims at finding out the target of a compound using a tertiary structural change of a protein to which the compound is bound as the index, so that it can be necessarily expected that the thus found protein is not a simple binding protein of the compound but a protein molecule whose function is greatly changed by said compound.
(2) Cloning of DHFR Gene and Preparation of GST Fusion Type DHFR Expression Plasmid
The DHFR described in the aforementioned Example 3 is a protein whose ability to bind to MTX is known (Proc. Natl. Acad. Sci. USA, vol. 87, no. 8: pp. 2955-2959, 1990). In order to use in the following example as the tool for detecting biochemical binding of ATP5B and biguanide, complete length cDNA of human DHFR gene (RefSeq accession number NM—000791) was cloned under the same conditions of Example 2(1) using a human lymphocyte cDNA library (Clontech) as the template and using primers of the nucleotide sequences represented by SEQ ID NO:84 and SEQ ID NO:85. After cloning the thus obtained DHFR cDNA fragment of about 560 base pairs into pcDNA3.1/V5-His-TOPO, the DHFR cDNA fragment was cut out making use of the restriction enzyme sites BamHI and XhoI added to both termini of the aforementioned primers. At the same time, a GST fusion protein expression vector pGEX-6P-1 (Amersham) was converted into a straight chain form by digesting it with the restriction enzymes BamHI and XhoI. A mixture of both of them was mixed with a DNA ligase liquid (DNA ligation kit II; Takara Shuzo) and treated at 16° C. for 3 hours, thereby preparing a plasmid in which the DHFR cDNA is inserted into the multi cloning site of pGEX-6P-1 (to be referred to as pGEX-DHFR hereinafter). By carrying out determination of nucleotide sequence in the same manner as in the aforementioned example using the oligonucleotide shown in SEQ ID NO:86 as the primer, a plasmid into which the coding region of DHFR cDNA and the GST tag translation frame of pGEX vector were simultaneously inserted was selected.
(3) Expression and Purification of GST Fusion Type DHFR Protein
The pGEX-DHFR prepared in the Example 5(2) described in the above was transferred into Escherichia coli BL 21 through its transformation by a heat shock method. After overnight shaking culture using 2.4 ml of a culture liquid, its entire volume was transferred into 400 ml of the culture liquid and cultured at 37° C. for 3 hours on a shaker, and then IPTG (Sigma) was added thereto to a final concentration of 2.5 mM and the shaking culture was further carried out for 3 hours to induce expression of a GST fusion DHFR protein (to be referred to as GST-DHFR hereinafter). The cells were recovered, and the GST-DHFR was purified on glutathione Sepharose bawds in accordance with the conventionally known GST pull-down method. As the control, a protein of the GST tag moiety alone (to be referred to as GST protein hereinafter) was expression-induced from the Escherichia coli BL 21 transformed with pGEX-6P-1 and purified, in the same manner as described in the above. By carrying out separation by SDS polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining in accordance with the conventionally known methods, it was confirmed that the protein having expected molecular weight (GST-DHFR; 45 kDa, GST protein; 26 kDa) was purified.
(4) Biochemical Binding of Biguanide and ATP5B
Making use of the GST-DHFR protein prepared in the Example 5(3) described in the above, the presence or absence of biochemical binding of biguanide and ATP5B protein was examined. Illustratively, making use of the property of DHFR to bind to methotrexate (MTX), a compound in which methotrexate was fused to a part of the molecular structure of a biguanide species phenformin, (2S)-5-[(3-{[{[amino(imino)methyl]amino}(imino)methyl]amino}propyl)amino]-2-({4-[[(2,4-diaminopteridin-6-yl)methyl](methyl)amino]benzoyl}amino)-5-oxopentanonic acid dihydrochloride (to be referred to as MTX-phenformin hereinafter), was prepared, this compound was fixed to the GST-DHFR purified on glutathione Sepharose beads by binding to its MTX moiety, and the presence or absence of the binding with ATP5B on the protruding phenformin side was verified by the GST pull-down method.
Firstly, the MTX-phenformin was prepared using conventionally known organic synthesis techniques in accordance with the following reaction scheme.
Subsequently, binding of phenformin and ATP5B was examined by the GST pull-down method using the aforementioned purified MTX-phenformin and GST-DHFR protein. Firstly, using 1.0 ng of the aforementioned pcDNA-ATP5B prepared in Example 5(1) as the template, radioisotope-labeled human ATP5B protein was prepared by in vitro transcription and translation by mixing 40 μl of a TNT system (TNTR Quick Coupled Transcription/Translation System; Promega) and 0.74 MBq of a radioisotope (redivue Pro-mix L-[35S]; Amersham) and in accordance with the protocols attached thereto. A 15 μl portion of this human ATP5B protein preparation liquid and 1 μg for each of the GST protein or GST-DHFR, purified on the glutathione Sepharose beads, were mixed and shaken at 4° C. for 1 hour after adding 0.3 ml of buffer A thereto. Thereafter, the protein binding to the GST protein or GST-DHFR on beads was co-precipitated by centrifugation. After repeating this operation 4 times, proteins in the precipitate were separated by SDS polyacrylamide gel electrophoresis in accordance with a conventionally known method, and the protein bound to the probe was detected by an autoradiography. In all steps of this test, 10 μM in final concentration of the aforementioned MTX-phenformin was added or not added to the buffer, and the results were compared. As a result, as shown in
(5) Prediction of Biguanide Binding Site Existing on the ATP5B Molecule Surface
From the results of the aforementioned Example 5(4), it was confirmed that the human ATP5B protein as one of the polypeptides for tool use directly binds to phenformin. Accordingly, whether or not a keyhole structure to which biguanide can bind is present on the surface of ATP5B protein molecule was examined on a computer based on the data of X-ray crystalline structure analysis of bovine ATP5B. Illustratively, prediction was carried out about a region (site) which is judged that binding of biguanide is possible, on the protein surface of ATP5B as one of the subunits of F1F0-ATP synthase (F1 indicates extramembrane polymer, and F0 indicates intramembrane polymer).
The crystal structure data were obtained from “RCSB Protein Data Bank” as an ID of 1BMF. Heptamer molecules (3 α subunits (A, B and C chains), 3 β subunits (D, E and F chains) and 1 γ subunit (G chain)) of bovine mitochondrial F1-ATP synthase (bovine mitochondrial F1-ATPase) are contained in the 1BMF. ATP5B is β subunit of F1-ATPase as the extracellular region of F1F0-ATP synthase (Nature, 1997; 386: 299-302, Nature, 1994; 370 (6491): 621-628), and the bovine ATP5B has 99% of identity with human ATP5B in terms of amino acid residues (human ATP5B has 529 residues and bovine ATP5B has 482 residues, and human ATP5B is longer by 46 residues in the N-terminal and by 1 residue in the C-terminal). Among the heptamer molecules of the aforementioned bovine mitochondrial F1-ATP synthase, only the D chain which is bovine ATP5B and one of the β subunits and the A chain and C chain as α subunits adjacent thereto (to be referred to as complex αβα hereinafter) were used in the analysis. Ph4Dock which is a function prepared in the MOE (molecular operating environment) software of Chemical Computing Group Inc. (CCG) was used in the analysis. The Ph4Dock is a function to automatically screen a stable complex structure including its binding position making use of a computer, by merely providing a tertiary structure of a ligand and a receptor (J. Med. Chem. 2004 Dec. 30; 47 (27): 6804-11). By providing structural information of metformin and phenformin which are species of biguanide and structural information of the complex αβα making use of this function, sites on the complex αβα, to which these compounds bind, are screened. The results of carrying out screening of the sites on the surface of bovine ATP5B protein molecule, which render possible formation of a stable complex body through the binding of each of metformin and phenformin and the complex αβα, are shown in the following Table 2.
Table 2 shows the sites to which metformin and phenformin are respectively bound and the energy values on the binding at that time (U_ele+U_vdw). The site in the table indicates binding site, and the binding sites are numbered in order of larger and higher hydrophobic nature for the sake of convenience. The U_ele represents electrostatic interaction energy, and the U_vdw van der Waals energy, respectively, and 20 candidates were arranged and inscribed in order of smaller values of U_ele-+U_vdw. In the molecular mechanics calculation, mmff94s force field was used. In this case, when it is assumed that metformin and phenformin use the same molecule as the medicinal target, the binding sites to which metformin and phenformin bind in common are only the site 20 and site 29. In addition, since the human ATP5B prepared in the aforementioned Example 5(4) by carrying out in vitro transcription and translation bound to phenformin, it can be seen that ATP5B can bind to biguanide, not only under a state of complex but also by itself alone. Thus, when it is limited to a site where ATP5B by itself forms a binding site, the site 20 alone remains as the candidate. Based on the above, it was predicted that metformin and phenformin bind to the site 20 of the complex αβα.
In order to inspect the prediction described in the above, namely direct binding of phenformin to the site 20, it may be effected by finding the most important residue for the binding of phenformin, and experimentally showing that the binding ability disappears when the residue is mutated. Accordingly, when binding of phenformin and site 20 was observed, the presence of 3 patterns of binding mode was predicted as a result. It was found that a cooperative molecular recognition is carried out in each of these binding modes by 3 residues of Glu 125, Glu 241 and Asp 245 (each numeral shows position of the amino acid residue counting from the amino-terminal side of bovine ATP5B molecule) as amino acid residues in the ATP5B molecule. Accordingly, a mutation type human ATP5B was actually prepared in accordance with the method shown in the following example, by respectively substituting Glu 175 and Asp 295 on the human ATP5B molecule, which corresponds to the Glu 125 and Asp 245 among the aforementioned 3 amino acid residues of bovine ATP5B, and its ability to bind to phenformin was examined.
(6) Preparation of Mutation Type Human ATP5B Expression Plasmid and Inspection of Biguanide Binding Ability of Mutation Type Human ATP5B
Using the aforementioned expression plasmid pcDNA-ATP5B as the template and using respective sets of primers of the nucleotide sequences represented by SEQ ID NO:87 and SEQ ID NO:81, and SEQ ID NO:80 and SEQ ID NO:88, two DNA fragments of about 530 base pairs of 5′-side and about 1080 base pairs of 3′-side of cDNA having a mutation in which the 175th position of amino acid, Glu, of human ATP5B was replaced by Val were amplified by PCR. All of the PCR reaction, cloning stem and determination of nucleotide sequence were the same as the methods shown in Example 5(1). Using an equivalent mixture of the thus obtained two PCR products as the template, PCR was carried out using the primers represented by SEQ ID NO:80 and SEQ ID NO:81 this time, thereby obtaining a complete length cDNA of about 1600 base pairs coding for a Glu 175 Val mutation type ATP5B. The thus obtained cDNA fragment was cloned into an expression vector (pcDNA3.1/V5-His-TOPO; Invitrogen), and then the nucleotide sequence inserted into the vector was identified using the synthetic primer represented by SEQ ID NO:90 to confirm that the Glu 175 Val mutation was formed in ATP5B. The thus obtained expression plasmid is referred to as pcDNA-ATP5B(E175V) hereinafter. Next, using respective sets of primers of the nucleotide sequences represented by SEQ ID NO:91 and SEQ ID NO:81, and SEQ ID NO:80 and SEQ ID NO:92, a complete length cDNA coding for an Asp 295 Val mutation type ATP5B was obtained by the same method, and an expression vector pcDNA-ATP5B(D295V) was prepared.
Using the pcDNA-ATP5B(E175V) and pcDNA-ATP5B(D295V) prepared in the above, binding ability of the Glu 175 Val or Asp 295 Val mutation type ATP5B protein with phenformin was inspected by the pull-down assay in accordance with the method shown in the aforementioned Example 5(4). As a result, as shown in
(7) Detection of AMPK Activation Ability of Biguanide in Cell Over-Expressing Human ATP5B or LKB1
Though direct target protein of biguanide has not been found yet, it has been revealed that it activates intracellular AMP activation kinase (AMPK) through its phosphorylation (Zhou G. et al. J. Clin. Invest. 2001 October; 108 (8): 1167-74). Since the activity of AMPK is to improve glucose metabolism by accelerating uptake of glucose, it is considered that this activation of AMPK is the main reaction pathway which produces diabetes treating effect which is the dug effect of biguanide. In case that the ATP5B found by the identification method of the present invention is the true target molecule which carries the drug effect (principal effect) of biguanide, the ATP5B protein should be positioned at further upstream of the activation of AMPK in the intracellular signal transduction system which is functioned by biguanide. Accordingly, in order to inspect by an experiment that the ATP5B molecule binding to biguanide is actually positioned at upstream of AMPK activation, whether or not the activation of AMPK by biguanide undergoes an influence under a condition in which ATP5B is excessively expressed in a cell was examined.
For the aforementioned purpose, cloning of a gene cDNA coding for LKB1 which has been shown as one of the enzymes that phosphorylate AMPK (Hardie D G, J. Cell Sci. 2004 Nov. 1; 117 (Pt 23): p. 5479-5487) and preparation of an LKB1 expression plasmid were firstly carried out.
In the same manner as the method described in Example 5(1), DNA oligo primers represented by SEQ ID NO:82 and SEQ ID NO:83 were prepared, and a complete length cDNA of human LKB1 gene was cloned by PCR using a human kidney-derived cDNA library (Clontech) as the template. The PCR was carried out under the same conditions of the aforementioned Example 5(1), and the thus amplified DNA fragment of about 1300 base pairs was inserted into the expression vector pcDNA3.1/\75-His-TOPO. As a result of determining nucleotide sequence of the insertion DNA fragment of the accomplished plasmid, it was confirmed that it is a clone consisting of the complete length cDNA sequence of human LKB1 shown by RefSeq accession number NM—000455. Hereinafter, this expression plasmid is referred to as pcDNA-LKB1.
This expression plasmid pcDNA-LKB1 and the pcDNA-ATP5B prepared in the aforementioned Example 5(1) or an empty vector (pcDNA3.1) (Invitrogen) were transferred into HeLa S3 cell. The HeLa S3 cell (ATCC) was cultured in a 6 well culture plate (35 mm in well diameter) until it reached a state of 70% confluent, by adding 2 ml of a minimum essential medium DMEM (Gibco) containing 10% fetal bovine serum (Sigma) to each well of the culture plate. The medium was substituted by 1 ml per well of a serum-free medium OPTI MEM I (Invitrogen), and 3.0 μg/well of each of the empty vector (pcDNA3.1), pcDNA-LKB1 and pcDNA-ATP5B was transiently transferred using Lipofect amine 2000 (Invitrogen). After 12 hours of the culturing, this was substituted by 2 ml/well of DMEM containing 10% of fetal bovine serum from which low molecular substances had been removed by a charcoal treatment, and further cultured for 36 hours. This was further cultured for 1 hour (1 hr) under a condition of adding phenformin (Sigma) to a final concentration of 1 mM or the solvent (DMSO) alone. The medium was discarded, the cells were washed with a phosphate buffer (to be referred to as PBS hereinafter, and then the cells were lysed by adding 0.15 ml of the aforementioned buffer A (however, the NaCl concentration was 150 mM, and various phosphatase inhibitors; 2 mM Na3VO4, 10 mM NaF, 25 mM β-glycerophosphate, 0.2 mM Na2MoO4, 20 nM okadaic acid, were added). The activity of AMPK in the cells was detected at the level of phosphorylation of molecules by a conventionally known SDS electrophoresis and a western blotting which uses an anti-phosphorylation AMPK antibody (Phospho-AMPK-α(Thr172) Antibody, Daiichi Pure Chemicals). In this case, in order to confirm that there is no difference in the amount of protein of AMPKα (an AMPK subunit which undergoes phosphorylation) among samples, a western blotting which uses an anti-AMPKα antibody (Daiichi Pure Chemicals) was simultaneously carried out. As a result, as shown in
These mean that the AMPK activation ability contributing to the drug effect of phenformin, metformin or the like biguanide changes when the amount of ATP5B protein presenting in the cell is changed, and show that ATP5B is evidently positioned at the upstream of AMPK in the intracellular signal of biguanide. When both of this fact and another fact that biochemical binding of ATP5B and phenformin was shown by the aforementioned example are considered, it can be concluded that ATP5B binds to biguanide, and is the true target protein which contributes to the drug effect of said compound. In this connection, the phenomenon in which activation of AMPK by biguanide, namely acceleration of phosphorylation, was obstructed by the over-expression of ATP5B can be explained by either one of the following theories. It can be explained by a mechanism in which ATP5B originally has an action to suppress phosphorylation of AMPK, and biguanide activates AMPK by binding to ATP5B and thereby preventing its AMPK suppressing action. Alternatively, it can be explained that since the ATP5B molecule which acts by forming a complex with an endogenous molecule was over-expressed alone, biguanide was purged (removed) by binding to excess monomer of ATP5B, and as a result, acceleration of phosphorylation of AMPK originally generated by biguanide via a complex including ATP5B was not found. In this connection, such a phenomenon in which actions of compounds are offset each other by over-expression of a target protein is generally known and is also used as a means for identifying that a specified protein is a target protein of a compound (Curr. Genet. 2002; 41 (3): p. 142-149, J. Biol. Chem. 2005; 280 (13): p. 12231-12238, Proc. Natl. Acad. Sci. USA 1996; 93 (21): 11919-11924, Yeast, 1998 14 (10): 935-942). By either one of the above explanations of theories, there is no change in the fact that ATP5B is the target protein which contributes to the drug effect of biguanide, and that the substance which binds to ATP5B has the same drug effect (principal effect) of biguanide.
Based on the above results, it was shown that a compound which can induce the same principal effect of the drug effect produced by biguanide as an agent for treating diabetes, by making use of the same intracellular signal pathway, (namely an agent for treating diabetes) can be selected by making use of the polypeptides for tool use including ATP5B as the target protein of biguanide as an agent for treating diabetes (namely using as a screening tool of an agent for treating diabetes).
In addition, it was proved that a target protein of an agent to be tested, which contributes to the principal effect of the compound, can be identified by the identification method of the present invention.
(1) Screening Method
A screening method which can select a substance to be tested (namely an agent for treating diabetes) that binds to a polypeptide, using biguanide and ATP5B as one of the polypeptide for tool use, and using a change in the binding of said polypeptide and biguanide as the index, is shown.
Binding of ATP5B and biguanide can be detected in accordance with the aforementioned Example 5(4). As sown in
(2) Screening of an Agent for Treating Diabetes
In accordance with the screening method of the aforementioned (1), screening was carried out by adding various compounds as the substances to be tested instead of free phenformin under a condition of 10 μM in concentration of MTX-phenformin (MTX-PH), and 2 compounds, 2-[(E)-(1H-1,2,4-triazol-3-ylimino)methyl]phenol (Maybridge, to be referred to as compound A hereinafter) and 6-chloro-9H-purine-2-amine (Aurora, to be referred to as compound B hereinafter), were found as a result thereof as hit compounds. That is, as shown in
As described in the aforementioned Example 6, the compound A and compound B found by the screening method of the present invention have the ability to activate AMPK in cells. Next, whether or not these compounds have the in vivo hypoglycemic action similar to the case of biguanide was examined. At the same time, action of the aforementioned 2 compounds on the increase of blood lactic acid value as an adverse side effect of biguanide was also examined. A total of 15 animals of 11 weeks of age of db/db mouse (BKS.Cg-+Leprdb/+Leprdb/Jcl; CLEA Japan) as a diabetes model mouse were divided into 3 groups, each consisting of 5 animals. Each of metformin (Sigma) and the aforementioned compound A was dissolved in a solvent (5% Cremophor, 0.2% methyl cellulose: MC) to a concentration of 30 mg/ml or 10 mg/ml. Metformin was administered at a dose of 300 mg/kg body weight, and the compound A at 100 mg/kg body weight, to the abdominal cavities of respective 5 animals of the aforementioned mouse, and compared with a group to which the same volume of the vehicle (5% Cremophor, 0.2% MC) alone was administered. They were subjected to fasting at the same time with the administration, and blood samples were collected from the tail of each animal 0 minute, 90 minutes and 180 minutes thereafter to measure blood sugar level and blood lactic acid level. The blood sugar level was measured using a simplified blood sugar analyzer (Acu Check Active II; Roche), and the blood lactic acid level was measured using a simplified lactic acid analyzer (Lactate Pro; Arkray Marketing), respectively. In the same manner, 12 animals of the db/db mouse of 18 weeks of age were divided in 3 groups, 4 animals for each. Each of metformin (sigma) and the aforementioned compound B was dissolved in physiological saline to a concentration of 30 mg/ml or 9 mg/ml. Metformin was administered at a dose of 300 mg/kg body weight, and the compound A at 100 mg/kg body weight, to the abdominal cavities of respective 4 animals of the aforementioned mouse, and compared with a group to which the same volume of the vehicle (physiological saline) alone was administered. In the same manner as the above, they were subjected to fasting at the same time with the administration, and the blood sugar level and blood lactic acid level were respectively measured 0 minute, 90 minutes and 180 minutes thereafter in the same as described in the above.
As a result, in comparison with the vehicle administration group, metformin shoed significant hypoglycemic action after 90 minutes and 180 minutes in each test. Each of the compound A and compound B showed the hypoglycemic action after 90 minutes and 180 minutes having a significant difference (
Thalidomide has significant drug effects for sleep inducing agents, multiple myeloma, HIV, Hansen's disease and the like, but direct target protein of thalidomide has so far been unclear. Accordingly, an attempt was made to screen a target protein of thalidomide using the method shown in the aforementioned “Example 2(2). A human monocytic cell line THP-1 (ATCC) was cultured and suspended in 60 ml of a minimum essential medium (RPMI 1640, Invitrogen) containing 10% fetal calf serum (FCS) and cultured on a plate of 10 cm in diameter (Asahi Techno Glass) until reached 106 cells/ml. The cells were collected by 3 minutes of centrifugation at 1200 rpm, washed once with 10 ml of ice-cooled PBS and then lysed by adding 2.0 ml of the aforementioned buffer A, and the cell extract was collected. This cell extract was centrifuged at 1500 rpm for 5 minutes to discard the precipitate, and the soluble fraction of supernatant was collected. Under a condition of adding or not adding 100 μM in final concentration of thalidomide ((−)-thalidomide, Sigma), a pull-down test was carried out by mixing this soluble fraction of cell extract with 8 species of GST fusion chaperone proteins purified on the glutathione Sepharose beads (a mixture of GST-HSPA1A, GST-HSPH1, GST-HSPCA, GST-HSPD1, GST-DNAJA1, GST-HSPB1, GST-HSPE1 and GST-HSPA4; respective proteins were mixed in 0.5 μg portions). In this connection, steps and conditions of the pull-down test were the same as the aforementioned Example 2(2). That is, after shaking at 4° C. for 1 hour, proteins binding to the GST fusion chaperone protein mixture on beads were co-precipitated. In the same manner as in the aforementioned Example 4, each molecular chaperone protein and beads were used by chemically crosslinking them in advance by a conventionally known method. After co-precipitation of the proteins binding to the chaperone protein mixture, the residue was suspended in 0.5 ml of the aforementioned buffer A′ to which 100 μM in final concentration of thalidomide had been added or not added, and again co-precipitated by centrifugation. After repeating this operation 4 times, proteins in the precipitate were separated by SDS polyacrylamide gel electrophoresis in accordance with a known method, and the proteins were detected by a negative staining method (Wako Pure Chemical Industries. As a result, the presence of two or more protein bands which are present only when thalidomide was added was detected. These proteins are a group of proteins in which binding of said proteins and the molecular chaperone protein mixture was changed by the addition of thalidomide, namely a group of proteins in which their tertiary structures were changed by the addition of thalidomide. These bands were cut out, the proteins were digested into fragments using trypsin, and then the thus formed peptide mixtures were recovered from the gel and subjected to the identification of proteins by mass spectrum analysis in the same manner as the method of Example 4. As a result, it was revealed that the protein contained in a band of about 45 kDa which is present only when thalidomide was added is TARDBP (RefSeq accession number NP—031401).
It is known that TARDBP is a transcription factor having the activity to bind to DNA, RNA and the like nucleic acids and has an action to suppress HIV (Ou S H et al., Virol 1995 June; 69 (6): 3584-3596). Since a significant anti-HIV action of thalidomide is known (Franks M E et al., Lancet 2004; 363 (9423): 1802-1811), there is no inconsistency in thinking that TARDBP is a target protein of thalidomide.
Though a protein as direct target of thalidomide has not been known yet, it has been reported that it suppresses production of tumor necrosis factor α (TNF-α) or the like cytokine from cells (Franks M E et al., Lancet 2004; 363 (9423): 1802-1811). Thus, an attempt was made to inspect whether or not the target protein of thalidomide, TARDBP, found by the method of the present invention in the aforementioned Example 8 is the true target molecule carrying the principal effect of thalidomide, by carrying out an experiment using a change in the production of TNF-α from a cell as the index.
(1) Cloning of TARDBP Gene and Preparation of TARDBP Expression Plasmid
In accordance with the gene sequence of human TARDBP in the RefSeq accession number NM—007375, primers having the nucleotide sequences represented by SEQ ID NO:93 and SEQ ID NO:94 were synthesized, and an attempt was made to amplify complete length cDNA of human TARDBP by PCR from a human lymphocyte-derived cDNA library (Clontech) using said primers. The PCR was carried out using a DNA polymerase (TAKARA LA Taq; Takara Shuzo), by heating at 94° C. (3 minutes) and then repeating 35 times of a cycle consisting of 94° C. (30 seconds), 58° C. (1.5 minutes) and 72° C. (4 minutes). As a result of separating the PCR product by an agarose gel electrophoresis, it was confirmed tat a DNA fragment of about 1250 base pairs was amplified. Accordingly, this DNA fragment in the reaction liquid was cloned into an expression vector (pcDNA3.1/V5-His-TOPO; Invitrogen) using TOPO TA Cloning System (Invitrogen). The primer used in this case, represented by SEQ ID NO:94, was designed in such a manner that the stop codon of said gene was removed so that a vector-derived V5 epitope (derived from the V protein of paramyxovirus SV5, Southern J A, J. Gen. Virol. 72, 1551-1557, 1991) and a His 6 tag (lindner P, BioTechniques 22, 140-149, 1997) are continued in the same frame of TARDBP gene triplet on the 3′ side after the cloning. Nucleotide sequence of the inserted DNA fragment in the thus obtained plasmid was determined using primers which bind to the T 7 promoter region on the vector (TOPO TA Cloning kit; Invitrogen; SEQ ID NO:89) and a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer; Applied Biosystems). As a result, it was confirmed that it is a clone comprising the complete length cDNA coding for the human TARDBP, shown by the RefSeq accession number NM—007375 (SEQ ID NO:95). Hereinafter, this expression plasmid is referred to as pcDNA-TARDBP.
(2) Preparation of TARDBP High Expression Cell and Measurement of TNF-α Expression in Said Cell
The pcDNA-TARDBP or an empty vector (pcDNA3.1) (Invitrogen) was transferred into HeLa S3 cell. Illustratively, the HeLa S3 cell (ATCC) was firstly cultured in a 12 well culture plate until it reached a state of 70% confluent, by adding 1 ml of a minimum essential medium DMEM (Gibco) containing 10% fetal bovine serum (Sigma) to each well of the culture plate. The medium was substituted by 0.5 ml per well of a serum-free medium OPTI MEM I (Invitrogen), and 0.8 μg/well of the pcDNA-TARDBP or pcDNA3.1 was transiently transferred using Lipofect amine 2000 (Invitrogen). After 12 hours of the culturing, this was substituted by 1 ml/well of DMEM containing 10% of fetal bovine serum and further cultured for 12 hours. This was further cultured for 16 hours under a condition of adding or not adding okadaic acid (Wako Pure Chemical Industries) to a final concentration of 50 nM. In this case, 100 of thalidomide was added a part of the cells simultaneously with the okadaic acid treatment. The medium was discarded, the cells were washed twice with ice-cooled PBS, and then these cells were frozen and stored at −80° C.
(3) Measurement of TNF-α Expression in Cells
Total RNA was prepared from each cell frozen in the aforementioned Example 9(2), using a reagent for RNA extraction use (Isogen; Nippon Gene) and in accordance with the instructions attached thereto. The thus prepared each total RNA was then treated with a deoxyribonuclease (Nippon Gene), subjected to phenol/chloroform treatment and ethanol precipitation and dissolved in sterile water. Using 1 μg of this total RNA, its reverse transcription into a single-stranded cDNA was carried out in a system of 20 μl using a kit for reverse transcription reaction use (Advantage™ RT-for-PCR Kit; Clontech).
Six oligonucleotides (SEQ ID NO:97 to SEQ ID NO:102) were designed and synthesized as primers of PCR for the measurement of gene expression quantity. A combination of SEQ ID NO:97 and SEQ ID NO:98 was used for human β-actin gene, and a combination of SEQ ID NO:99 and SEQ ID NO:100 for human TNF-α gene, and a combination of SEQ ID NO:101 and SEQ ID NO:102 for human TARDBP gene, respectively.
Real time measurement of PCR amplification by PRISM™ 7700 Sequence Detection System was carried out in a system of 25 μl using the aforementioned 6 species, 3 sets of primers and in accordance with the instructions attached thereto. In each system, 5 μl of single-stranded cDNA, 12.5 μl of 2×SYBR Green reagent and 7.5 μmol of each primer were used. In this case, the single-stranded cDNA stored in (2) was used bi diluting it 100 times. In this connection, 0.1 of a human genomic DNA (Clontech) was diluted and a 5 μl portion thereof was used instead of the single-stranded cDNA. The PCR was carried out, after heating at 50° C. for 10 minutes and subsequent 95° C. for 10 minutes, by repeating 45 cycles of a process consisting of 2 steps of 95° C. for 15 seconds and 60° C. for 60 seconds.
The expression quantities of human TNF-α gene and human TARDBP gene in each sample were corrected by the expression quantity of β-actin gene based on the following equation.
[Corrected expression quantity of TNF-α or TARDBP]=[expression quantity of TNF-α or TARDBP gene (raw data)/[expression quantity of β-actin gene (raw data)]
In comparing the expression quantities of TNF-α and TARDBP genes, relative amounts were calculated by regarding the expression quantity in a cell, into which an empty vector (pcDNA3.1) was transferred and which was treated only with the solvent (DMSO), as 100, with the results sown in
As shown in
As shown in
On the other hand, in the case of the TARDBP high expression cell, the action to accelerate expression of TNF-α from the cell by the addition of okadaic acid was almost the same in comparison with the control cell, but the action to suppress expression of TNF-α by the addition of thalidomide was not observed. This result can be easily explained based on an assumption that the thalidomide which should act originally was purged (removed) from inside the cell due to the binding of thalidomide in the cell and excess amount of TARDBP protein caused by the over-expression of TARDBP. It is known that TARDBP is a transcription factor which has the activity to bind to DNA, RNA and the like nucleic acids. That is, since TARDBP is considered to be one molecule in a functional complex consisting of two or more transcription factors essential for the expression induction of TNF-α, it is considered that expression induction of TNF-α is not accelerated when the molecule alone is over-expressed, but when the function of said molecule is inhibited by thalidomide, function of the transcription complex essential for the expression induction of TNF-α is lost, and as a result, production of TNF-α is suppressed.
Based on the above results, it was considered that the protein TARDBP which binds to molecular chaperon only when thalidomide is added, found by the identification method of the present invention, is a molecule concerned in the expression control of TNF-α considered to be one of the molecular mechanisms carrying the drug effect of thalidomide.
Based on this, it was proved that the identification method of the present invention can be used for the identification of target proteins without applying modification of agents to be tested, in addition to the identification of the biguanide target ATPSB shown in the aforementioned example.
The screening method of the present invention can be applied to the screening of an agent for treating diabetes. The screening tool of the present invention can be used in the aforementioned screening.
The identification method of the present invention is useful as an identification method of target proteins which are useful in studying improvement of existing agents.
While the invention has been describe with reference to specific embodiments thereof, changes and modifications obvious to those skilled in the art are included in the scope of the invention.
Explanation of “Artificial Sequence” is described in the numerical heading <223> in the following Sequence Listing. Illustratively, respective nucleotide sequences represented by SEQ ID NOs:28 to 77, 84 to 89, 91 and 92 of the Sequence Listing are artificially synthesized primer sequences.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-234673 | Aug 2005 | JP | national |
| 2005-279582 | Sep 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/315745 | 8/9/2006 | WO | 00 | 9/18/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2007/020853 | 2/22/2007 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5585277 | Bowie et al. | Dec 1996 | A |
| 5679582 | Bowie et al. | Oct 1997 | A |
| 6207401 | Plowman et al. | Mar 2001 | B1 |
| 6579868 | Asano et al. | Jun 2003 | B1 |
| 6790958 | Lum et al. | Sep 2004 | B2 |
| 7244577 | Todd et al. | Jul 2007 | B2 |
| 20020055123 | Pakula et al. | May 2002 | A1 |
| 20040072368 | Martinez | Apr 2004 | A1 |
| 20040191835 | Pakula et al. | Sep 2004 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20090041754 A1 | Feb 2009 | US |
