JPH10330286A - Use and screening of abnormal gene product agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine capable of preventing and treating Alzheimer's disease, schizophrenic disorder, etc., based on an analyzed information about causal relation between transgenic and disease obtained by analyzing a disease- related structural gene. SOLUTION: This medicine contains an agent of an abnormal gene product, which is an abnormal receptor, an abnormal channel, an abnormal transporter or an abnormal enzyme, and is used for preventing and treating the disease caused by the product. The agent is screened (specified), for example, by bringing the abnormal gene product into contact with a test specimen and examining the activity of the material to the product. The approach for making a medicine from the cause of a disease is enabled.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to uses of abnormal gene product agonists and screening methods. More specifically, the present invention uses a completely different approach, that is, a disease, by using information obtained from the analysis of structural genes related to human diseases, for example, information for elucidating the causal relationship between gene mutations and diseases. Drug discovery approach from cause "
And a use of an abnormal gene product agonist prepared based on such a novel drug discovery method.

[0002]

2. Description of the Related Art Conventional drug discovery technology has mainly studied the pathological mechanism of a disease and examined the possibility of developing a new drug from the mechanism of action. This pathological mechanism was the result of the pathogenesis, not the cause. for that reason,
Development of the lead compounds obtained from the screening system has often been interrupted due to the lack of expected clinical effects or unexpected toxicological manifestations. On the other hand, genome research has been attracting attention since 1990 as a new technology in the field of natural science.
This is a study of the analysis of genetic information that is widely involved in biology and medicine in general, and the vast amount of genetic information obtained through this genomic study provides a clue to elucidating the causal relationship between genetic mutations and diseases. This is because it is expected that the drug discovery method from Japan will be possible. The development of a drug derived from genetic information and acting on the pathogenesis of the disease will proceed in the next step. It is a new theoretical drug discovery approach that involves: • identification of the sequence and function of disease-related genes • determination of disease-causing genes and functional analysis of genetic information • selection of candidate drugs derived from the information of disease-causing genes. At present, drug discovery approaches based on genetic information related to the genome project are still at the earliest stage, "Identifying the sequence and function of disease-related genes", and efforts are being made to elucidate the relationship between disease and genetic abnormalities. Worldwide.

[0003] Although several disease-related genes have been reported in Alzheimer's disease and hypertension, elucidation of various risk factors has been awaited, and elucidation of the full picture still requires considerable time. In obesity, the cause of the pathogenesis is approaching elucidation, and it is highly possible that antiobesity drugs will be developed in the near future. In non-insulin dependent diabetes, the pathogenesis of diabetes will soon be revealed at the genetic level. In heart disease, there is a high possibility that a drug with a new mechanism of action will be found in the near future, including the discovery of genes related to myocardial infarction. In cancer, efforts to find genes for disease causes or risk factors have steadily been successful. However, various types of cancers are so diverse that it is a fact that much knowledge is still needed in order to clarify the overall picture of the causes of each cancer.

[0004] Most cases of Alzheimer's disease (AD) are sporadic cases of unclear inheritance. However, familial AD (famili
al Alzheimer's disease: FAD) There are cases. This FAD
It is thought that elucidation of the etiological gene of Escherichia coli will lead to elucidation of the pathogenesis of spontaneous AD and further development of therapeutic agents therebehind, and research on the FAD gene has been actively conducted in recent years. As a result, to date, early-onset FA
Chromosome 14 was strongly linked in many of D's families, and a small number of families were found to have abnormalities in the β-amyloid precursor protein gene on chromosome 21. In contrast, the molecular etiology of arctic Alzheimer's disease, which accounts for the majority of Alzheimer's disease, or familial late-onset Alzheimer's disease, has not been elucidated until recently. However, in recent years, research from the standpoint of whether these diseases are multifactorial genetic diseases involving multiple genetic risk factors has attracted attention. 1993 Apolipoprotein E by Corder et al.
One of the polymorphisms, APOE4, was reported as a genetic risk factor for Alzheimer's disease (Science, 261 , p92
1, 1993). They analyzed APOE in a number of Alzheimer's disease patients and pointed out that among the polymorphisms in these proteins, APOE4 is a genetic risk factor. According to their report, the frequency of APOE4 is significantly higher in the group of patients with familial late-onset Alzheimer's disease than in the healthy group. In addition, when the Alzheimer's disease group was divided into three groups: a group without APOE4, a group with APOE4 in a heterozygote, and a group with APOE4 in a homozygote, the onset age became younger in the group with more APOE4. It was revealed. As a risk factor other than APOE4, gene polymorphism of VLDL receptor, which is an apolipoprotein E receptor, has been actively studied.

With respect to obesity, the ob protein has recently been identified (Nature, 372 , p425, 1994). When obesity is observed, the expression and secretion of the ob protein in adipocytes increases, which stimulates the satiety center of the hypothalamus. It has been found that there is a negative feedback circuit that suppresses eating and energy consumption. Currently, studies are being actively conducted on obese obesity model mice called ob / ob mice, and studies are being carried out to determine how the ob protein (leptin, Leptin) is involved in human obesity. In addition, the relationship between obesity and the β3 adrenergic receptor has attracted attention. That is, obesity is caused by a decrease in energy consumption as well as an excess of energy intake. In humans as well as rats and mice, overeating and obesity stimulate the activity of the β3 receptor in the sympathetic nervous system, and it is thought that energy expenditure, particularly in brown adipocytes, may increase, resulting in heat production. I have. This pathway can be considered as a feedback mechanism that suppresses the progression of obesity. Therefore, when such a feedback route does not work due to a congenital abnormality in the β3 receptor, energy consumption is not increased even by obesity or overeating, and obesity is promoted. In fact, a mutation in which the Trp residue at position 64 of the β3 adrenergic receptor is changed to an Arg residue has been reported in an ethnic group called obesity and diabetes, which frequently occurs in obesity and diabetes [N. Engl. J. Med., 333] . ,
343, (1995)]. In addition, the same mutation has been identified in Finns and Caucasians. This mutation differs from the known mutations in diabetes (insulin, insulin receptor, glucokinase, and mitochondrial gene mutations), and is very frequent, with about 8% in Caucasians and 31% in Pima Indians. Mutations have been observed. Thus, the notion that obesity is merely a matter of habit and culture has been recognized as a disease caused by genetic levels.

It is known that non-insulin dependent diabetes mellitus (NIDDM) is caused by the interaction between genetic factors and environmental factors. And this genetic factor is not necessarily a single gene, but multi-factor inheritance in which multiple genes are involved in the onset. At present, the actual status of genetic factors is not yet clear, but the existence of several candidate genes is known. If we could elucidate the risk factors for the development of diabetes and lead to early diagnosis, early treatment, and prevention of the development of diabetes, it would lead to a reduction in medical costs required when diabetes became severe and complications occurred. However, it is highly desirable from the medical economic standpoint. Genes that cause abnormal insulin secretion and genes that cause insulin resistance are considered as candidate genes that are deeply involved in NIDDM. Among them, genes involved in insulin secretion include insulin glucokinase and mitochondrial gene abnormalities. As genes involved in the decrease in insulin sensitivity (insulin resistance) of skeletal muscle and liver, abnormalities of insulin receptor and glucokinase involved in glucose uptake in liver have been identified. Glucokinase abnormality and MODY (Matur
Detailed studies have been conducted on the causal relationship of ity-Onset Diabetes in the Young) disease. Glucokinase is the rate-limiting enzyme in glucose metabolism in pancreatic B cells and liver,
It is a very interesting candidate gene that can explain the pathological condition of NIDDM such as insulin secretion abnormality and insulin resistance by one gene abnormality. MODY is an early-onset subtype of NIDDM that develops before the age of 25 and shows autosomal dominant inheritance, has glucokinase mutations,
It has been concluded that this is the cause. Separately, studies are being conducted on the relationship between insulin receptor abnormalities and NIDDM. The insulin receptor consists of a total of 1355 amino acid residues, and it has been shown that point mutations cause insulin resistance. In the case of insulin receptor abnormalities, even heterozygotes appear as phenotypes, and homozygotes show a stronger expression system.
It takes the form of inheritance called ant (co-dominant). Studies have also been conducted on the relationship between mitochondrial gene abnormalities and NIDDM, but these three genetic abnormalities are diabetic genes in the sense that a single genetic abnormality causes a special subtype of diabetes. It can be said that there is. However, although the β3 adrenergic receptor described in the section on obesity is a powerful NIDDM-causing factor, it cannot be said to be a pathogenic gene. This factor, together with other genes, is thought to be involved in the development of obesity or NIDDM under the influence of environmental factors.

It is widely known that environmental factors are involved in the onset and progression of hypertension, but recently, hereditary predisposition has been elucidated. However, with respect to essential hypertension, which is said to be 90% or more of hypertension, the genetic factors related to the onset are gradually understood, and it takes much time to elucidate the entire disease state. Characteristics of human hypertension include multifactorial inheritance, incomplete penetrance, and a large effect of environmental factors. Currently, methods of gene analysis using human populations include (1) linkage analysis using DNA of family members in cases of apparent familialness, (2) sick siblings, Affected siblings using only sister DNA (affec
ted sub-pair method), (3) association
study) method. Among them, linkage analysis is effective in identifying the causative gene of a special form of hypertension with a strong genetic background, and the method of detecting affected sibs and related detection methods are used for analysis of genetic risk factors for essential hypertension. Have been. Under these circumstances, essential hypertension, which is considered to be the cause of most patients, has a late onset,
It is difficult to collect and no large-scale linkage analysis has been performed. In such a situation, the most effective in recent years is the affected sib-versus method. In 1992, Jeunemaitre et al. Identified 15 mutations in the angiotensinogen gene and used the affected sib-pair method (brothers / sisters with hypertension) in Utah, U.S.A. and the U.S.A. to detect the presence of M235T in exon 2.
(Single-base substitution where codon 235 changes from Met to Thr) Mutation and hypertension were shown to be significantly correlated (Cell, 7
1 , p169, 1992). This correlation was more pronounced in severe hypertension and was found to be a mutation that increases human angiotensinogen blood levels of the TT genotype as well as the ACE / DD gene polymorphism. A point mutation of the AII type 1 receptor gene (A1166C) was reported to correlate with hypertension (Hypertension, 24 , p63, 1994) and the AT1-R gene k
Nock-out mouse lowers blood pressure, AII type 2 receptor gene kn
Report of increased blood pressure in ock-out mouse (Proc. N
atl. Acad. Sci. USA, 92 , p3521, 1995; Nature, 37
7 , p744, 1995) suggest that the AII receptor gene is also genetically involved in regulating blood pressure in some way. In addition, Zee et al. Reported that abnormalities in the low-density lipoprotein receptor gene were associated with obesity in hypertensive patients (Biochem. Biophys. Res. Commun., 189 , p965,
1992) and Ogiwara et al. Also added GJP (gap junction
protein) locus is reported to be involved (Hypertens. Res., 18, p63, 1995). Thus, research at the genetic level for hypertension is steadily progressing, but long-term large-scale genetic immunology is also needed to elucidate the essential etiological factors. It will take some time.

[0008] Genetic studies of heart disease factors have also been actively conducted, and Bringham and Woman's Hospital, USA
al group has two genes that suppress myocardial infarction,
It has been reported that the location of one of the genes that increase the risk on the chromosome has been identified (Nature Genetics, 13 , p429, 19
96). Although it is a malignant tumor considered to be highly hereditary,
Inherited tumors exhibiting the so-called autosomal dominant form have a limited number of cases compared to common cancers, and their association with chromosomes and genes has been elucidated and only several dozen are known . The majority of cancers are diseases that do not fit into the Mendelian form of inheritance based on single-gene mutations, or that do not allow the candidate gene approach to be taken at the genetic level. However, this does not mean that common cancers are not necessarily spontaneous or hereditary. Genetic epidemiological studies indicate that relatives of certain cancer patients are at increased risk of developing similar tumors. In other words, from a multifactorial genetic standpoint, it is appropriate to consider that a plurality of environmental factors and genetic factors are conditions for developing cancer. Although cancer is a gene to which a multifactorial genetic model is applied, cancer is still a genetic disease, and carcinogenesis accumulates multiple abnormalities of oncogenes and tumor suppressor genes, which occur synergistically at multiple stages. It has been revealed that it will go. For colorectal cancer, its multi-step developmental process has been studied in detail. Due to mutations in oncogenes such as Ras,
It is thought that the proliferation of cells becomes active, and that the function of the normal gene is inactivated due to the abnormality of the tumor suppressor gene p53, and it becomes impossible to control the proliferation of the cells. Recently, a group at Columbia University in the United States succeeded in cloning PTI-1, a gene that promotes malignant transformation of cells from benign prostatic hyperplasia to prostate cancer. PTI-1 acts as a switch that turns a benign glandular type into a malignant tumor. This factor is a gene that regulates the translation process and makes translation abnormal. A 46 kd protein with homology to the polypeptide chain elongation factor EF1a (50 kd). PTI-1 is thought to have a point mutation and deletion in EF1. The group's study found that patients with advanced prostate cancer
Expression of PTI-1 has been confirmed in 15 of the humans.

As described above, although efforts to discover genes for the cause or risk factor of each disease have been steadily successful, much knowledge is still needed to clarify the overall picture of disease onset. The next step of “identification of the sequence and function of disease-related genes”, “Determination of disease-causing genes and functional analysis of gene information”, is whether the abnormalities of the genes are the result of disease progression or the cause of the disease. Is an important task to determine if However, the findings in the previous step (finding of a disease-related gene) are merely substance information of the gene sequence, and there is hardly any method for accurately estimating the function of a gene or the function of a gene product from this information. Techniques for generating knockout transgenic mice are currently considered the most feasible approach, but at the state of the art, genetic manipulations can cause serious and unexpected damage to living organisms, resulting in stillbirth or death shortly after birth. Often does not progress. If new progress accumulates in the future, the technology for producing knockout transgenic mice will greatly contribute to the analysis of gene function in the future. Bioinformatics has the potential to elucidate the function of genes, but it is considered that basic information is currently being prepared. The speed of gene sequencing has been remarkable, and human cDNA sequences have already been analyzed to a considerable extent. Although it is predicted that many difficulties will be caused in the last few years, it is expected that gene analysis will be advanced by many universities and genomic ventures. The increase in the amount of basic information has a synergistic effect, and the functional analysis of genes by bioinformatics may proceed rapidly at some point in the future, and is expected to exert its potential in the future as a means of gene function analysis. The sequence and function of the disease-related gene are analyzed, and as a result, at the stage where the disease-causing gene has been identified, the development of a new drug starts based on the information as a third step. However, there is no established drug discovery technology.

[0010]

SUMMARY OF THE INVENTION Under the circumstances described above, a new drug discovery technology has been established based on information for elucidating the causal relationship between a gene mutation and a disease obtained from the analysis of human disease-related structural genes. Is desired. The present invention provides such a novel drug creation method and a novel use of an abnormal gene product agonist prepared based on the method.

[0011]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a cDNA library prepared from human cells or a commercially available cDNA derived from human cells can be used.
Abnormal gene products from the NA library (abnormal receptors, etc.)
The present invention was completed by using a cell transformed with this gene. The present invention relates to a drug discovery technology that enables a drug discovery approach from the cause of the onset, unlike the conventional new drug development approaching from the onset results such as the pathological mechanism of the disease and the mechanism of action of the drug. The use of drug technology makes it possible to create a new drug based on a direction of drug discovery approach that is completely different from the conventional one, that is, a “drug approach from disease causes”. In particular, it has a significant impact on the creation of therapeutics for diseases related to the brain and nervous system in which natural ligands are small molecules.

More specifically, the present invention relates to (1) an agent for preventing or treating a disease caused by an abnormal gene product agonist, which comprises an abnormal gene product agonist; The prophylactic / therapeutic agent according to the above (1), which is a channel, an abnormal transporter or an abnormal enzyme; and (3) the preventive / therapeutic agent for a disease caused by an abnormal receptor, which comprises an abnormal receptor agonist. (4) the prevention or treatment agent according to (1), wherein the disease caused by the abnormal receptor is a disease caused by a substantial decrease in the transmission system of cells having the abnormal receptor. -Therapeutic agent; (5) The prevention / treatment according to the above (3), wherein the disease caused by the abnormal receptor is a disease caused by a natural ligand not substantially operating a transmission system of a cell having the abnormal receptor. Agent; (6) caused by abnormal receptor The prophylactic / therapeutic agent according to the above (3), wherein the disease is a disease caused by a substantial decrease in the affinity of the natural ligand for the abnormal receptor; The prophylactic / therapeutic agent according to the above (5), which is a transmission system based on a change in the intracellular concentration of the responding substance caused by the binding; (8) the prophylactic or therapeutic agent according to the above (7), wherein the responding substance is cAMP, inositol phosphate or calcium ion. -Therapeutic agent; (9) The preventive / therapeutic agent according to (3), wherein the abnormal receptor agonist is a substance that activates a normal receptor; (10) The abnormal receptor agonist does not activate a normal receptor The prophylactic / therapeutic agent according to the above (3), which is a substance; (11) Use of the product agonist for treating a disease caused by an abnormal gene product; (12) The abnormal gene product is an abnormal receptor The (1)
(13) a method for screening for an abnormal gene product agonist, which comprises contacting an abnormal gene product with a test substance and assaying the operability of the substance for the product; The above-mentioned (1), wherein the abnormal gene product is an abnormal receptor
(15) for treating a disease caused by an abnormal gene product, which comprises contacting the abnormal gene product with a test substance and assaying the operability of the substance for the product; (16) contacting an abnormal receptor with a test substance and assaying the operability of the substance with respect to the abnormal receptor, wherein the mammal is afflicted with a disease caused by the abnormal receptor. A method for screening a drug for substantially operating a transmission system of a cell having an abnormal receptor in an animal; (17) an abnormal receptor prepared by expressing a gene encoding the abnormal receptor in a cell; A screening method according to the above (15), which is a receptor; (18) a gene encoding an abnormal receptor is prepared from cells of a mammal suffering from a disease caused by the abnormal receptor. The screening method according to the above (17), which is a gene encoding an abnormal receptor identified by comparative analysis of the produced gene and a gene prepared from cells of the same kind of mammal that does not have the abnormal receptor. (19) contacting an abnormal gene product with a test substance, testing the operability of the substance with respect to the product, and preparing a drug which is determined to substantially activate the abnormal gene product; A method for preparing a drug for treating a disease caused by an abnormal gene product; (20) bringing an abnormal receptor into contact with a test substance, assaying the operability of the substance for the abnormal receptor, and determining the abnormal receptor A method for preparing a substance for treating a disease caused by an abnormal receptor, which comprises preparing a substance determined to substantially activate a cell transmission system having the cell; The method according to the above (20), which is an abnormal receptor prepared by expressing a gene encoding the abnormal receptor in a cell; (22) the method wherein the gene encoding the abnormal receptor is used in a disease caused by the abnormal receptor; A gene encoding an abnormal receptor identified by comparing and analyzing a gene prepared from cells of an affected mammal and a gene prepared from cells of a mammal of the same species that do not retain the abnormal receptor. (21) the method according to (23); use of the abnormal gene product obtained by expressing a gene encoding the abnormal gene product in cells for screening a substance for treating a disease caused by the product; 24) The abnormal gene product is (2), which is an abnormal receptor.
(25) a use of an abnormal gene product obtained by expressing a gene encoding an abnormal gene product in a cell to screen a substance agonist; and (26) an abnormal gene product is abnormal. The receptor (2)
5) It provides the stated use.

As used herein, the term “abnormal gene product” includes, for example, abnormal receptors, abnormal channels, abnormal transporters, abnormal enzymes and the like. The above “abnormal” refers to a mutation in a structural gene corresponding to a gene product, in particular, a mutation naturally occurring in a living body, and as a result, a substance that activates a normal gene product (eg, activates a normal receptor). And a mutation that can cause disease due to the difference in reactivity of a natural ligand present in a living body, etc.) from the reactivity of the substance to a normal gene product. In addition, the function of the abnormal gene product is the same as the function of the normal gene product, that is, after the agonist for the abnormal gene product activates the abnormal gene product, when the agonist for the normal gene product activates the normal gene product (E.g., a change in the intracellular concentration of a response substance in the transmission system of a cell having a normal receptor).
Diseases caused by abnormal gene products include Alzheimer's disease (eg, familial Alzheimer's disease, sporadic Alzheimer's disease), schizophrenia, depression, hypertension (eg, essential hypertension, etc.), obesity , Diabetes (eg, non-insulin dependent diabetes mellitus, etc.), heart disease (eg, myocardial infarction, etc.), cancer (eg, colorectal cancer, prostate cancer, etc.), rheumatism, allergic disease (eg, atopy, etc.), arteriosclerosis Among them, Alzheimer's disease, schizophrenia, depression, hypertension, obesity, diabetes, and cancer are preferred.

As used herein, the term “abnormal receptor” refers to a substance in which the affinity of a natural ligand is substantially changed (eg, decreased, enhanced; preferably reduced) as a result of mutation of the structural gene of the receptor in vivo. Receptors which cause disease due to a substantial change in the affinity of the natural ligand are preferred. Here, the “substantial change” indicates that, when comparing the affinity of the natural ligand for the normal receptor and the aberrant receptor, the change is such that the disease can be caused. Also, any change that is not significant or may cause a disease may be used. In addition, the function of the abnormal receptor is the same as that of the normal receptor, that is, after the agonist for the abnormal receptor activates the abnormal receptor, the natural ligand for the normal receptor activates the normal receptor. It is preferable to show a response similar to the response that occurs in the cell (for example, a change in the intracellular concentration of a response substance in the transmission system of a cell having a normal receptor), and the transmission system of a cell having an abnormal receptor has a normal receptor More preferably, it is identical to the cell's transmission system. That is, the affinity of the natural ligand is substantially reduced due to the mutation of the receptor gene, even though the cell having the abnormal receptor has the same transmission system as the cell having the normal receptor. If it is, the transmission system of cells with abnormal receptors does not substantially operate, which may cause disease.However, it binds with abnormal receptors and activates the transmission system of cells with abnormal receptors. The use of an agonist of a possible abnormal receptor makes it possible to effectively prevent or treat such a disease. The disease caused by the abnormal receptor may be any disease as long as the affinity of the natural ligand for the receptor is substantially changed, and the affinity of the natural ligand for the receptor is substantially reduced. Diseases caused by natural ligands substantially inactivating the transmission system of cells having abnormal receptors, Diseases caused by substantially reduced transmission systems of cells having abnormal receptors And diseases caused by abnormal signals or excessive transduction in the transmission system of cells having abnormal receptors. More specifically, for example, Alzheimer's disease (eg, familial Alzheimer's disease, sporadic Alzheimer's disease, etc.), schizophrenia, depression, hypertension (eg,
Essential hypertension, etc.), obesity, diabetes (eg, non-insulin dependent diabetes, etc.), heart disease (eg, myocardial infarction, etc.), cancer (eg, colorectal cancer, prostate cancer, etc.), rheumatism, allergic disease (eg, , Atopy, etc.), arteriosclerosis, etc., among which Alzheimer's disease,
Schizophrenia, depression, hypertension, obesity, diabetes, cancer and the like are preferred. The abnormal receptor agonist includes an agonist of the abnormal receptor, an antagonist of the abnormal receptor, and the like. These are appropriately selected and used depending on the target disease. For example, a disease caused by a substantial decrease in the affinity of a natural ligand for a receptor,
In diseases such as diseases caused by the natural ligand not substantially operating the transmission system of cells having abnormal receptors and diseases caused by substantially reduced transmission system of cells having abnormal receptors, An agonist of the abnormal receptor is preferably used, and in a disease caused by an abnormal signal or excessive transmission in a transmission system of a cell having the abnormal receptor, an antagonist of the abnormal receptor is preferably used. The abnormal receptor agonist may be a substance that activates a normal receptor or a substance that does not activate a normal receptor, and is appropriately selected and used depending on the target disease. Substances that do not substantially act on the body are preferably used. The above-mentioned transduction system includes a response substance (eg, cA) generated by binding of a natural ligand to a receptor.
MP, inositol phosphate, calcium ion, etc.).

As used herein, the term “abnormal channel” refers to a channel in which the affinity of a substance (eg, a blocker, an opener, etc.) that operates a normal channel is substantially changed as a result of mutation of the structural gene of the channel. Among others, channels that cause disease due to a substantial change in the affinity of the agonist are preferred. Also,
The function of the abnormal channel and that of the normal channel are the same, that is, after the active substance for the abnormal channel activates the abnormal channel, the response generated when the active substance for the normal channel activates the normal channel (for example, the gate Closed or open)
It is preferable to show the same response as. Diseases caused by abnormal channels include Alzheimer's disease (eg,
Familial Alzheimer's disease, sporadic Alzheimer's disease, etc.), schizophrenia, depression, hypertension (eg, essential hypertension), obesity, diabetes (eg, non-insulin dependent diabetes mellitus), heart disease (Eg, myocardial infarction, etc.), cancer (eg, colorectal cancer, prostate cancer, etc.), rheumatism, allergic diseases (eg, atopy, etc.), arteriosclerosis, etc., among which Alzheimer's disease, schizophrenia Preferred are depression, hypertension, obesity, diabetes, and cancer.

As used herein, the term “abnormal transporter” refers to the affinity of a substance that activates a normal transporter (eg, a substance that promotes or inhibits transport by the transporter) as a result of mutation of the structural gene of the transporter. And transporters that cause a disease due to a substantial change in the affinity of the agonist. In addition, the function of the abnormal transporter and the normal transporter is the same, that is, after the agonist of the abnormal transporter activates the abnormal transporter, when the agonist of the normal transporter activates the normal transporter It is preferable to show a response similar to a response (for example, transport of ions, nutrients and the like) occurring in the above. Diseases caused by abnormal transporters include Alzheimer's disease (eg, familial Alzheimer's disease, sporadic Alzheimer's disease), schizophrenia, depression, hypertension (eg, essential hypertension), obesity , Diabetes (eg, non-insulin dependent diabetes), heart disease (eg, myocardial infarction),
Cancer (eg, colorectal cancer, prostate cancer, etc.), rheumatism, allergic diseases (eg, atopy, etc.), arteriosclerosis, etc., among which Alzheimer's disease, schizophrenia, depression, hypertension, obesity , Diabetes, cancer and the like are preferred.

As used herein, “abnormal enzymes” include:
Mutations in the structural gene of the enzyme include enzymes in which the affinity of a substance (eg, an activator, an inhibitor, etc.) that activates a normal enzyme has been substantially changed. Enzymes that cause disease due to changes are preferred. Further, the function of the abnormal enzyme and the normal enzyme is the same, that is, after the agonist for the abnormal enzyme activated the abnormal enzyme, the response generated when the agonist for the normal enzyme activated the normal enzyme (for example, (E.g., activation of a substrate protein). Diseases caused by abnormal enzymes include Alzheimer's disease (eg, familial Alzheimer's disease, sporadic Alzheimer's disease), schizophrenia, depression, hypertension (eg, essential hypertension), obesity, Diabetes (eg, non-insulin dependent diabetes, etc.), heart disease (eg, myocardial infarction, etc.), cancer (eg, colorectal cancer, prostate cancer, etc.), rheumatism, allergic disease (eg, atopy, etc.), arteriosclerosis, etc. Among them, Alzheimer's disease, schizophrenia, depression, hypertension, obesity, diabetes, and cancer are preferred.

In order to identify a gene encoding an abnormal gene product (eg, an abnormal receptor), for example, a mammal (eg, a rat, rabbit, sheep, pig, cow, cat, By comparing and analyzing genes prepared from cells of dogs, monkeys, humans, etc., preferably humans) with genes prepared from cells of the same species of mammal that do not carry the abnormal gene product,
Genes encoding abnormal gene products can be prepared and identified. Analyze the sequence and function of disease-related genes,
As a result, an abnormal gene specified as a disease-causing gene (for example, a gene encoding an abnormal gene product derived from a human cell, preferably a gene encoding an abnormal receptor derived from a human cell) can be obtained by, for example, the following method. Can be prepared. First, encode the abnormal gene product
RNA is prepared from cells of an animal species from which the gene product is derived (eg, human cells, preferably cells of an animal suffering from a disease caused by an abnormal gene product). Methods for preparing RNA from these materials include the guanidine thiocyanate method [JM Chirgwin et al., Biochemistry, 18, 5294 (1979)]. Oligo dT is added to the RNA thus obtained.
After adding primers or random oligonucleotides, reverse transcriptase is added to cDNA
Can be synthesized. CDNA obtained based on the previously reported sequence of the abnormal gene product or the sequence identified and analyzed
A sense primer and an antisense primer for amplifying the product cDNA from the standard can be added, and PCR can be carried out according to the instructions of a commercially available kit (for example, a kit of Cetus / Perkin-Elmer). After separating the amplified cDNA by a method known per se, for example, agarose electrophoresis,
Can be recovered from the gel. The nucleotide sequence of this cDNA was determined by the dideoxynucleotide synthesis chain termination method [T. Messing et al.
Nucl. Acids Re
s.), Vol. 9, p. 309 (1981)]. The plasmid having the cloned cDNA can be used as it is or, if desired, cut out with an appropriate restriction enzyme and inserted into another vector. Any vector can be used as long as it can be replicated corresponding to the host. The host is Escherichia
a., a plasmid derived from E. coli, such as pBR322 [F. Bolivar et al., Gene,
2, p. 95 (1979)], pBR325, pUC12, pUC13 and the like. When the host is yeast, a yeast-derived plasmid such as pSH19 [S. Harashima et al., Molecular and Cellular Biology (Mol. Cell. B.
iol.), vol. 4, p. 771 (1984)], and pSH19-1 (European Patent Application Publication EP-A-0235430). When the host is an animal cell, for example, SV40 is added to pBR322.
pSV2-X with ori inserted [RC Mulligan and P. Berg,
Procedurals of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sc
i. USA), vol. 78, p. 2072 (1981)], pcD-X [H.
Okayama and P. Berg, Mol. Cell. Biol., Volume 3, 28
0 (1983)]. When the host is an insect cell, for example, Baculovirus transfer vector pVL1392, pVL1
393 [Manufacturer (Invitrogen Corporation, CA, USA) manual (MAXBAC ^™ Baculovirus expression system,
Manual version 1.4)].

The cloned cDNA has a translation initiation codon (ATG) at the 5 'end and a translation stop codon (TTG) at the 3' end.
AG, TGA or TAA). Further, the cDNA
It is preferred to connect a promoter sequence upstream in order to express The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. When the host is E. coli, T7 promoter, trp promoter, t
Examples thereof include an ac promoter, a lac promoter, a λPL promoter and the like, and a T7 promoter is particularly preferable.
When the host is yeast, a GAPDH promoter, a PGK promoter, a PHO5 promoter, an ADH promoter and the like can be mentioned, and a GAPDH promoter is particularly preferable.
When the host is an animal cell, examples include an SV40-derived promoter, a retrovirus promoter, a human cytomegalovirus promoter, and the like. When the host is an insect cell, the nuclear polyhedrosis virus polyhedrin (po
lyhedrin) promoter and the like. A promoter can be prepared from the corresponding gene. Also,
It can also be chemically synthesized. The expression vector may contain a signal sequence, a pre-pro sequence and the like, and any of these may be used as long as they function in the host. A transformant is produced using the recombinant expression plasmid containing the DNA thus constructed.

Examples of the host include Escherichia,
Examples include yeast, animal cells, insect cells, and the like. Escherichia bacteria include Escherichia coli K12 DH1 [B. Lo
Natl. Acad. Sci. USA, 60, 160 (196
8 years)], C600 [RK Appleyard, Genetics, Vol. 39, p. 440 (1954)], MM294
[K. Backman et al., Proc. Natl. Acad. Sci. USA, 73rd.
Volume, 4174 (1976)], N4830 [ME Gottesman
Journal of Molecular Biology (J. Mol. Biol.), Vol. 140, p. 57 (1980)]. As yeast, for example Saccharomyces cerevisiae (Saccharomyces cerevisiae) AH22R ^- [A.
Miyanohara et al., Proc. Natl. Acad. Sci. USA, Vol. 80,
1st page (1983)], NA87-11A, DKD-5D, NA74-3A, NA74
-3Aρ ^- [Y. Kaisho et al., East (Yeast), Vol. 5, No.
91 (1989)] and Schizosaccharomyces pombe (Sc
hizosaccharomyces pombe) ATCC38399 (h-leu1-32), T
H168 (h90 ade6-M210 ura1 leu1) [M. Kishida and
C. Shimada, Current Genetics
tics), Vol. 10, p. 443 (1986)]. As animal cells, for example, monkey COS-
7 cells, monkey Vero cells, Chinese hamster ovary (CHO) cells, mouse L cells, human FL cells, and mouse myeloma cells (such as Sp2 / 0 cells) that are suspension cells, mouse YAC-1 cells, mouse MethA cells, Mouse P388 cells, mouse EL-4 cells and the like. As insect cells, Sf
9 cells and the like. To transform Escherichia, for example, T. Maniatis et al. [Molecular Cloning, Cold Spring Harbor Laboratories]
y), page 249 (1982)]. To transform yeast, for example, A. Hinnen et al. [Proc. Natl. Acad. Sci. USA, Vol. 75, p. 1929 (19
78)]. Transformation of animal cells is described, for example, in M. Wigler et al., Cell, No. 14.
Volume, page 725 (1978).
To transform insect cells, the manufacturer (Invitrogen C
orpotation) manual (MAXBAC ^TM Baculovirusexpr)
ession system, Manual version 1.4). The transformant thus obtained is cultured by a method known per se.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [JH Miller, Experiments in Molecular Genetics (Experiments)
in Molecular Genetics), p. 431, Cold Spring Harbo
r Laboratory, (1972)]. If necessary, to make the promoter work efficiently, for example, isopropylthiogalactoside (IPTG) or indolyl-3-
An agent such as acrylic acid can be added. The cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or stirring can be added. When culturing a transformant in which the host is yeast, for example, a medium such as Burkholder's minimum medium [KL Bostain et al., Pr.
Natl. Acad. Sci. USA, 77, 4504 (1980
Year)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added. When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal calf serum [H. Eagle, Science, Vol. 130, p. 432 ( 1959)],
DMEM medium [R. Dulbecco and G. Freeman, Virology, Vol. 8, p. 396 (1959)], RPMI-164
0 medium [GE More et al., Journal of the American Medical Association (J. Am. Med.
oc.), vol. 199, p. 519 (1967)], 199 medium [J.
F. Morgan et al., Proc. Of the Society for Experimental Biology and Medicine (Proc. Soc. Exp. Biol. Med.), Chapter 73.
Volume, Page 1 (1950)], ASF104 Medium [Ajinomoto Co., Ltd.]
And the like. The cultivation is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added. When culturing a transformant whose host is an insect cell, as a medium,
For example, TNM-FH medium [WF Hink et al., Nature, Vol.
466 (1990)]. Culture is usually about 1
Perform at about 5 to 30 ° C for about 24 to 72 hours, adding aeration and stirring as needed.

According to the present invention, to isolate an expression product (an abnormal gene product such as an abnormal receptor) from the above culture,
The separation and purification methods known per se can be appropriately combined. These known separation / purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Methods that mainly use differences in molecular weight, such as methods that use differences in charge such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, and hydrophobic methods such as reverse-phase high-performance liquid chromatography A method using a difference, a method using an isoelectric point difference such as an isoelectric focusing method, and the like can be given. According to the present invention, the abnormal gene product is E. coli,
Gene expression by genetic engineering using animal cultured cells, insect cultured cells, and the like has made it possible to produce high-purity, large-scale products.

When the abnormal gene product thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto. It can be converted to a free form or another salt by a method or a method analogous thereto. It is also possible to use cDNAs of abnormal gene products (eg, abnormal receptors in humans, etc.) to express them in eukaryotic cells and use the resulting abnormal gene products to search for compounds that operate them. Alternatively, by using a gene encoding the specified abnormal gene product as a DNA probe, the mRNA content of the abnormal gene product expressed in the living body can be measured by Northern blotting. Furthermore, an antibody against the abnormal gene product can be prepared, and the product can be measured in vivo by in situ hybridization. The abnormal gene product can be used to screen for an abnormal gene product agonist, a drug for treating a disease caused by the abnormal gene product (eg, having an abnormal receptor in a mammal suffering from a disease caused by the abnormal receptor). And the like, and is particularly useful for screening for agonists or antagonists of abnormal receptors associated with diseases in warm-blooded animals such as humans. .

The above-mentioned screening will be described more specifically below. The abnormal gene product is useful for searching for its agonist. That is, it is possible to provide a method for screening for an abnormal gene product agonist, which comprises contacting the product with a test substance.
As the test compound, in addition to known ligands, synthetic compounds, peptides, proteins, etc., for example, tissue extracts of warm-blooded mammals (eg, mouse, rat, pig, cow, sheep, monkey, human, etc.), cell culture A supernatant or the like is used.
For example, the tissue extract, the cell culture supernatant, and the like are added to the abnormal gene product, and fractionated while assaying the agonistic activity, etc., to finally obtain a single agonist. Specifically, by using an isolated abnormal gene product, or by constructing an expression system for an abnormal gene product (eg, an abnormal receptor, etc.) and using the expression system, the product has an agonistic activity of the product. Methods for determining compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) are included. In particular, when screening for an agonist of an abnormal receptor, it is preferable to use an abnormal receptor prepared by expressing a gene encoding the abnormal receptor in cells.

More specifically, the present invention measures the binding amount of a labeled test substance to an abnormal gene product (eg, an abnormal receptor) when the labeled test substance is brought into contact with the product. When the labeled test substance is brought into contact with a cell containing an abnormal gene product (eg, an abnormal receptor or the like) or a membrane fraction of the cell, the labeled test substance reacts with the cell or the membrane fraction. The amount of binding was measured, and the labeled test substance was brought into contact with the product expressed on the cell membrane by culturing a transformant containing a DNA encoding an abnormal gene product (eg, an abnormal receptor). Measuring the amount of binding of the labeled test substance to the product in the case where the test substance is an abnormal gene product (eg, an abnormal receptor, etc.)
When the cells are brought into contact with cells containing, the activity via the product (eg, the activity of promoting or inhibiting the growth promotion, phosphorylation of intracellular protein, etc.) is measured, and the operability of the substance is assayed. The test substance and the abnormal gene product (eg, abnormal receptor)
When the transformant containing the DNA encoding is contacted with an abnormal gene product expressed on the cell membrane by culturing, the activity mediated by the abnormal gene product (for example, growth promotion, phosphorylation of intracellular protein, etc. (A promoting or suppressing activity, etc.) and assaying the operability of the substance, or a combination of these as needed. Examples of the labeled test substance include substances labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ¹³⁵ S], and the like.

A specific example for screening for an abnormal receptor agonist will be described below. First, a cell containing the abnormal receptor or a membrane fraction of the cell is suspended in an appropriate buffer to prepare an abnormal receptor standard. As the buffer, a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8), a Tris-hydrochloric acid buffer, and the like are used.
Any buffer may be used as long as it does not inhibit the binding between the test substance and the abnormal receptor. In addition, in order to reduce non-specific binding, CHAPS, Tween-80 (trade name) (Kao-Atlas), digitonin, surfactants such as deoxycholate, and various proteins such as bovine serum albumin and gelatin are used as buffers. Can be added. Furthermore, PMSF is used to reduce the degradation of receptors and test substances by proteases.
Protease inhibitors such as (phenylmethanesulfonyl fluoride), leupeptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can also be added. 0.01ml ~ 10
A fixed amount (5000 cpm to 500,000 cpm) of a test substance labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C] or the like is allowed to coexist in ml of the receptor solution. Prepare a reaction tube containing a large excess of unlabeled test substance to determine the amount of non-specific binding (NSB). The reaction is carried out at 0 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction,
After filtration through a glass fiber filter or the like and washing with an appropriate amount of the same buffer, the radioactivity remaining on the glass fiber filter is measured by a liquid scintillation counter or a γ-counter. A test substance in which the count (B-NSB) obtained by subtracting the non-specific binding (NSB) from the total binding amount (B) exceeds 0 cpm can be selected as a ligand for the abnormal receptor.

In order to test the operability of a test substance for an abnormal receptor, first, cells containing the abnormal receptor are cultured in a multiwell plate or the like. Before determining the ligand, replace the medium with a fresh medium or an appropriate buffer that is not toxic to the cells, add the test substance, etc., incubate for a certain period of time, and then extract the cells or collect the supernatant to generate the ligand. The quantified product is quantified according to each method. If the production of a substance as an indicator of the cell stimulating activity is difficult to be assayed by a degrading enzyme contained in the cell, the assay may be performed by adding an inhibitor against the degrading enzyme. When the test substance is brought into contact with cells containing the abnormal receptor, the activity mediated by the product (eg, the activity of promoting growth, promoting or suppressing phosphorylation of intracellular proteins, etc.) or cAMP, inositol phosphorus Expression on the cell membrane by measuring changes in the concentration of responding substances in the intracellular transmission system such as acids and calcium ions, or by culturing transformants containing the test substance and DNA encoding the abnormal receptor An activity through an abnormal gene product when contacted with an abnormal receptor (eg, an activity to promote growth, promote or suppress phosphorylation of intracellular protein, etc.) or c
The operability of the substance can be assayed by measuring changes in the concentration of a responding substance in an intracellular transmission system such as AMP, inositol phosphate, and calcium ion.

The present invention also relates to a method of contacting an abnormal gene product (eg, an abnormal receptor) with a test substance, testing the operability of the substance with respect to the product, and substantially activating the abnormal gene product. A method for preparing a drug for treating a disease caused by an abnormal gene product, which comprises preparing a drug to be determined, by bringing a test substance into contact with an abnormal receptor,
Treating a disease caused by an abnormal receptor, comprising assaying the operability of the substance with respect to the abnormal receptor, and preparing a substance that is determined to substantially act on the transmission system of cells having the abnormal receptor. Based on information elucidating the causal relationship between genetic mutations and diseases obtained from the analysis of human disease-related structural genes, such as a method of preparing a substance for
It is intended to provide an established new drug creation method or preparation method. The drug thus prepared is used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, if necessary, for use as a prophylactic / therapeutic agent for a disease caused by an abnormal gene product. It can be used parenterally, or in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or in the form of an injection such as a suspension. For example, by mixing with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of the formulation, Can be produced.

Examples of additives which can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry, and the like. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice of dissolving or suspending the active substance in vehicles, such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like, and suitable solubilizing agents, for example, Alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, polysorbate)
80 (trade name), HCO-50), etc. The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. Also, a buffer (for example, phosphate buffer,
Sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants And the like. The prepared injection is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity because it is effective even at a low dose, for example, a warm-blooded mammal (for example,
Rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, humans, etc.). Dosage may vary depending on the target disease and its symptoms, but when administered orally as a therapeutic agent for hypertension, generally 0.1 mg / day for adults (as 60 kg) is used.
~ 100 mg, preferably about 1.0-50 mg, more preferably about 1.
0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc. For example, in the case of injection, usually for adults (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.

Furthermore, the present invention encodes the use of the agonist of the product and the abnormal gene product (eg, abnormal receptor) for the treatment of a disease caused by the abnormal gene product (eg, abnormal receptor). Use of an abnormal gene product obtained by expressing a gene in a cell, for screening a substance for treating a disease caused by the product, and use of a gene encoding an abnormal gene product (eg, an abnormal receptor) in a cell The present invention also provides a use of an abnormal gene product obtained by expression in E. coli for screening for an agonist of the product.

In the specification of the present application, when bases and amino acids are indicated by abbreviations, IUPAC-IUBCommision on Bioche
This is based on the abbreviation by mical Nomenclature or the abbreviation commonly used in the art, and examples thereof are as follows. In addition, when there is an optical isomer with respect to an amino acid, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine SDS: sodium dodecyl sulfate Gly: glycine (G) Ala: alanine (A) Val: valine (V) Leu: leucine (L) Ile: isoleucine (I) Ser): Serine (S) Thr: Threonine (T) Cys: Cysteine (C) 1/2 Cys: Half cystine Met: Methionine (M) Glu: Glutamic acid (E) Asp: Aspartic acid (D) Lys: Lysine (K Arg: arginine (R) His: histidine (H) Phe: phenylalanine (F) Tyr: tyrosine (Y) Trp: tryptophan (W) Pro: proline (P) Asn: asparagine (N) Gln: glutamine (Q) Apr: Ampicillin resistance gene Tcr: Tetracycline resistance gene

[0032]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the following examples, which are merely examples and do not limit the present invention in any way.

【Example】

Example 1 cDNA Cloning of Human β3 Adrenergic Receptor In order to amplify human β3 adrenergic receptor cDNA by the PCR method, the nucleotide sequence of a previously reported human β3 adrenergic receptor [JM Lelias et al., FEBS Letters (FEBS Lets)
t.), vol. 324, p. 127 (1993)], the following two types of primers (NO. 1 and NO. 2) were synthesized. Sense primer No. 1: 5'-ATTTGGGAGACCCCCTCCTTCCTTCTTTCC-3 '(SEQ ID NO: 1) Antisense primer No. 2: 5'-ACAGAGTTGTTGCTTCTTGTCCTTCAGGCC-3' (SEQ ID NO: 2) TaKaRa Ex Taq [Takara Shuzo Co., Ltd.] The following PCR reaction was performed using the buffer and the attached buffer. Human adipocyte-derived cDN
A library (CLONTECH Laboratories, Inc.) 0.5 μl
5 μl of PCR reaction buffer (10 times concentration) attached to the template
dNTP mixture 4 μl, TaKaRa Ex Taq 0.5 μl (2.5 U), two types of primers (No. 1 and λgt11 forward primer [Takara Shuzo Co., Ltd.] and No. 1 and λgt11 reverse primer [Takara Shuzo ( Co., Ltd.]; 100 each
pmol) was added to each separate tube, and 5
0 μl was used. The PCR reaction at 94 ° C. for 2 minutes, 61 ° C. for 1 minute, 72 ° C. for 1 minute was repeated 25 times each, and the two were mixed.
No. 1 and No. 2; 100 pmol each), and the PCR reaction at 94 ° C. for 2 minutes, 55 ° C. for 1 minute, 72 ° C. for 1 minute was repeated 30 times. When the PCR product was separated by 1% agarose gel electrophoresis, a DNA fragment amplified at a position corresponding to the size (1310 bp) predicted from the nucleotide sequence of the human β3 adrenergic receptor was confirmed. This DNA fragment was recovered from the agarose gel, and plasmid vector pUC19 [Takara Shuzo Co., Ltd.]
Was inserted into the SmaI site to generate p19-hβ3R. The nucleotide sequence of the cDNA portion was determined by the dideoxynucleotide synthesis chain termination method [J.
Messing et al., Nucleic Acids Research
ic Acid Res.), Vol. 9, p. 309 (1981)], and confirmed that the sequence was the same as the previously reported sequence.

Example 2 Preparation of Mutant (Trp64Arg) DNA of Human β3 Adrenergic Receptor A synthetic oligonucleotide for mutagenesis was synthesized and Mutan-Su
Performed as follows using per Express Km [Takara Shuzo Co., Ltd.]. Synthetic oligonucleotide for mutation introduction No. 3: 5'-TGGCCATCGCCCGGACTCCGAG-3 '(SEQ ID NO: 3) Plasmid p19-hβ3R described in Example 1 was replaced with restriction enzyme EcoRI.
A DNA fragment obtained by digesting with XbaI and XbaI was recovered from an agarose gel, and inserted into the EcoRI and XbaI sites of the plasmid vector pKF18k [Takara Shuzo Co., Ltd.] attached to the kit to prepare p18k-hβ3R. 10 ng of this DNA was used as a template, 5 pmol of the selection primer attached and 5 pmol of the No. 5 primer, 5 μl of reaction buffer (10-fold concentration), 4 μl of dNTP mixture, and TaKaR
a Add 0.5 μl (2.5 U) of LA Taq, and add 50 μl with sterile distilled water.
PC at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 3 minutes
After the R reaction was repeated 25 times, DNA was recovered by ethanol precipitation. This DNA was dissolved in 5 µl of sterile distilled water, and 2 µl of the DNA was used to introduce it into the attached Escherichia coli MV1184 strain to obtain a transformant growing on an LB plate containing kanamycin (50 µg / ml). The nucleotide sequence of the mutation site was determined for several clones among these transformants, and the clone confirmed to have the mutation was named p18k-hβ3 (W64R) R.

Example 3 Preparation of Recombinant DNA for Expressing Human β3 Adrenergic Receptor Gene in Animal Cells The plasmid p19-hβ3R described in Example 1 was digested with EcoRI and XbaI, and then the human β3 adrenergic receptor cDNA was digested. Was recovered from an agarose gel. Next, the vector pME18S for transient expression in animal cells [R. Sasada et al., Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Commun.), No.
190, p. 1173 (1993)], the above cDNA fragment was inserted into the EcoRI and XbaI sites to prepare an expression plasmid phβ3R201. Next, a neomycin resistance gene (neo), which is a drug resistance marker, was incorporated as follows for selection of stable expression strains. First, the KpnI site of plasmid phβ3R201 and S
Between the spI site, the SV40 early promoter and neo gene, SV
A fragment consisting of the 40 polyA addition signal was inserted to prepare a plasmid phβ3R203. Plasmid p described in Example 2
After digesting 18k-hβ3 (W64R) R with EcoRI and XbaI, human β3
A fragment of the adrenergic receptor mutant cDNA was recovered from the agarose gel. Performed in the same manner as described above,
β3 (W64R) R201 and phβ3 (W64R) R203 were produced.

Example 4 Expression of the Human β3 Adrenergic Receptor Gene in Animal Cells The plasmids described in Example 3 (phβ3R203 and phβ3 (W64
R) R203) CHO cells (Chinese hamster ovary cells)
Mammalian Transfection Kit (STRATAG)
ENE, CA, USA). Ham F containing 10% (v / v) FCS (fetal calf serum) in 80 cm ² flask
-12 medium (LIFE TECHNOLOGIES, INC.)]
5 × 10 ⁵ O cells were seeded. This culture was cultured overnight at 37 ° C. in the presence of 5% carbon dioxide, and then replaced with 10 ml of the above medium. Plasmid (phβ3R203) 30 described in Example 1
μg to 450 ml with sterile water.
50 μl of on # 1 was added, and 500 μl of solution # 2 was further added and mixed. After standing at room temperature for 15 minutes, 1 ml of the mixture was dropped on the cells and mixed, and 37 ° C in the presence of 3% carbon dioxide.
Overnight. The next day, remove the medium and remove the F-12 medium (serum free)
After washing the cell surface with, 15 ml of a complete medium (10% FCS / F-12) was added, and the cells were cultured overnight at 37 ° C in the presence of 5% carbon dioxide. The next day, about 200 cells per well were seeded in a 12-well plate in which a D-MEM / F-12 medium containing 10% FCS [Nikken Biomedical Research Institute Co., Ltd.] was dispensed (1 ml / well). After overnight culture, colony formation was observed while changing the medium every 3 to 4 days in a medium (10% FCS / D-MEM / F-12) containing 400 μg / ml of G418 (LIFE TECHNOLOGIES, INC.). Until they were ready. After a large number of colonies were formed, one to several colonies per well were placed in a 24-well plate in which 10% FCS / D-MEM / F-12 medium containing 400 μg / ml G418 was dispensed (2 ml / well). Was seeded and cultured to obtain G418-resistant cells (primary cloning).
Next, the concentration of G418 was increased to 800 μg / ml for cells in 2 to 3 wells among the cells of the primary cloning, and 10 colonies per well were seeded and cultured in a 24-well plate.
18 resistant cells were obtained. From these cells, a human β3 adrenergic receptor highly expressing strain was selected by the method shown in Example 5 below (secondary cloning).

Example 5 Measurement of cAMP activity in cells expressing human β3 adrenergic receptor cAMP activity was measured using the cAMP ELA SYSTEM (Amersham, UK).
Was carried out as follows. Cells expressing human β3 adrenergic receptor (CHO / CHO) on the 24-well plate obtained in Example 4
phβ3R203 and CHO / phβ3 (W64R) R203)
Medium (10% FCS / D-MEM / F-12)
BMX [3-isobutyl-1-methylxanthine; Wako Pure Chemical Industries, Ltd.] was added. After culturing at 37 ° C for 30 minutes in the presence of 5% carbon dioxide, 10-5 M isoproterenol [(±) -isoprotereno
l; Funakoshi Co., Ltd.], followed by further culturing for 30 minutes.
Next, the cell surface was washed three times with 4 ° C PBS (phosphate buffer), 300 µl of 0.1N hydrochloric acid was added, and the mixture was boiled at 95 ° C for 10 minutes.
Take 25 μl from each well and use the assay buffe
r Dissolved in 75 μl. From the lysate, 50 μl was taken, and dispensed together with 100 μl of anti-cAMP rabbit antibody into each well of a 96-well microtiter plate on which an anti-rabbit IgG donkey antibody attached to the kit was immobilized, and left at 4 ° C. for 1 hour. Next, the HRP label c
After adding 100 μl of AMP and further standing at 4 ° C. for 1 hour, each well was washed four times, and TMB (3,3,5,5-tetramethylbenzidin) was added.
e) was added to each well, and left at room temperature for 60 minutes.
100 μl of N sulfuric acid was added, and the absorbance was measured at a wavelength of 450 nm. Cell lines in which cAMP activity was increased by isoproterenol, a beta-adrenergic receptor agonist, were selected.

Example 6 Measurement of Binding Activity of Mutant Human β3 Adrenergic Receptor CH, a Chinese Hamster Ovary-Derived Cell Line Expressing Trp64Arg Mutant Human β3 Adrenergic Receptor
O cells were prepared to search for compounds that specifically bind to Trp64Arg mutant β3 adrenergic receptor. Trp64Arg mutant β
(3) Wash the CHO cells expressing the adrenergic receptor three times with ice-cold phosphate buffer, then immerse them in ice-cold hypotonic solution (1 mM Tris-HCl buffer, pH 7.2) for 10 minutes, and detach and collect the cells After that, the cell membrane fraction containing the receptor was collected by centrifugation at 18,000 rpm for 15 minutes at 4 ° C. TM is added to the obtained cell membrane fraction.
E buffer (75 mM Tris-HCl buffer, pH 7.4, 12.5 mM magnesium chloride, 1.5 mM EDTA, 4 μM desipramine, 5 μg / m
l leupeptin, 1 µg / ml benzamidine, 5 µg / ml trypsin inhibitor and 40 µg / ml bacitracin) were added thereto, and the mixture was uniformly suspended using a 25-gauge injection needle to prepare a receptor sample.
The prepared receptor preparation (containing 10-30 μg as protein) and [ ¹²⁵
I] -Iodocyanopindolol (240pM, DuPont-NEN, US
A) and the compound, mix to a final volume of 250 μlTM
The volume was adjusted with E buffer and allowed to stand at room temperature for 90 minutes. Glass fiber filter (GF / B, Packard Instrument
Co., Inc., USA) and cell harvesters (Filter Mate
(Cell Harvester, Packard Instrument Co., Inc. USA)
Filtration is performed using a suction filter, and the radioactivity of the filter is measured using a scintillation counter (TopCount Microplate Scintillati
onCounter, Packard Instrument Co., Inc. USA). Non-specific binding was determined by adding (S)-(−)-propranolol (Sigma, USA) to a final concentration of 100 μM.

Example 7 Measurement of cAMP Elevation Activity in CHO Cells Expressing Mutant Human β3-Adrenergic Receptor CH, a Chinese Hamster Ovary-Derived Cell Line Expressing Trp64Arg Mutant Human β3-Adrenergic Receptor
O cells were seeded in 96-well microtiter plates (1x
10 ⁴ cells / well), the test compounds were added after confluency reached after 72 hours of culture, 40 minutes, allowed to stand at 37 ° C.
(100 μl / well). After washing the cells three times with a phosphate buffer at 4 ° C., 0.1N hydrochloric acid was added, and the mixture was boiled at 95 ° C. for 10 minutes. 25 μl was collected from each well and cAMP EIA SYSTEM (Amersham, U.S.A.)
K) Dissolve in 75 μl of the supplied assay buffer, 50 of which
Using the μl as a sample, quantification was performed using the above cAMP EIA SYSTEM. The method is as follows. The above sample (50 μl) and 100 μl of the anti-cAMP rabbit antibody were added to a 96-well microtiter plate immobilized with an anti-rabbit IgG donkey antibody, and the mixture was allowed to stand at 4 ° C. for 2 hours, and further labeled with horseradish peroxidase (HRP). Add 100 μl of cAMP, and add
Let stand for hours. After aspirating each well, the well was washed four times with a washing solution (400 μl / well). Next, 150 μl of tetramethylbenzidine, a substrate of HRP, was added to each well, and the mixture was incubated at room temperature with shaking for 60 minutes. Finally, after adding 100 μl of 1.0 N sulfuric acid to each well to terminate the reaction,
The amount of cAMP was quantified by measuring the absorbance at a wavelength of 450 nm. As a result, the cAMP concentration in the CHO cells increased depending on the concentration of the added compound, and the increase was significant compared to the case where no compound was added.

Example 8 Searching for Compounds By searching for compounds according to the method of Example 6,
Compounds with high affinity for the Trp64Arg mutant human β3 adrenergic receptor can be found. By conducting the experiment according to the method of Example 7, the compound is expected to increase the cAMP concentration in CHO cells expressing the Trp64Arg mutant human β3 adrenergic receptor depending on the added concentration.

[0040]

According to the present invention, an agent for preventing or treating a gene-related disease can be advantageously provided. That is,
New drug discovery technologies and new drugs that enable drug discovery approaches from disease causes by using information obtained from analysis of disease-related structural genes and information elucidating the causal relationship between gene mutations and diseases An object of the present invention is to provide a use of an abnormal gene product agonist prepared based on a creation method.

[0041]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single-stranded Sequence type: Sense Sequence: ACAGAGTTGT TGCTTCTTGT CCTTCAGGCC

SEQ ID NO: 2 Sequence length: 30 Sequence type: Number of nucleic acid strands: Single-stranded Sequence type: Antisense Sequence: ACAGAGTTGT TGCTTCTTGT CCTTCAGGCC

SEQ ID NO: 3 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Sequence type: sense Sequence: TGGCCATCGC CCGGACTCCGAG

Claims (26)

[Claims] (1) an abnormal gene product agonist,
An agent for preventing or treating a disease caused by the product. 2. The preventive / therapeutic agent according to claim 1, wherein the abnormal gene product is an abnormal receptor, an abnormal channel, an abnormal transporter or an abnormal enzyme. 3. The preventive / therapeutic agent according to claim 1, which is a preventive / therapeutic agent for a disease caused by an abnormal receptor, which comprises an abnormal receptor agonist. 4. The preventive / therapeutic agent according to claim 3, wherein the disease caused by the abnormal receptor is a disease caused by a substantial decrease in the transmission system of cells having the abnormal receptor. 5. The preventive / therapeutic agent according to claim 3, wherein the disease caused by the abnormal receptor is a disease caused by a natural ligand not substantially operating the transmission system of a cell having the abnormal receptor. 6. The preventive / therapeutic agent according to claim 3, wherein the disease caused by the abnormal receptor is a disease caused by a substantial decrease in affinity of the natural ligand for the abnormal receptor. 7. The preventive / therapeutic agent according to claim 5, wherein the transmission system is a transmission system based on a change in the intracellular concentration of a response substance caused by the binding between the natural ligand and the receptor. 8. The prophylactic / therapeutic agent according to claim 7, wherein the responsive substance is cAMP, inositol phosphate or calcium ion. 9. The preventive / therapeutic agent according to claim 3, wherein the abnormal receptor agonist is a substance which activates a normal receptor. 10. The preventive / therapeutic agent according to claim 3, wherein the abnormal receptor agonist is a substance that does not activate a normal receptor. 11. Use of a product agonist for treating a disease caused by an abnormal gene product. 12. The use according to claim 10, wherein the abnormal gene product is an abnormal receptor. 13. A method for screening for an abnormal gene product agonist, comprising contacting an abnormal gene product with a test substance and assaying the operability of the substance with respect to the product. 14. The screening method according to claim 13, wherein the abnormal gene product is an abnormal receptor. 15. A method for screening a substance for treating a disease caused by an abnormal gene product, comprising bringing the abnormal gene product into contact with a test substance and assaying the operability of the substance for the product. . 16. An abnormal receptor of a mammal afflicted with a disease caused by the abnormal receptor, wherein the abnormal receptor is brought into contact with a test substance and the operability of the substance with respect to the abnormal receptor is assayed. A method for screening a drug for substantially operating a transmission system of a cell having a body. 17. The screening method according to claim 15, wherein the abnormal receptor is an abnormal receptor prepared by expressing a gene encoding the abnormal receptor in a cell. 18. A gene encoding an abnormal receptor, wherein the gene is prepared from a cell of a mammal afflicted with a disease caused by the abnormal receptor and a gene prepared from cells of a mammal of the same kind which does not have the abnormal receptor. 18. The screening method according to claim 17, which is a gene encoding an abnormal receptor identified by comparative analysis with the gene. 19. A method comprising bringing a test substance into contact with an abnormal gene product, assaying the operability of the substance with respect to the product, and preparing a drug which is determined to substantially activate the abnormal gene product. , A method of preparing a drug for the treatment of a disease caused by an abnormal gene product. 20. An abnormal receptor and a test substance are brought into contact with each other, the operability of the substance with respect to the abnormal receptor is assayed, and a substance which is determined to substantially activate the transmission system of cells having the abnormal receptor is determined. A method for preparing a substance for treating a disease caused by an abnormal receptor, which comprises preparing the substance. 21. The method according to claim 20, wherein the abnormal receptor is an abnormal receptor prepared by expressing a gene encoding the abnormal receptor in a cell. 22. The gene encoding the abnormal receptor is prepared from a gene prepared from a cell of a mammal suffering from a disease caused by the abnormal receptor and a gene prepared from a cell of the same kind of mammal not having the abnormal receptor. 22. The method according to claim 21, wherein the gene encodes an abnormal receptor identified by comparative analysis with the gene. 23. Use of an abnormal gene product obtained by expressing a gene encoding an abnormal gene product in a cell for screening a substance for treating a disease caused by the product. 24. The use according to claim 23, wherein the abnormal gene product is an abnormal receptor. 25. Use of an abnormal gene product obtained by expressing a gene encoding an abnormal gene product in a cell, for screening an agonist of the product. 26. The use according to claim 25, wherein the abnormal gene product is an abnormal receptor.