Information
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Patent Application
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20040214231
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Publication Number
20040214231
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Date Filed
July 01, 200321 years ago
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Date Published
October 28, 200419 years ago
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Inventors
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Original Assignees
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CPC
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US Classifications
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International Classifications
Abstract
Differential expression of genes whose expression is different in the activated eosinophils of atopic dermatitis patients was measured by comparative analysis using a gene chip. As a result, the TR3 and TINUR genes, whose expression is significantly elevated in activated eosinophils, were successfully identified. The present inventors discovered that these genes can be used to test for allergic disease and to screen candidate compounds for therapeutic agents for allergic disease.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of testing for allergic diseases, and methods of screening candidate compounds for therapeutic agents for allergic diseases, using the expression of the allergic disease-related TR3 or “TINUR genes as an index. The present invention is also directed to pharmaceutical agents for treating allergic diseases.
BACKGROUND OF THE INVENTION
[0002] Allergic diseases such as atopic dermatitis are considered to be multifactorial diseases. Multifactorial diseases are caused by the interaction of many different genes, the expression of each of which is independently influenced by multiple environmental factors. Thus, determining the specific genes that cause a specific allergic disease is extremely difficult.
[0003] Allergic diseases are generally presumed to be associated with the expression of genes having mutations or defects, and/or with the overexpression or reduced expression of specific genes. To determine the role of gene expression in a disease, it is necessary to understand how genes are involved in the onset of that disease, and how gene expression is altered by external stimulants such as drugs.
[0004] Recent developments in gene expression analysis techniques have enabled analysis and comparison of gene expression in multiple clinical samples. As an example of such methods, the differential display (DD) method is useful. Liang and Pardee originally developed this method in 1992 (Science, 1992, 257: 967-971). By using this method, several dozen or more samples can be screened at one time, thereby enabling the detection of genes whose expression in one sample differs from other samples. Information essential to determining the causative gene(s) of a disease is expected to be obtained by examining genes with mutations, or genes whose expression changes depending on time and the environment, including genes whose expression is influenced by environmental factors.
[0005] Recently, patient interviews and patient medical and family history have become important factors in the diagnosis of allergic disease. More objective methods of diagnosing allergies include testing patient blood samples and observing patient immune response to allergen(s). Examples of the former method include allergen-specific IgE measurement, the leukocyte histamine release test and the lymphocyte blast transformation test. The presence of allergen-specific IgE is evidence of an allergic reaction against an allergen. However, allergen-specific IgE is not always detected in every patient. Furthermore, in principle, IgE assaying requires tests to be performed on all of the allergens necessary for diagnosis. The leukocyte histamine release test and the lymphocyte blast transformation test are methods for observing immune system reaction towards a specific allergen in vitro. Operation of these methods is complex.
[0006] Another known method useful in allergy diagnosis is based on the immune response observed when a patient contacts an allergen (i.e., the latter method). Such tests include the prick test, scratch test, patch test, intradermal reaction and induction test. These tests allow direct diagnosis of a patient's allergic reaction, but are highly invasive as patients are actually exposed to allergens.
[0007] Methods of confirming the involvement of an allergic reaction, regardless of allergen type, are also being trialed. For example, a high serum IgE titer indicates an allergic reaction in a patient. The serum IgE titer corresponds to the total amount of allergen-specific IgE. Determining the total amount of IgE is simple, regardless of the type of allergen; however, IgE titer may be reduced in some patients, for example, in those with non-atopic bronchitis.
[0008] The number of eosinophils and the level of eosinophil cationic protein (ECP) are diagnostic items for delayed-type reactions following Type I allergy-and allergic inflammatory reactions. The number of eosinophils is considered to reflect the progress of allergic symptoms. ECP, a protein contained in eosinophil granules, is also strongly activated in patients having an asthma attack. Although allergic symptoms can indeed be identified using these diagnostic items, the extent to which they can actually be used as diagnostic indices is limited.
[0009] Therefore, diagnostic indices useful in understanding pathological conditions in patients with allergic diseases, and in determining treatment regimens for such diseases, regardless of the type of allergen, have been greatly sought after. Allergic disease markers that are less risky for patients and capable of readily providing information required for diagnosis would be of great use. If genes associated with allergic disease can be identified, the expression of such genes can be used as an index to test for allergic diseases. Furthermore, if the cellular function of proteins encoded by these genes can be elucidated, observations regarding these functions can be used as a base to promote the development of therapeutic methods and pharmaceutical agents for treating allergic diseases.
SUMMARY OF THE INVENTION
[0010] The present invention was achieved in light of the above context. An objective of the present invention is to identify genes associated with allergic diseases. Furthermore, using expression of these genes as an index, another objective of the present invention is to provide methods of testing for allergic diseases, and methods of screening candidate compounds for therapeutic agents for allergic diseases, as well as pharmaceutical agents for treating allergic diseases.
[0011] The present inventors performed extensive analyses to achieve the above-mentioned objectives. Eosinophils commonly serve as typical clinical indicators of atopic dermatitis. Thus, the present inventors considered that if a gene whose expression level changes with eosinophil levels could be isolated, it could lead to the isolation of a gene directly involved in atopic dermatitis.
[0012] The present inventors first attempted to identify a gene whose expression level differs with a specific allergic disease. Differential expression comparative analysis using a gene chip was carried out on genes expressed in the peripheral blood eosinophils of healthy subjects, and of atopic dermatitis patients with various pathological conditions (light, severe and steroid sensitive, and severe and steroid resistant). Genes showing a greater than 3-fold variation were sorted, and the TR3 gene was selected from among approximately 12,000 A-chip genes, wherein the chip was mainly loaded with known genes. Two cases of eosinophil RNA from each group, including the healthy subjects, were applied to the gene chip, and expression comparison between two groups was carried out by comparing gene expression in four combinations of two cases from each group. Comparison of expression between healthy subjects and subjects with severe symptoms (steroid sensitive) showed that TR3 expression varied by more than three-fold (enhanced in severe symptoms) in all four combinations. To confirm those observations, RT-PCR was carried out on panels of peripheral blood eosinophils having a larger number of patients from healthy subjects and atopic dermatitis patients. These results showed that TR3 expression in atopic dermatitis patients was enhanced as compared to that in healthy subjects, thus reproducing the results obtained using the gene chip.
[0013] TR3 is known as an α-type of the nuclear orphan receptor subfamily; however, to date it has not been reported as being related to allergic disease.
[0014] TINUR is a β-type of the nuclear orphan receptor subfamily, and predicted to be functionally similar to TR3. In the same manner as for TR3, the present inventors carried out a comparison of TINUR expression between healthy subjects and patients, using ABI7700 and the same panel of patient peripheral blood eosinophils, in which there were more than ten samples per group. The results confirmed that, regardless of symptom severity, TINUR gene expression was significantly enhanced in atopic dermatitis patients as compared to healthy subjects. Like the TR3 gene, a relationship between the TINUR gene and allergic disease has not yet been reported.
[0015] Genes suggestive of apoptotic character are found in the peripheral blood eosinophils of atopic dermatitis patients. This may be because negative feedback regulation acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition.
[0016] Allergic diseases may be tested by using the expression level of the TR3 or TINUR gene of this invention as an index.
[0017] The TR3 and TINUR receptors are orphan receptors, and hitherto, neither their native ligands nor activators have been found. The present inventors developed a high-throughput system for searching for ligands, and using this system, succeeded in obtaining compounds that may function as activators of TR3 or TINUR transcription. These compounds are prostaglandins (PGA derivatives) comprising a cyclopentenone structure, and may be native ligands of the TR3 or TINUR receptor. Experiments using mutants in which a receptor's ligand-binding domain (LBD) region had been deleted indicated that the prostaglandin derivatives function by acting on this region. Moreover, experiments utilizing BIAcor demonstrated that PGA derivatives bind to TR3 and TINUR.
[0018] Thus, the present inventors found that it is possible to screen candidate compounds for a therapeutic agent for an allergic disease, and that PGA derivatives are TR3 or TINUR ligand activators.
[0019] The present inventors used a pharmacophore model to simulate the binding site of a PGA derivative TR3 ligand binding domain. The present inventors selected compounds from the database based on structure-activity relationship information for the PGA derivative reporter system. The present inventors selected compounds other than PGA derivatives that matched the binding pocket. These compounds are expected to function as TR3 receptor ligands.
[0020] Compounds that induce TR3 or TINUR gene expression, or compounds that bind to the TR3 or TINUR receptor and promote transcription activity (for example, ligand activators) are expected to have therapeutic effects on allergic diseases.
[0021] Furthermore, the present inventors discovered for the first time that the expression of TR3 and TINUR in cultured peripheral blood eosinophils is dramatically induced by apoptosis stimulation of cells via an anti-CD30 antibody comprising agonist activity towards eosinophil CD30. Therefore, a therapeutic agent for allergic diseases can be provided, wherein such an agent increases TR3 or TINUR gene expression using eosinophil CD30 ligand stimulation, and induces eosinophil apoptosis by regulating the expression of genes downstream of TR3 or TINUR occurring in eosinophils.
[0022] The present invention relates to a method of testing for allergic diseases, and a method of screening candidate compounds for therapeutic agents for allergic diseases. These methods are performed using, as an index, expression of the TR3 or TINUR gene, which are genes highly expressed in activated eosinophils during allergic disease. The present invention also relates to pharmaceutical agents for treating allergic diseases. Specifically, the present invention provides:
[0023] [1] a method of testing for an allergic disease, said method comprising the steps of:
[0024] a) measuring the expression level of a TR3 or TINUR receptor protein, or a gene encoding the TR3 or TINUR receptor protein, in eosinophil cells of a test subject; and
[0025] b) comparing the expression level of the protein or gene in the eosinophil cells of the test subject with an expression level in eosinophil cells of a healthy subject.
[0026] [2] the testing method of claim 1, wherein the gene expression level is measured by cDNA PCR.
[0027] [3] the testing method of claim 1 or 2, wherein the allergic disease is atopic dermatitis.
[0028] [4] a reagent for testing for an allergic disease, said reagent comprising an oligonucleotide of at least 15 nucleotides in length that comprises a nucleotide sequence complementary to a polynucleotide encoding a TR3 or TINUR receptor protein, or to its complementary strand.
[0029] [5] a method of detecting the influence of a candidate compound on the expression level of a polynucleotide of (a) or (b) below, wherein said method comprises the steps of:
[0030] (1) contacting the candidate compound with a cell that expresses a polynucleotide of (a) or (b):
[0031] (a) a polynucleotide encoding a TR3 or TINUR receptor protein; and
[0032] (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein; and
[0033] (2) measuring the expression level of the polynucleotide of (a) or (b).
[0034] [6] the method of claim 5, wherein the cell is from a leukocyte cell line.
[0035] [7] a method of detecting the influence of a candidate compound on the expression level of a polynucleotide of (a) or (b) below, wherein said method comprises the steps of:
[0036] (1) administering the candidate compound to a test animal; and
[0037] (2) measuring the expression intensity of a polynucleotide in the eosinophil cells of the test animal, wherein the polynucleotide is selected from (a) or (b):
[0038] (a) a polynucleotide encoding a TR3 or TINUR receptor protein; and
[0039] (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein.
[0040] [8] a method of screening for a compound that increases the expression level of the polynucleotide (a) or (b), wherein said method comprises the steps of detecting the influence on expression level by the method of any one of claims 5 to 7, and selecting a compound that increases that expression level as compared to a control.
[0041] [9] a method of detecting the influence of a candidate compound on the expression level of a polynucleotide encoding a TR3 or TINUR receptor protein, wherein said method comprises the steps of:
[0042] (1) contacting a candidate compound with a cell or cell extract containing a DNA comprising a structure such that a reporter gene and the transcription regulatory region of a gene encoding a TR3 or TINUR receptor protein are operably linked; and
[0043] (2) measuring the activity of the reporter gene.
[0044] [10] a method of screening for a candidate compound that increases the expression level of a gene encoding a TR3 or TINUR receptor protein, wherein said method comprises the steps of detecting the influence of a compound on the activity of the reporter gene by the method of claim 9, and selecting a compound that increases the activity compared to a control.
[0045] [11] a method of screening candidate compounds for a therapeutic agent for an allergic disease, wherein said method comprises the steps of:
[0046] 1) contacting a test compound with a TR3 or TINUR receptor protein;
[0047] 2) measuring the binding activity between the test compound and the TR3 or TINUR receptor protein; and
[0048] 3) selecting the compound that binds to the TR3 or TINUR receptor protein.
[0049] [12] a method of screening candidate compounds for a therapeutic agent for an allergic disease, wherein said method comprises the steps of:
[0050] 1) providing cells transfected with (a) a DNA that can express a fusion protein of a TR3 or TINUR receptor protein or its ligand binding domain and a transcription regulatory region binding protein, and (b) a DNA having a reporter gene is operably linked downstream of a DNA sequence to which the transcription regulatory region binding protein binds;
[0051] 2) contacting the cell with the test compound;
[0052] 3) measuring the activity of the reporter gene; and
[0053] 4) selecting the compound that changes this activity.
[0054] [13] a therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a compound obtainable by the screening method of any one of claims 10 to 12.
[0055] [14] a therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of any one of claims 10 to 12.
[0056] [15] a therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a ligand of a TR3 or TINUR receptor.
[0057] [16] the therapeutic agent for an allergic disease of claim 15, wherein the ligand of a TR3 or TINUR receptor is a prostaglandin comprising a cyclopentenone structure.
[0058] [17] the therapeutic agent for an allergic disease of claim 16, wherein the prostaglandin having a cyclopentenone structure is selected from the group consisting of prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, and 8-isoprostaglandin A1.
[0059] [18] the therapeutic agent for an allergic disease of claim 15, wherein the ligand of a TR3 receptor is any one of the compounds listed in Tables 14 to 49.
[0060] [19] the therapeutic agent for an allergic disease of any one of claims 13 to 18, wherein the allergic disease is atopic dermatitis.
[0061] [20] an animal model for an allergic disease, wherein the animal is a transgenic non-human vertebrate in which the expression intensity of polynucleotide (a) or (b) below is decreased in eosinophil cells:
[0062] (a) a polynucleotide encoding a TR3 or TINUR receptor protein; and
[0063] (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein.
[0064] [21] the animal model of claim 20, wherein the transgenic animal is a knockout animal.
[0065] [22] a method of inducing cell apoptosis, said method comprising activation of a TR3 or TINUR receptor protein in the cell.
[0066] [23] the apoptosis induction method of claim 22, which comprises the step of contacting a cell with a compound that is obtainable by the screening method of any one of claims 10 to 12, or a prostaglandin comprising a cyclopentenone structure.
[0067] [24] the apoptosis induction method of claim 22 or 23, wherein said cell is an eosinophil cell.
[0068] [25] an apoptosis-inducing agent, which comprises a compound or a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of any one of claims 10 to 12.
[0069] [26] an apoptosis-inducing agent comprising a ligand of a TR3 or TINUR receptor as an active ingredient.
[0070] [27] the apoptosis-inducing agent of claim 26, wherein the ligand of the TR3 or TINUR receptor is a prostaglandin comprising a cyclopentenone structure.
[0071] [28] the apoptosis-inducing agent of claim 27, wherein the prostaglandin comprising a cyclopentenone structure is selected from the group consisting of prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, and 8-isoprostaglandin A1.
[0072] [29] the apoptosis-inducing agent of claim 26, wherein the ligand of the TR3 receptor is any one of the compounds listed in Tables 14 to 49.
[0073] [30] a TR3 or TINUR gene expression-inducing agent, which comprises a ligand of an eosinophil CD30 receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074]
FIG. 1 shows a graph of Table 6.
[0075]
FIG. 2 shows a schematic illustration of a ligand searching system for a TR3 or TINUR receptor constructed by the present inventors. A TR3 or TINUR ligand-binding site is inserted into X, and the full-length retinoic acid X receptor (RXR)α gene is inserted into Y. These constructs are transfected into NIH3T3 cells, and the activity of induced luciferase is measured.
[0076]
FIG. 3 shows a schematic illustration of the structure of the TR3 and TINUR receptor proteins.
[0077]
FIG. 4 shows a graph demonstrating the transcription-activating function of TR3 in a series of cyclopentenone prostaglandins using the system of FIG. 2.
[0078]
FIG. 5 shows a graph demonstrating the results of measuring the expression levels of the TINUR gene in healthy subjects and patients using ABI7700.
[0079]
FIG. 6 shows a graph demonstrating the transcription-activating function of the TINUR gene in a series of cyclopentenone prostaglandins using the system of FIG. 2.
[0080]
FIG. 7 shows diagram of prostaglandin A2 in alpha model, in which the binding position of the PGA derivative for the TR3 ligand binding domain has been simulated using the Pharmacophore model.
[0081]
FIG. 8 shows a graph demonstrating the decrease of prostaglandin A2 transcriptional activity by the LBD deletion mutant. ALBD denotes the deletion mutant.
[0082]
FIG. 9 shows diagrams showing PGA1 and PGA2 bound to TR3 LBD or TINUR LBD, revealed using BIAcor S51. Glutathione S-transferase (GST) was used as a comparison control, and 13,14-Dihydro-15-keto-PGA2 was used as a negative control.
[0083]
FIG. 10 shows graphs demonstrating the results of TR3 expression induction in apoptotic stimulation of peripheral blood eosinophils using an anti-CD30 or anti-Fas antibody. Beta-actin-corrected values and GAPDH-corrected values are shown.
[0084]
FIG. 11 shows graphs demonstrating the results of TINUR expression induction in apoptotic stimulation of peripheral blood eosinophils using anti-CD30 or anti-Fas antibody. Beta-actin-corrected values and GAPDH-corrected values are shown.
[0085]
FIG. 12 is a graph indicating apoptosis induction after treating the eosinophil-specific cell line, AML14.3D10, with anti-CD30 antibody or anti-Fas antibody.
[0086]
FIG. 13 is a graph indicating TR3 expression induction after treating the eosinophil-specific cell line, AML14.3D10, with anti-CD30 antibody or anti-Fas antibody.
[0087]
FIG. 14 shows a graph indicating TINUR expression induction after treating the eosinophil-specific cell line, AML14.3D10, with anti-CD30 antibody or anti-Fas antibody.
[0088]
FIG. 15 shows a working hypothesis for allergic disease treatment via eosinophil cell death, caused by a member of the nuclear receptor Nur subfamily, including TR3 and TINUR.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The present inventors discovered that the expression level of the TR3 and/or TINUR genes increases in the eosinophils of atopic dermatitis patients. Therefore, using TR3 and/or TINUR gene expression level as an index, tests for allergic disease can be performed on test subjects.
[0090] The present invention provides methods for testing for an allergic disease, which comprise the step of measuring the expression level of the TR3 or TINUR gene.
[0091] A preferred embodiment of the present invention includes the following steps:
[0092] (a) measuring the expression level of a gene encoding the TR3 or TINUR receptor protein in the eosinophil cells of a test subject; and
[0093] (b) comparing this measured value to that measured in the eosinophils of a healthy subject.
[0094] The TR3 and TINUR receptors are a and 0-type orphan nuclear receptors respectively, wherein orphan nuclear receptors are composed of three subfamilies. As shown in Table 1, orphan nuclear receptors have various names, and the terms “TR3 gene” and “TINUR gene” as used in the context of the present invention should not necessarily be construed as being limited to human-derived genes.
1TABLE 1
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HumanMouseRat
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αNAK-1 (TR3)nur77NGFI-B
βTINUR/NOTNurr1RNR-1
γMINOR/CHNTECNOR-1
|
[0095] Information relating to the amino acid sequences of these TR3 and TINUR receptor proteins, and the nucleotide sequences of genes encoding these proteins, can be readily obtained from various gene databases available to those skilled in the art. Specifically, the nucleotide sequence of a gene encoding the human TR3 receptor protein (TR3 gene) is shown in SEQ ID NO: 1; and the amino acid sequence of the human TR3 receptor protein is shown in SEQ ID NO: 2. The nucleotide sequence of a gene encoding the human TINUR receptor protein (TINUR gene) is shown in SEQ ID NO: 3; and the amino acid sequence of the human TINUR receptor protein is shown in SEQ ID NO: 4.
[0096] Herein, the general phrase “allergic disease” refers to a disease involving allergic reactions. More specifically, an “allergic disease” is defined as a disease for which an allergen is identified, where there is a strong correlation between exposure to that allergen and the onset of pathological change, and where that pathological change has been proven to have an immunological mechanism. Herein, an immunological mechanism means that leukocytes show an immune response to allergen stimulation. Examples of allergens include mite antigens and pollen antigens.
[0097] Representative allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis, and pollen and insect allergies. Allergic diathesis is a genetic factor that can be inherited by the children of allergic parents. Familial allergic diseases are also called atopic diseases, and the causative, genetically transmitted factor is atopic diathesis. “Atopic dermatitis” is a general term for an atopic disease, especially diseases accompanied by dermatitis symptoms.
[0098] The tests for allergic diseases of the present invention can include, for example, a test for determining whether a subject is affected with an allergic disease, a test for determining whether a subject comprises the trait of being easily affected by an allergic disease, and a test for assessing whether allergic symptoms are improving. The TR3 or TINUR gene of this invention showed increased expression level in the activated eosinophils of atopic dermatitis patients. Since eosinophils are a representative clinical marker for atopic dermatitis, a clinical marker associated with their decrease is useful for assessing therapeutic effects. More specifically, increased TR3 or TINUR gene expression indicates improvement of the allergic disease, accompanied by a decrease in eosinophils.
[0099] There is a correlation between atopic dermatitis severity and the number of eosinophils, such that active reduction of eosinophil number may lead to curing the disease. Measurement of these genes, whose specific induction in eosinophils is accompanied by a decrease in eosinophil numbers, along with discovery of methods or substances that actively induce these genes from outside the cell, may lead to novel methods of atopic dermatitis therapy, and diagnostic methods for evaluating these therapeutic methods.
[0100] Herein, the expression level of the TR3 or TINUR gene includes transcription of the gene to mRNA, as well as translation into their protein. Therefore, a method of testing for an allergic disease according to the present invention can be performed by comparing the expression intensity of mRNA corresponding to the particular gene, or the expression level of the protein encoded by that gene.
[0101] Measurement of TR3 or TINUR gene expression level in a method of testing for allergic diseases of the present invention may be conducted according to gene analytical methods known to those skilled in the art. More specifically, a hybridization technique using as a probe a nucleic acid that hybridizes to either the TR3 or TINUR gene, or a gene amplification technique using as a primer a DNA that hybridizes to a gene of this invention, or such can be utilized.
[0102] Primers or probes that can be used as reagents for testing for an allergic disease according to the present invention include a polynucleotide comprising at least 15 nucleotides that is complementary to the nucleotide sequence of SEQ ID NO: 1 or 3, or the complementary strand thereof. Herein, the term “complementary strand” refers to the other strand of one strand of a double stranded DNA, which is composed of A:T (or A:U for RNA) and G:C base pairs. In addition, “complementary” means not only those sequences completely complementary to a region of at least 15 continuous nucleotides, but also those having a homology of at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or higher. The degree of homology between nucleotide sequences can be determined using a known algorithm, such as BLASTN.
[0103] Such polynucleotides are useful as probes to detect and isolate a polynucleotide encoding a protein of the present invention, or as primers to amplify a polynucleotide of the present invention. When used as a primer, these polynucleotides have a chain length of usually 15 bp to 100 bp, and preferably 15 bp to 35 bp. When used as a probe, DNAs comprising the entire sequence of a polynucleotide of the present invention, or its partial sequence containing at least 15-bp, are used. When used as a primer, the 3′ region must be complementary to a polynucleotide of the present invention, however the 5′ region can be linked to a restriction enzyme-recognition sequence, tag, or the like.
[0104] A “polynucleotide” of the present invention may be either DNA or RNA. These polynucleotides may be either synthetic (isolated) or naturally occurring. In addition, DNA used as a hybridization probe is preferably labeled. Examples of labeling methods are described below. Herein, the term “oligonucleotide” refers to polynucleotides with a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides. Exemplary labeling methods are as follows:
[0105] nick translation labeling using DNA polymerase I;
[0106] end labeling using polynucleotide kinase;
[0107] fill-in end labeling using the Klenow fragment (Berger, SL, Kimmel, AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames, B D, Higgins, S J (1985) Genes Probes: A Practical Approach. IRL Press; Sambrook, J, Fritsch, E F, Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press);
[0108] transcription labeling using RNA polymerase (Melton, D A, Krieg, P A, Rebagkiati, M R, Maniatis, T, Zinn, K, Green, M R. (1984) Nucleic Acid Res., 12, 7035-7056); and
[0109] non-radioisotopic labeling of DNA by incorporating modified nucleotides (Kricka, L J. (1992) Nonisotopic DNA Probing Techniques. Academic Press).
[0110] When testing for allergic diseases using hybridization techniques, for example, Northern hybridization, dot blot hybridization or DNA microarray techniques may be used. Gene amplification techniques such as RT-PCR may also be used. During the gene amplification step of RT-PCR, PCR amplification monitoring can be used to quantitatively analyze expression of the gene of the present invention.
[0111] In PCR gene amplification monitoring, the detection target (the DNA or reverse transcript of RNA) is hybridized to probes that are dual-labeled at both ends with different fluorescent dyes, whose fluorescence cancels each other out. As the PCR proceeds and the Taq polymerase degrades the probe due to its 5′-3′ exonuclease activity, the two fluorescent dyes become distant from each other and fluorescence is detected. Fluorescence is detected in real time. By simultaneously measuring a standard sample in which the target copy number is known, it is possible to use cycle number to determine the target copy number of the subject sample, when PCR amplification is linear (Holland, P. M. et al., 1991, Proc. Natl. Acad. Sci. USA 88: 7276-7280; Livak, K. J. et al., 1995, PCR Methods and Applications 4(6): 357-362; Heid, C. A. et al., 1996, Genome Research 6: 986-994; Gibson, E. M. U. et al., 1996, Genome Research 6: 995-1001). For example, ABI PRISM7700 (PE Biosystems) may be used for the PCR amplification monitoring method.
[0112] A method of testing for allergic diseases of the present invention can also be carried out by detecting a protein encoded by the TR3 or TINUR gene. Test methods that may be employed include those using an antibody that binds to a protein encoded by the TR3 or TINUR gene, such as Western blotting, immunoprecipitation and ELISA.
[0113] Antibodies that bind to the TR3 or TINUR protein used in the detection step may be produced by techniques well known to those skilled in the art. Antibodies used in the present invention may be polyclonal or monoclonal (Milstein, C. et al., 1983, Nature 305 (5934): 537-40). For example, polyclonal antibodies against a protein of the present invention may be produced by collecting blood from mammals sensitized with an antigen, and separating serum from this blood using known methods. Serum containing polyclonal antibodies may be used as polyclonal antibodies. A fraction containing polyclonal antibodies can be further isolated from this serum as required. Alternatively, monoclonal antibodies may be obtained by isolating immune cells from mammals sensitized with an antigen, fusing these cells with myeloma cells or the like, cloning the hybridomas thus obtained, and collecting the antibodies from the culture for use as monoclonal antibodies.
[0114] These antibodies may be appropriately labeled to detect the TR3 or TINUR protein. Alternatively, instead of labeling these antibodies, a substance that specifically binds to these antibodies, for example, protein A or protein G, may be labeled to indirectly detect the protein. ELISA is one example of such an indirect detection method.
[0115] A protein or its partial peptide to be used as an antigen may be obtained by: 1) inserting the TR3 or TINUR gene, or a portion of the TR3 or TINUR gene, into an expression vector, 2) introducing the vector into an appropriate host cell to produce a transformant, 3) culturing the transformant to express the recombinant protein, and 4) purifying the expressed recombinant protein from the culture or the culture supernatant. Alternatively, oligonucleotides consisting of a partial amino acid sequence of the amino acid encoded by the TR3 or TINUR gene can be chemically synthesized and used as the immunogen.
[0116] The samples of this invention are preferably eosinophils derived from test subjects. Eosinophils can be prepared from peripheral blood using conventional methods. For example, leukocytes are isolated by fractionating heparinized blood using centrifugation. Granulocytes can then be fractionated by, for example, Ficoll centrifugation of the leukocytes. Eosinophils can be then isolated by neutrophil depletion using the CD16 antibody. A sample for immunological assays of the aforementioned proteins can then be obtained by disrupting these isolated eosinophils to produce a lysate. Alternatively, a sample for measuring mRNA corresponding to the aforementioned gene can be obtained by extracting mRNA from this lysate. The use of a commercially available kit is useful in extracting mRNA or preparing eosinophil lysate.
[0117] In the present invention, the expression level of the gene serving as the index can be measured from whole blood or a peripheral blood leukocyte population, without isolating eosinophils. In this case, the change of gene expression level in cells can be determined by correcting measured values. For example, the measured expression level of an index gene of the present invention can be corrected based on the measured expression level of a housekeeping gene, that is, a gene specifically expressed in eosinophils, and whose expression level does not significantly change, regardless of cellular conditions.
[0118] Alternatively, where the protein to be detected is a secretory protein, comparison of the expression level of a gene encoding the protein can be accomplished by measuring the amount of the target protein in a sample of the subject's body fluid, such as blood or serum.
[0119] When the result of a test for allergic disease of this invention shows elevated expression of a gene of this invention, allergic symptoms are presumed to be improving together with a decrease in eosinophils. This is especially the case for patients with an allergic disease such as atopic dermatitis Furthermore, this invention also relates to an allergic disease animal model, wherein said animal is a transgenic non-human animal having decreased expression of the polynucleotide of (a) or (b) in eosinophil cells:
[0120] (a) a polynucleotide encoding the TR3 or TINUR receptor protein; and
[0121] (b) a polynucleotide encoding a protein whose expression in the eosinophils of atopic dermatitis patients is increased, wherein the polynucleotide hybridizes under stringent conditions with a polynucleotide encoding the TR3 or TINUR receptor protein.
[0122] According to this invention, a decrease in expression level includes a knockout condition in which gene function has been substantially repressed. Herein, substantial repression of gene function refers to a condition in which neither expression of the gene, nor activity of the protein encoded by that gene, can be observed. Gene expression level can be confirmed by quantitative PCR, such as that shown in the Examples. Comparison with normal conditions can be used to confirm that translation product protein activity is virtually undetectable.
[0123] Such transgenic animals include animals that are incapable of expressing the original protein activity due to, for example, the introduction of a mutation into the coding region of the gene, which artificially causes an amino acid sequence mutation, or the introduction of a stop codon. Examples of amino acid sequence mutations include substitution, deletion, insertion and addition of amino acid(s). In addition, by mutating the transcriptional regulatory region of the gene, the actual expression of the gene of this invention can be controlled.
[0124] Methods for obtaining transgenic animals comprising a particular target gene are known. For example, a transgenic animal can be obtained by a method wherein a gene and an ovum are mixed and treated with calcium phosphate; a method wherein the gene is directly introduced into the nucleus of an pronuclear-stage oocyte using a micropipette under a phase contrast microscope (microinjection method, U.S. Pat. No. 4,873,191); a method wherein embryonic stem cells (ES cells) are used; etc. Other methods have also been developed, including a method for infecting ovum with a retroviral vector in which a gene has been inserted, and a method for transducing a gene into ovum via sperm. This latter sperm vector method is a gene recombination technique whereby an exogenous gene is introduced into an ovum by fertilization with a sperm, wherein that exogenous gene has been incorporated into the sperm by adhesion, electroporation, or the like (M. Lavitranoet, et al. Cell, 57, 717, 1989).
[0125] Transgenic animals of the present invention can be produced using any vertebrate except humans. Transgenic animals comprising various gene insertions and modified gene expression levels are currently being produced using vertebrates such as mice, rats, rabbits, miniature pigs, goats, sheep or cattle.
[0126] An example of a transgenic animal of this invention includes a knockout animal in which expression of a non-human homologue of the human TR3 or TINUR gene (described in SEQ ID NO: 1 and 3 respectively) is inhibited. Observation of the knockout animal phenotype enables knowledge of the specific function of the knocked out gene. The gene comprising the nucleotide sequence of SEQ ID NO: 1 or 3 showed increased expression in the eosinophils of human atopic dermatitis patients. Therefore, an animal in which a homologue of this gene is knocked out is useful as an animal model for allergic diseases.
[0127] For example, if a knockout animal of this invention develops dermatitis, or exhibits a change in measured values relating to some sort of allergic disease, a screening system can be constructed to search for a compound that comprises the function of facilitating recovery from that change.
[0128] Methods for producing knockout animals are well known. Using the example of a mouse, a known method for the production of a knockout animal is by homologous recombination using embryonic stem cells, and then selection of embryonic stem cells in which one of the alleles is modified or destroyed. A chimeric animal containing cells derived from an embryonic stem cell together with cells derived from an embryo can be obtained, for example, by inserting a genetically manipulated embryonic stem cell into a fertilized egg. When this chimeric animal (chimera refers to a single individual formed from somatic cells derived from two or more fertilized eggs) is crossed with a normal mouse, a heterozygote in which one of the alleles is modified or destroyed in its entirety, can be produced. Furthermore, a homozygote can be produced by crossing heterozygotes. The transgenic animals of this invention include both heterozygotes and homozygotes.
[0129] Homologous recombination refers to a mechanism of genetic recombination that occurs between two genes comprising the same or very similar nucleotide sequences. PCR can be used to select cells that have undergone homologous recombination. A portion of an inserted gene, and a portion of the region in which insertion is expected, can be used as primers in a PCR reaction carried out to confirm homologous recombination in cells that produce amplification products. Furthermore, when inducing homologous recombination of a gene expressed in an embryonic stem cell, cell selection can be easily carried out using neomycin resistance, wherein a neomycin resistance gene has been linked to a transgene and introduced into a cell. This and other known methods, and modified methods thereof, can be used to select cells.
[0130] In addition to use in the screening of pharmaceutical agents for the treatment or prevention of allergic diseases, described below, transgenic animals of this invention are also useful for elucidating the mechanisms of allergic diseases, and for testing the safety of screened compounds.
[0131] The present invention revealed that expression of the TR3 and TINUR genes increases in the eosinophils of atopic dermatitis patients. This may be because negative feedback regulation acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition. Therefore, animals that can be used as allergic disease model animals include animals in which the expression level of the TR3, the TINUR gene, or a gene functionally equivalent to the TR3 or TINUR gene, has been artificially lowered in eosinophil cells. A decrease of gene expression level in eosinophils includes a decrease in the expression level over the entire leukocyte population. In other words, this phrase includes decreased expression of the aforementioned genes not only in eosinophils but also over the general leukocyte population. In the present invention, a functionally equivalent gene normally refers to a gene of either (a) or (b), described above. More specifically, examples of functionally equivalent genes of this invention include genes that hybridize under stringent conditions to a gene that encodes TR3 or TINUR. Generally, the following conditions can be indicated as the stringent conditions of this invention. For example, hybridization in 4×SSC at 65° C. followed by washing with 0.1×SSC at 65° C. for one hour. The temperature conditions for hybridization and washing greatly influence stringency and can be adjusted using melting temperature (Tm). Tm varies with the ratio of constitutive nucleotides in the hybridizing base pairs, and with the composition of the hybridization solution (concentrations of salts, formamide and sodium dodecyl sulfate). Therefore, on considering these conditions, one skilled in the art can select appropriate conditions to achieve an equal stringency based experience or experimentation.
[0132] For example, the aforementioned transgenic animals may be used as the animal model of this invention.
[0133] Furthermore, the present invention provides a method for detecting the influence of a candidate compound on the expression level of a polynucleotide of this invention. According to this invention, TR3 or TINUR gene expression level is significantly increased in the eosinophils of atopic dermatitis patients. This is thought to be due to negative feedback regulation that acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition. Therefore, based on these methods for detecting influence on gene expression level, compounds that increase gene expression level can be selected, and therapeutic drugs for allergic diseases can be obtained. Herein, compounds that increase the expression level of a gene refer to compounds that comprise the function of inducing any one of the steps selected from gene transcription, translation and expression of protein activity. The present invention further provides a method for detecting the activity of the TR3 or TINUR gene product protein (transcriptional activation ability), as well as TR3 or TINUR gene expression level. Therapeutic drugs for allergies can be devised by selecting compounds that increase the activity of the TR3 or TINUR gene product protein (transcriptional activation ability).
[0134] The method for detecting the influence of a candidate compound on the expression level of a polynucleotide of this invention can be performed in vivo or in vitro. To detect in vivo influence, an appropriate test animal should be used. Test animals that can be used include, for example, an allergic disease animal model, or an allergic disease animal model that is a transgenic non-human animal in which the expression of the aforementioned (a) or (b) gene is inhibited in eosinophils. In vivo influence on expression level based on the present invention can be detected, for example, according to the following steps:
[0135] (1) administering a candidate compound to a test animal; and
[0136] (2) measuring the expression level of the polynucleotide of the above-described (a) or (b) in the eosinophils of the test animal.
[0137] A test animal for the method of detection of this invention can also include, for example, transgenic animals in which TR3 or TINUR gene expression has been decreased through the expression of a TR3 or TINUR antisense gene. Such transgenic animals may be produced by first constructing an antisense RNA expression vector by inserting the full-length TR3 or TINUR gene, or partial sequence thereof, in the reverse direction and downstream of an appropriate promoter sequence. This expression vector is then introduced into the nucleus to express a TR3 or TINUR antisense gene. Thus, a transgenic animal with reduced TR3 or TINUR gene expression can be obtained. When the expression vector contains a promoter whose transcription is regulated by an appropriate pharmaceutical substance, TR3 or TINUR gene expression level in the transgenic animal can be controlled by administering that substance.
[0138] The influence of a pharmaceutical agent candidate compound on TR3 or TINUR gene expression can be detected by administering that compound to an animal model in which TR3 or TINUR gene expression has been reduced as described above, and then monitoring the effect of that compound on TR3 or TINUR gene expression in the eosinophils of that animal model.
[0139] The method of screening of this invention allows selection of pharmaceutical agents involved in TR3 or TINUR gene expression in various ways. For example, this invention enables the discovery of pharmaceutical agent candidate compounds having any of the following functions:
[0140] Activating a signal transduction pathway that drives TR3 or TINUR gene expression;
[0141] Increasing TR3 or TINUR gene transcription activity;
[0142] Inhibiting degradation or stabilization of the TR3 or TINUR gene transcription product; etc.
[0143] In vitro detection can be performed using a method wherein a candidate compound is contacted with cells expressing one of the above-described (a) or (b) genes, and the expression level of that gene is detected. More specifically, the method may be carried out according to the following steps:
[0144] (1) contacting a candidate compound with cells that express a polynucleotide of the above-described (a) or (b); and
[0145] (2) measuring the expression level of that polynucleotide of the above-described (a) or (b).
[0146] In this invention, cells to be used in step (1) can be obtained by inserting such a polynucleotide into an appropriate expression vector, and then transfecting suitable host cells with that vector. Vector or host cells capable of expressing a gene of this invention should be used. Examples of host cells in the host-vector system are Escherichia coli, yeast cells, insect cells, animal cells and the like. Vectors for use with each of these cells can be routinely selected.
[0147] Vectors may be transfected into a host by biological, physical or chemical methods. Biological methods include, for example, methods using viral vectors; methods using specific receptors; and cell-fusion methods (HVJ (hemagglutinating virus of Japan; Sendai virus) method, polyethylene glycol (PEG) method, electric cell fusion method, and microcell fusion method (chromosome transfer)). Examples of physical methods include microinjection, electroporation and the use of a gene particle gun. Chemical methods are exemplified by the calcium phosphate precipitation method, liposome method, DEAE-dextran method, protoplast method, erythrocyte ghost method, erythrocyte membrane ghost method, and microcapsule method.
[0148] In a detection method of this invention, leukocyte cell lines can be used as cells for expressing the polynucleotide of the aforementioned (a) or (b). Examples of leukocyte cell lines are cell lines derived from leukocytes, such as Eol, YY-1, HL-60, TF-1 and AML14.3D10. Among the leukocyte cell lines, cell lines derived from eosinophils are preferred for a detection method of this invention. Examples of cell lines derived from eosinophils include Eol, YY-1 and AML14.3D10.
[0149] Eol (Eol-1: Saito H et al., Establishment and characterization of a new human eosinophilic leukemia cell line. Blood 66, 1233-1240, 1985) can be obtained from the Hayashibara Research Institute. YY-1 (Ogata N et al., The activation of the JAK2/STAT5 pathway is commonly involved in signaling through the human IL-5 receptor. Int. Arch. Allergy Immunol., Suppl 1, 24-27, 1997) is available from the Institute of Cytosignal Research. AML14.3D10 (Baumann M A et al., The AML14 and AML14.3D10 cell lines: a long-overdue model for the study of eosinophils and more. Stem Cells, 16, 16-24, 1998) is commercially available from Paul CC at Research Service, VA Medical Center, Dayton, Ohio, USA.
[0150] HL-60 clone 15 (ATCC CRL-1964), an undifferentiated leukocyte cell line, will differentiate into eosinophils to produce an eosinophil cell line when cultured for about a week in the presence of butyric acid. Eosinophils are polymorphonuclear and exhibit eosinophilic granules, and can thus be detected by their morphological characteristics. Morphological observations are performed using Giemsa staining and Difquick staining. Generally, a human leukocyte cell line containing eosinophils can be established by cloning an immortalized cell sample from a leukemia patient. Therefore, one skilled in the art can use a conventional method to obtain an eosinophil cell line, as necessary. The method of screening involves the addition of a candidate compound to the aforementioned leukocyte cell line, measurement of the expression levels of the polynucleotides of (a) or (b) in the leukocyte cell line, and selection of a compound that increases the gene expression level.
[0151] Transformed cells in which the expression of the polynucleotide of the aforementioned (a) or (b) is modified can be used as cells for the in vitro detection method. Examples of such transformed cells include cells transformed with an expression vector for the polynucleotide antisense. Cells transformed with an antisense expression vector can be obtained according to a principle similar to that used in the production of the aforementioned transgenic animal. Using the transformed cell thus obtained, the influence of the candidate compound on gene expression level can be detected.
[0152] In a method of the present invention, the expression levels of the polynucleotide of the above-described (a) or (b) can be compared by detecting the expression levels of not only proteins encoded by these genes, but also of their corresponding mRNAs. When comparing expression level using mRNA, the step of preparing an mRNA sample as described above is conducted instead of preparing a protein sample. Protein and mRNA detection can be carried out according to known methods, such as those described above.
[0153] By obtaining the transcriptional regulatory region of the TR3 or TINUR gene, a reporter assay system can be constructed. A reporter assay system is a system of screening for a transcriptional regulatory factor that acts on the transcriptional regulatory region. Such a system uses the expression level of a reporter gene located downstream of the transcriptional regulatory region, and expressed under the control of that regulatory region, as an index.
[0154] A transcriptional regulatory region is exemplified by a promoter and an enhancer, as well as a CAAT box, TATA box or the like, usually found in the promoter region. Examples of suitable reporter genes include the chloramphenicol acetyltransferase (CAT) gene, luciferase gene and growth hormone genes.
[0155] A transcriptional regulatory region of the TR3 or TINUR gene can be obtained using conventional methods as follows. First, a genomic DNA clone comprising the cDNA sequence based on a nucleotide sequence described in SEQ ID NO: 1 or 3, is screened by a method using PCR or hybridization from a human genomic DNA library, such as the BAC or YAC libraries. Based on the resulting genomic DNA sequence, the transcriptional regulatory region of the TR3 or TINUR gene is predicted and obtained. A reporter construct is prepared by cloning the obtained transcriptional regulatory region upstream of a reporter gene. The resulting reporter construct is introduced into a cultured cell strain to prepare transformants for screening. By contacting a candidate compound with a transformant and detecting reporter gene expression, the effect of that candidate compound on the transcriptional regulatory region can be assessed.
[0156] Based on the methods for detecting influence on the expression level of the polynucleotides of the present invention, a compound that alters the expression level of these polynucleotides can be screened. The present invention relates to a method of screening for a compound that alters the expression level of a polynucleotide of above-described (a) or (b), comprising the steps below.
[0157] The present invention provides a method of screening for a compound that increases the expression level of a polynucleotide of above-described (a) or (b), the method comprising the steps of: 1) detecting the influence of a candidate compound on the expression level of the polynucleotide in vivo and/or in vitro, and 2) selecting the compound that increases expression level as compared to a control.
[0158] This invention also relates to a method of screening for a compound that acts on the transcriptional regulatory region, wherein that method uses a reporter assay which utilizes the transcriptional regulatory region of the TR3 or TINUR gene. A compound that increases reporter gene expression level as compared to a control can be selected based on the results of the reporter assay of the present invention, and a compound that induces TR3 or TINUR gene expression can thus be obtained. Thus, the present invention relates to a method of screening for agonists or antagonists that bind to the ligand-binding domain.
[0159] The TR3 and TINUR receptor proteins, discovered by the present inventors as proteins associated with allergic diseases, are orphan receptors and hitherto, their native ligand activators have not been found. TR3 or TINUR protein ligand activators are considered to directly activate TR3 or TINUR in eosinophils, and to promote apoptosis. Therefore, TR3 or TINUR receptor ligand activators are expected to serve as therapeutic agents for allergic disease. Generally, a receptor ligand can be obtained by searching for compounds that bind to the receptor protein.
[0160] The present invention provides a method of screening candidate compounds for therapeutic agents for allergic disease, wherein such a method comprises selecting compounds that can bind to the TR3 or TINUR protein. In this method, the TR3 or TINUR receptor protein is contacted with a test compound, binding activity between each receptor protein and the test compound is measured, and a compound that binds to a receptor protein is selected. Agonists and antagonists can be selected by measuring this binding as well as by measuring TR3 or TINUR transcription activity.
[0161] The TR3 and TINUR receptor proteins of this method include their partial peptides. One skilled in the art can use known methods to measure binding activity between the TR3 or TINUR receptor protein and a test compound of the above-described method.
[0162] For example, if the compound that binds to TR3 or TINUR is a protein, West-Western blotting can be performed as the screening method of the present invention. Specifically, a cDNA library that uses a phage vector (λgtll, ZAPII, etc.) is constructed from tissues or cells predicted to express a protein (test protein) that binds to the TR3 or TINUR protein. This library is then expressed on LB-agarose, and expressed proteins are immobilized onto a filter. The TR3 or TINUR protein is purified as a biotin labeled protein, or as a fusion protein with the GST protein, and reacted with the above-mentioned filter. Binding activity can be evaluated by using streptavidin, anti-GST antibodies, or the like to detect plaques that express the test protein.
[0163] Another embodiment of the method for screening a candidate compound for an allergic disease therapeutic agent of this invention includes the steps of:
[0164] (1) providing cells transfected with (a) a DNA that can express a fusion protein of the TR3 or TINUR receptor protein or a ligand binding domain thereof, and a transcription regulatory region binding protein, and (b) a DNA comprising a reporter gene operably linked downstream of the DNA sequence to which the transcription regulatory region binding protein binds;
[0165] (2) contacting the above-mentioned cells with a test compound;
[0166] (3) measuring the activity of the above-mentioned reporter gene; and
[0167] (4) selecting the compound that changes the above-mentioned activity.
[0168] The phrase “operably linked” in the above-mentioned method refers to a condition in which the reporter gene is bound such that it can be expressed when the TR3 or TINUR receptor protein, or the ligand binding domain of that protein, binds to a ligand of the receptor protein or to a ligand-like compound. The GAL4 protein can be preferably used as the “transcription regulatory region binding protein” in the above-mentioned method. Furthermore, the “DNA sequence to which a transcription regulatory region binding protein binds” can be, for example, a GAL4-binding DNA region. The screening method of the present invention can be performed using a high throughput method.
[0169] In a preferred embodiment of the screening method of the present invention, screening may be performed using the “two-hybrid system” (for example, “MATCHMAKER Two-Hybrid System”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (all of which are manufactured by Clontech), “HybriZAP Two-Hybrid Vector System” (Stratagene), and methods reported in the literature (Dalton S, and Treisman R (1992) “Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element.” Cell 68, 597-612”). More specifically, the method of the present invention may be performed as described below, though it is not to be construed as being limited thereto, and those skilled in the art can appropriately modify the method illustrated below to achieve this invention.
[0170] In the two-hybrid system, the TR3 or TINUR protein or a partial peptide thereof is normally fused with the GAL4 DNA binding domain and expressed in yeast cells. Using cells that are predicted to express a protein that binds to the TR3 or TINUR protein or to the partial peptide thereof, a cDNA library is constructed which expresses the protein as a fusion protein fused with a VP16 or GAL4 transcriptional activating region. The library is then introduced into yeast cells, and library-derived cDNAs are isolated from detected positive clones. (A positive clone can be detected by reporter gene activation caused when a protein that binds to the TR3 or TINUR protein, or their partial peptides including their ligand binding domain, is expressed in yeast cells, and that protein binds to the TR3 or TINUR protein or the partial peptide.) Proteins encoded by the isolated cDNAs can be obtained by transfecting and expressing these cDNAs in E. coli. Thus, proteins that bind to the TR3 or TINUR protein or their partial peptide, and genes encoding these proteins may be prepared. Examples of reporter genes that can be used in the two-hybrid system include, but are not limited to, the HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene and Plasminogen activator inhibitor type 1 (PAI-1) gene. Screening using the two-hybrid method can also be performed using mammalian cells or the like, in addition to yeast cells.
[0171] The present inventors utilized a two-hybrid system that uses mammalian cells, and constructed a high throughput system that can screen for ligands that increase the transcriptional activation function of the TR3 or TINUR protein. This system is an improvement over conventional mammalian two-hybrid systems, and is outlined in FIG. 2 (see Examples below).
[0172] In a preferred embodiment, the screening method of this invention is performed using the aforementioned high throughput system, developed by the present inventors.
[0173] TR3 or TINUR expression is induced under conditions of leukocyte hyperactivity, as in the peripheral blood during atopic dermatitis. As a result, there is a strong possibility that cell apoptosis will be induced. Ligands that exist in vivo can exist in locations where the nuclear receptor is highly expressed. Therefore, the present inventors screened according to the above-mentioned method, using small molecule lipid-soluble mediators predicted to be produced under such conditions as ligand candidate test compounds. Accordingly, the present inventors succeeded in obtaining from among the lipid-soluble mediators the following ligand activators for TR3: prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, 8-isoprostaglandin A1 and such; and for TINUR: prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin J2, 8-isoprostaglandin A1 and such. These compounds are prostaglandins comprising a cyclopentenone structure. This shows that ligand activators that up-regulate the transcriptional activating function of TR3 or TINUR can be obtained using a method of this invention.
[0174] Screening of compounds that bind to the TR3 or TINUR protein can also be performed using affinity chromatography. For example, the TR3 or TINUR protein can be immobilized on an affinity column carrier, and a test sample predicted to express a protein that binds to the TR3 or TINUR protein is applied thereto. Test samples that can be used in this case include cell extracts and cell lysates. After applying a test sample, the column is washed and any protein that has bound to the TR3 or TINUR protein can be prepared.
[0175] A DNA encoding a prepared protein can be obtained by analyzing that protein's amino acid sequence, synthesizing oligo DNAs based on the analyzed sequence, and then screening a cDNA library using those DNAs as a probe.
[0176] In the present invention, a biosensor utilizing the phenomenon of surface plasmon resonance may also be used to detect or measure the bound compound. A biosensor utilizing surface plasmon resonance (for example, BIAcore, Pharmacia) uses surface plasmon resonance signals to allow real-time observation of the interaction between the TR3 or TINUR protein and the test compound. Therefore, biosensors such as BIAcore can be used to evaluate binding between the TR3 or TINUR protein and a test compound.
[0177] Isolation of compounds that bind to the TR3 or TINUR protein can be routinely performed by those skilled in the art. Methods for screening molecules that bind to a protein of this invention, other than those mentioned above, include methods wherein synthetic compounds, natural product banks or random phage peptide display libraries are acted on the immobilized TR3 or TINUR protein.
[0178] A cell used to detect the influence of a candidate compound on the expression level and transcriptional activation mechanism of the TR3 or TINUR gene, and a polynucleotide or antibody for examining the expression level of this gene, can be combined as a detection kit using a method of the present invention. Candidate compound(s) for use as a positive or negative control, as well as instructions and the like, may be included in the kit. Based on the present invention, a kit for detecting the influence of a candidate compound on the expression level and transcriptional activation mechanism of the TR3 or TINUR gene, may be utilized as a kit for screening compounds that modify the expression level or transcriptional activation mechanism of the TR3 or TINUR gene.
[0179] Test candidate compounds that can be used in a screening method of this invention include, without limitation, compound preparations synthesized by chemical methods, such as steroid derivatives; compound preparations synthesized by combinatorial chemistry; mixtures containing multiple compounds, such as extracts from animal or plant tissues, or microbial cultures; purified proteins; expression products of gene libraries; and libraries of synthetic peptides. Furthermore, in a method of screening for compounds that bind to the TR3 or TINUR protein of the present invention, without limitation, it is preferable to use small molecule lipid-soluble mediators as test candidate compounds.
[0180] Compounds selected using a method of screening of the present invention are useful as therapeutic agents for allergic diseases. Expression of the TR3 or TINUR gene increases in the eosinophils of atopic dermatitis patients. These apoptosis associated genes may be induced due to negative feedback regulation which acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition. Therefore, compounds that can enhance the expression or function of these genes are expected to comprise the action of suppressing the symptoms of atopic dermatitis.
[0181] Compounds selected using a screening method of the present invention are expected to serve as allergic disease therapeutic agents that utilize a completely novel functional mechanism that involves TR3 or TINUR activation accompanied by eosinophil apoptosis induction. Therefore, the present invention provides allergic disease therapeutic agents comprising, as an active ingredient, a compound that can be obtained by a screening method of this invention.
[0182] The above-mentioned compound includes compounds in which a portion of the structure of the compound that may be isolated using a screening method of this invention is altered by addition, deletion and/or replacement. As described above, among lipid-soluble mediators, prostaglandins comprising a cyclopentenone structure were found by the present inventors to be compounds that enhance the transcriptional activation ability of TR3 or TINUR (TR3 or TINUR ligand activators). Therefore, examples of allergic disease therapeutic agents according to this invention preferably include those that comprise, as an active ingredient, a prostaglandin that comprises a cyclopentenone structure and that can be obtained using a screening method of this invention. Specific examples of prostaglandins for TR3 include prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, 8-isoprostaglandin A1 and such. Prostaglandins for TINUR include prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandinA2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin J2, 8-isoprostaglandin A1 and such.
[0183] Furthermore, substances having TR3 or TINUR receptor ligand activity of the present invention appear to induce eosinophil apoptosis and may have anti-allergic effects. Therefore, the present invention provides apoptosis-inducing agents comprising a TR3 or TINUR receptor ligand as an active ingredient, as well as allergic disease therapeutic agents comprising a TR3 or TINUR receptor ligand as an active ingredient. An apoptosis-inducing agent of the present invention is preferably an eosinophil apoptosis-inducing agent.
[0184] Examples of TR3 or TINUR receptor ligands include the above-mentioned prostaglandins comprising a cyclopentenone structure, and the compounds listed in Tables 14 to 49, shown below.
[0185] From docking studies of the three-dimensional structure of TR3 and TINUR respectively, one skilled in the art can readily infer, synthesize, and develop synthetic TR3 or TINUR ligands.
[0186] Generally, the term “docking study” refers to a computer-mediated search for compounds and conformations that fit into a ligand-binding domain, wherein these compounds and conformations are taken from a 3D database comprising several hundred thousand compounds, and wherein a 3D query pharmacophore model based on the 3D structure of a receptor is used. The docking study can be performed, for example, according to procedures (1) to (4):
[0187] (1) Construct a 3D protein structure (homology model) using Modeler;
[0188] (2) Search for a binding site using C2.LigandFit;
[0189] (3) Construct a pharmacophore query for the binding site using C2. SBF; and
[0190] (4) Search a 3D database using the pharmacophore query.
[0191] Literature relating to 3D pharmacophore searches includes, for example, Pharmacophore Perception, Development, and Use in Drug Design (1 ul Biotechnology Series, 2)-US-ISBN:0963681761 (Hardcover) Guner, Osman F. (Ed.)/Publisher: Intl. Univ. Line Published 1999/12.
[0192] Pharmaceutical agents containing such a synthetic ligand as an active ingredient are also included in the allergic disease therapeutic agents of this invention. Furthermore, by using the above-described synthetic ligands as test candidate compounds in an above-mentioned method of this invention, one can evaluate whether or not the synthetic ligand is a true ligand.
[0193] Having discovered that expression of the TR3 or TINUR receptor of this invention is specifically induced in eosinophils, the present inventors went on to search for small molecule ligands of these receptors. More specifically, they used a pharmacophore model to simulate the binding site of the PGA derivative of the TR3 ligand-binding region, and based on structure-activity relationship information on the PGA derivative reporter system, they selected from the database compounds other than PGA derivatives matching the binding pocket. Thus, compounds selected as described above are included as ligands of the TR3 or TINUR receptor of this invention. These compounds are shown in Tables 14 to 49. Such compounds may be more useful than agonist antibodies against the receptor of this invention.
[0194] The present inventors further discovered that eosinophil CD30 ligand stimulation increases the expression of the TR3 or TINUR gene. Thus, the present invention provides an expression-inducing agent for the TR3 or TINUR gene, which includes a ligand of the eosinophil CD30 receptor. The expression-inducing agent is expected to serve as an allergic disease therapeutic agent that functions by inducing eosinophil apoptosis by regulating expression of genes downstream of TR3 or TINUR in eosinophils.
[0195] The therapeutic agents, apoptosis-inducing agents, and gene expression-inducing agents for an allergic disease of this invention can be formulated by mixing an active ingredient with a physiologically acceptable carrier, excipient, diluent or such. The therapeutic agent for an allergic disease of this invention can be administered orally or parenterally, with the aim of improving allergic symptoms.
[0196] Oral drugs can be selected from dosage forms such as granules, powders, tablets, capsules, solutions, emulsions, suspensions and so on. Examples of parenteral agents include injections, suppositories and ointments. Injections may include subcutaneous injections, intramuscular injections and intraperitoneal injections.
[0197] The dosage of the therapeutic agent for allergic disease according to the present invention may vary depending upon patient age, sex, bodyweight and symptoms; treatment effects; administration method; treatment duration; and the type of active ingredient contained in the pharmaceutical composition, etc. Generally, the agent can be administered to an adult in the range of 0.1 mg to 500 mg per dose, and preferably 0.5 mg to 20 mg per dose. However, since dose changes with a variety of conditions, a dosage less than that described above may be sufficient in certain cases, and a dosage exceeding this range may be required in others.
[0198] The present inventors also discovered that cell apoptosis is induced by increased expression of the TR3 or TINUR receptor protein. Therefore, apoptosis can be induced by activating the TR3 or TINUR protein in cells. Thus, the present invention provides a method of inducing apoptosis of cells that comprises activation of the TR3 or TINUR receptor protein in these cells. The above method also includes a method wherein cell apoptosis is induced by the activation of TR3 or TINUR gene expression.
[0199] In a preferred embodiment of a method of the present invention, apoptosis is induced by contacting cells with a compound, or with a prostaglandin comprising a cyclopentenone structure, where these can be obtained by a screening method of this invention. The cells in a method of this invention are preferably eosinophils. The number of peripheral blood eosinophils is known to decrease in atopic dermatitis patients. Therefore, an allergic disease may be treated by specifically leading eosinophils to cell death, utilizing the method of the present invention. Thus, the present method is expected to lead to the development of novel methods for treating allergic disease.
[0200] Since compounds or prostaglandins comprising a cyclopentenone structure obtainable using a screening method of this invention are considered to comprise the function of inducing apoptosis, the present invention also provides apoptosis-inducing agents that comprise these compounds.
[0201] The present invention provides a gene whose expression differs in the activated eosinophils of atopic dermatitis patients. The use of the expression of a gene of this invention as an index enables testing for allergic disease and screening for candidate compounds for therapeutic agents.
[0202] The expression level of the allergic disease-associated genes of the present invention can be conveniently determined, regardless of allergen type. Therefore, the pathology of allergic reactions can be comprehensively understood.
[0203] The method of testing for allergic diseases of the present invention is less invasive for patients because gene expression level can be analyzed using peripheral blood eosinophils as samples. Every year, high throughput and cost effective gene analysis technology methods are being developed. Therefore, it is expected that in the near future, a method of testing for allergic diseases of the present invention will become an important bedside diagnostic tool. Accordingly, a method of the present invention is very valuable from a diagnostic perspective.
[0204] A screening method of the present invention is carried out using, as an index, a genetic function closely associated with eosinophil variation, which is a representative clinical marker for atopic dermatitis. Therefore, compounds that can be found using a screening method of the present invention are expected to be useful for pathological regulation of a wide variety of allergies.
[0205] The therapeutic agents for an allergic disease provided by the present invention are also useful as pharmaceutical agents that utilize a completely novel functional mechanism involving TR3 or TINUR activation along with eosinophil apoptosis induction.
[0206] Hereinafter the present invention is specifically illustrated with reference to Examples; however, is not to be construed as being limited thereto.
Differential Expression Analysis in the Peripheral Blood Eosinophils of Atopic Dermatitis Patients Using Affymetrix GeneChip
[0207] In order to discover novel therapy-associated genes with fluctuating expression, or genes useful in diagnosis, differential expression comparison analysis using GeneChip was carried out on genes expressed in the peripheral blood eosinophils of healthy subjects, and of atopic dermatitis patients with various pathologic conditions (light and severe steroid sensitivity, severe steroid resistance) This analysis is described below.
[0208] Table 2 shows the profiles of six atopic dermatitis patients and two healthy subjects from whom blood samples were drawn. Allergen non-specific (Total IgE), mite-specific and cedar-specific IgEs were measured using the EIA method. More specifically, test sera were allowed to react with an anti-human IgE antibody-bound cap, and allergen non-specific, mite-specific or cedar-specific IgE antibodies in the sera were bound. Next, β-D-galactosidase-labeled anti-human IgE antibody and a substrate solution (4-methylumbelliferyl-β-D-galactopyranoside) were added and reacted, producing a fluorescent substance. The reaction was quenched by adding a quenching solution, and antibody concentration was determined using the fluorescence intensity of a simultaneously measured standard IgE. L-lactate dehydrogenase (LDH) was measured using the UV method (Wroblewski-La Due method). The rate of NADH decrease caused by its reaction with pyruvic acid was calculated using decreases in absorbance. L-type Wako LDH (Wako Pure Chemicals) and a 7170-type automatic analyzer (Hitachi) were used to measure LDH values. The number of eosinophils was measured using microscopic examination and an automatic hemocyte analyzer SE-9000 (RF/DC impedance system, Sysmex), using 2 ml of EDTA-supplemented blood as the sample.
2TABLE 2
|
|
Severe ADSevere AD
subjectsubject
HealthyLight AD*(steroid(steroid
subjectsubjectsensitive**)resistant**)
Sex
MaleFemaleMaleFemaleFemaleMaleMaleMale
|
Age2317302512162416
Total452553802,40015,00014,00070,000
IgE (U/ml)
Cedar IgE<0.34<0.34<0.346.12<0.3494.860.6>100
Mite IgE<0.34<0.34<0.3418.2>100>100>100>100
LDH228241211296477465303595
|
*the dermatitis area <=10% the entire surface area
**sensitivity compared to standard local glucocorticoid therapy.
[0209] (1) RNA Extraction from Peripheral Blood Eosinophils for Use with a DNA Chip
[0210] A 3% dextran solution was added to whole blood drawn from a patient, and the mixture was left to stand at room temperature for 30 minutes to precipitate erythrocytes. The leukocyte fraction in the upper layer was collected, layered onto Ficoll solution (Ficoll-Paque PLUS; Amersham Pharmacia Biotech), and centrifuged at 1500 rpm for 30 minutes at room temperature. The granulocyte fraction collected in the lower layer was reacted with CD16 antibody magnetic beads at 4° C. for 30 minutes. Cells were separated using Magnetic cell sorting (MACS), and cells that eluted without being trapped were used in the experiment as eosinophils.
[0211] Eosinophils prepared as described above were dissolved in Isogen (Nippon Gene; Wako Pure Chemicals) and RNA was separated from this solution according to the Isogen protocol. Chloroform was added, the mixture was stirred and centrifuged, and the aqueous layer was collected. Next, isopropanol was added, the mixture was stirred and centrifuged, and the precipitated total RNA was collected. DNase (Nippon Gene; Wako Pure Chemicals) was added to the collected total RNA, the mixture was reacted at 37° C. for 15 minutes, and RNA was collected by phenol-chloroform extraction followed by ethanol precipitation. Using these RNAs and a gene chip, analyses were carried out according to the Affymetrix protocol, as detailed below.
[0212] (2) cDNA Synthesis for DNA Chip
[0213] Single stranded cDNA was prepared from 2 μg to 5 μg of total RNA, using reverse transcription with T7-(dT)24 (Amersham Pharmacia Biotech) as a primer, Superscript II Reverse Transcriptase (Life Technologies), and the method of the Affymetrix Expression Analysis Technical Manual. The T7-(dT)24 primer comprises a nucleotide sequence whereby d(T)24 has been added to a T7 promoter nucleotide sequence as shown below.
3|
T7-(dT)24 primer:
|
5′-GGCCAGTGAATTGTAATACGACTCACTATAG(SEQ ID NO:11)
GGAGGCGG-(dT)24-3′
[0214] Next, in accordance with the Expression Analysis Technical Manual, DNA ligase, DNA polymerase I, and RNase H were added to synthesize double-stranded cDNA. The cDNA was extracted by phenol-chloroform extraction, passed through Phase Lock Gels, and then purified using ethanol precipitation.
[0215] Biotin-labeled cRNA was synthesized using a BioArray High Yield RNA Transcription Labeling Kit. The cRNA was then purified using an RNeasy Spin column (QIAGEN), and fragmented by heat treatment.
[0216] One to 5 μg of this cRNA was added to a hybridization cocktail, in accordance with the Expression Analysis Technical Manual. This was placed into an array and hybridized for 16 hours at 45° C. The DNA chip used was a GeneChipR HG-U95A (Affymetrix). GeneChipR HG-U95A consists of probes comprising approximately 12,000 kinds of nucleotide sequence, derived from human cDNAs and ESTs.
[0217] The DNA chip was washed, and then streptavidin phycoerythrin was added for staining. After washing, a mixed antibody solution of normal goat IgG and biotinylated goat anti-streptavidin IgG antibody was added to the array. To enhance fluorescence intensity, streptavidin phycoerythrin was again added for staining. After washing, the chip was set into a scanner and analyzed using DNA chip analysis software.
[0218] (3) DNA Chip Analysis
[0219] Expression fluorescence sensitivity was measured and data analysis was performed using the DNA chip analysis software ‘Suite’. First, absolute analysis was performed on all chips, and gene expression level in each of the samples used was measured.
[0220] In the analysis of a chip's data, positives and negatives were determined by fluorescence intensity comparison with a probe set of perfect-match and m is match probes. Using Positive Fraction, Log Avg, and Pos/Neg values, results were evaluated into the three categories of Absolute Call: P (present), A (absent), and M (marginal). Definitions of these terms are shown below:
[0221] Positive Fraction: the ratio of positive pairs to probe pairs.
[0222] Log Avg: the average of the log of the fluorescence intensity ratio between perfect match and mismatch probe cells.
[0223] Pos/Neg: the ratio between the number of positive and negative probe pairs.
[0224] Average difference (Avg Diff), which is the average value of the difference in fluorescence intensities between perfect-match and mismatch probe cells, was also calculated.
[0225] Genes whose expression varied by more than threefold between patients and healthy subjects were screened, and TR3 was selected from approximately 12,000 HG-U95A chip genes. Since two eosinophil RNA samples from each group, including the healthy groups, were placed onto a gene chip, four combinations of expression comparison, 2×2 between each case, could be carried out. Expression comparison results indicated that TR3 showed a greater than three-fold variation (enhanced in severe cases) in all four combinations between healthy and severe (steroid sensitive) cases (Table 3).
4TABLE 3
|
|
ExperimentProbeAccessionAvgAbsDiffAvg DiffFold
NameSetNo.AnnotationDiffCallCallChangeB = AChange
|
C4E307-315280_g_atL13740TR3 orphan receptor1316PI1208*˜3.74(4I)
C4E307-340280_g_at1234PI1259*˜3.9
C4E309-315280_g_at2042PI1758*˜4.9
C4E309-340280_g_at1913PI1956*˜5.5
|
[0226] Primers and TaqMan probes used in ABI7700 were designed by Primer Express (PE Biosystems) from sequence information at the National Center for Biotechnology Information (NCBI), and based on accession numbers obtained using Suite. The 5′-end of the TaqMan probe was labeled with FAM (6-carboxy-fluorescein) and the 3′-end was labeled with TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) The primers and probe used in the TaqMan method are shown below.
[0227] Primer 1 (5′): CCACTTTGGGAAGGAAGATGCT (SEQ ID NO: 5)
[0228] Primer 2 (3′): ACTTTCGGATGACCTCCAGAGA (SEQ ID NO: 6)
[0229] TaqMan probe: ATGTACAGCAGTTCTACGACCTGCTCTCCG (SEQ ID NO: 7)
[0230] cDNA prepared from the total RNA by reverse transcription using poly-T (12 to 18 mer) as primers was used as the template. In order to make a standard curve for the calculation of copy number, a plasmid clone containing the nucleotide sequence amplified using both primers was prepared, and serial dilutions thereof were utilized as the template for reaction. The reaction mixture composition for monitoring PCR amplification is shown in Table 4.
5TABLE 4
|
|
Reaction mixture composition for
ABI-PRISM 7700 (amount per well)
|
|
Sterile distilled water25.66 (μl)
10× TaqMan buffer A 5
25 mM MgCl2 7
dATP (10 mM) 1.2
dCTP (10 mM) 1.2
dGTP (10 mM) 1.2
dUTP (10 mM) 1.2
Forward Primer (100 μM) 0.15
Reverse Primer (100 μM) 0.15
TaqMan Probe (6.7 μM) 1.49
AmpliTaq Gold (5 U/μl) 0.25
AmpErase UNG (1 U/μl) 0.5
Template solution 5
Total volume50
|
[0231] In order to correct differences in cDNA concentrations among the samples, the same quantitative analysis was carried out for the β-actin gene that was used as the internal standard. The copy number of the target gene was calculated by performing corrections based on the copy number of this β-actin gene. For β-actin gene quantification, human cDNA was used as the template.
[0232] The primers and probe accompanying the TaqMan β-actin Control Reagents (PE Biosystems) were used for the measurement of β-actin. Their nucleotide sequences are as follows:
6|
β-Actin forward primer:
|
TCA CCC ACA CTG TGC CCA TCT ACG A(SEQ ID NO:12)
|
β-Actin reverse primer:
|
CAG CGG AAC CGC TCA TTG CCA ATG G(SEQ ID NO:13)
|
β-actin TaqMan probe:
|
5′-(FAM)ATGCCC-T(TAMRA)-(SEQ ID NO:14)
CCCCCATGCCATCCTGCGTp-3′
[0233] FAM: 6-carboxy-fluorescein:
[0234] TAMRA: 6-carboxy-N,N,N′,N′-tetramethylrhodamine
[0235] GeneChip expression analysis is principally aimed at genetic screening, however as each group had only two samples, the reliability of this screening was confirmed by carrying out an expression comparison between healthy subjects and patients by ABI7700 in a large patient peripheral blood eosinophil panel, in which the number of samples per group was more than ten (Table 5).
7TABLE 5
|
|
Anti-
ToalAnti-mitecedarEosinophilEosinophil
No.Sample IDDonor IDTransfer IDSexAgeIgEIgEIgELDH(%)(mm3)
|
|
131BL10138V-00026 10138F265<0.34<0.34105080
healthy2BL10140V-00015 10140M52810.71<0.34782150
subjects3BL10141V-00040 10141F32590.37<0.34326040
4BL10142V-00032 10142F358314.611.21873250
5BL10143V-00029 10143F4529<0.341.75113290
6BL10144V-00027 10144F2917<0.341.5174290
7BL10145V-00034 10145F26120<0.3417.12723590
8BL10146V-00030 10146F30560<0.3463.22511120
9BL10147V-00001 10147M5044<0.3417.92654130
10BL10148V-00003 10148M4322043.542425250
11BL10149V-00028 10149M3211019.842453180
12BL10150V-00035 10150M6386<0.3412.62095300
13BL10151V-00019 10151M4842<0.34143001180
1514BL00058PA000799707311M05819.71390
light15BL00068PA000849708072F1316876.8365
cases16BL00112PA001129712051M25192.2151
17BL00123PA001209712252F1079912.91050
18BL00133PA001299712266M122741.6122
19BL00198PA000239807213M21963015.11080
20BL00207PA001819807273F66688635
21BL00217PA001909808033M577722.31790
22BL00221PA000429808061F814946.6378
23BL00234PA000299808311F57026.6510
24BL00252PA001769901071M1420967.2333
25BL00259PA001629902161M20262213.3846
26BL00270PA002139903292M152307.5368
27BL00317PA002400003282F141063.7724.72.8154
28BL00327PA001360004033M81178<0.35<0.354.4396
1529BL00095PA000999710031M31592.5190
moderate30BL00128PA001249712261M1271585.2361
cases31BL00145PA000489802192F923495.1193
32BL00268PA001799903261M95129.5906
33BL00278PA002179904061M15108222.11110
34BL00328PA001750004041M74775>10093.37.1638
35BL00089PA000989709092M735913.3638
36BL00110PA001109711281F311.56.1198
37BL00122PA001199712251F125289.7643
38BL00139PA000259801082M182261413.71140
39BL00156PA001439803264M626255551
40BL00287PA002219906231M1511493.7601
41BL00296PA000599908201M516396.8477
42BL00323PA002440003302M64532>10069.111909
43BL00335PA00259BL 18526369F141581>1005.4615.91820
1844BL00078PA000909708251F31353.8254
severe45BL00084PA000679709021M321499.81000
cases46BL00163PA001489803304M111373.5274
47BL00168PA001529804033F1927325.2261
48BL00180PA001639805151M171475813.61010
49BL00242PA000019810061M1913747131230
50BL00243PA002009810221F6109675.9662
51BL00247PA000719812211M161161013.4972
52BL00260PA002099902162M01362.5277
53BL00262PA001209902181F101203109
54BL00150PA001379803161F83714.9375
55BL00257PA002089902053M112687.6468
56BL00293PA002279907221F101830113.81750
57BL00298PA002299909141M119591>10018.211.9940
58BL00314PA002380002151M1923726>100306376
59BL00318PA002410003283F7131<0.35<0.355.7330
60BL00321PA002430003286F4232<0.35<0.359.1856
61BL00337PA002610005191F2947452.531.612.3797
|
[0236] TR3 expression in peripheral blood eosinophils was confirmed to be significantly enhanced in multiple comparisons of atopic dermatitis patients compared to healthy subjects, and this was largely independent of case severity (Table 6, FIG. 1).
8TABLE 6
|
|
L13740
L13740(raw)correction
C1E-2β-actin (raw)copy/copy/β-correctionraw/beta
L13740Bloodcopy/ng5 ng1 ngraw(/ng)/averagecorrection
|
13 healthy125312611192241.01130301221
subjects2541166563711272.16209434521
321423924544910.855938946573
4369621517610351.476729393701
5716536632412652.862741935442
6169173696913940.6758875082062
76013101142622852.40238633951
821306220974190.851236036493
937158912662531.484591854171
1064629719553912.582119848151
1120873721834370.833956352524
122121141313026260.847449033099
1337953912052411.516355526159
15 light1450875848939792.032618527481
cases15248937696213920.9945646911400
162218131292825860.8861986042918
173151681186223721.2591747961884
181418271190623810.5666367694202
192440281754235080.9749535843598
203480511494029881.3905523512149
213876932006340131.5489312342591
2226846842328461.072599907789
23206673584311690.8257099551415
241366521096821940.5459590334018
2521896346199240.8748123291056
2620927338797760.836097009928
2713197732966590.527282361250
281210642219144380.4836807979176
6 moderate2916590100.6628153310
cases in the301341191259525190.5358413464701
remission318634046939390.3449490822721
stage3247244037977591.887519071402
331709142451349030.6828452447180
3436781814972991.469525949204
9 moderate351622582369847400.6482612187311
cases in the369096900.3634432110
exacerbation372464602465249300.9846710425007
stage381468051280825620.5865223014367
391791791060321210.7158638182962
4013885848849770.5547713661761
41133317521010420.5326350511956
4217130852561105120.68441996615359
432852959041811.139827753159
10 severe4415490249949990.6188728761614
cases in the45789481299225980.3154187098238
remission4623161245959190.925346905993
stage47155564733714670.6215165842361
4838584814282861.541561787185
49264744437871.0577207883
501447153528370570.57817446512205
51205943754515090.8227950171834
52155395533510670.620841691719
531517032193343870.6060925057238
8 severe5439782120004001.589395971252
cases in the55446400505710111.783480045567
exacerbation562807248951791.121564845160
stage57161385650013000.6447752072016
581349781110322210.5392716244118
59247401971239420.098841439885
602417933748474970.9660239917760
61930682152043040.37183179911575
total15268113
|
[0237] (4) Statistical Analysis
[0238] Using the above-mentioned data, parametric and non-parametric multiple comparison tests were carried out. Statistical analyses were carried out using an SAS SYSTEM, Version 4.0 Preclinical Package (SAS Institute Inc.). The results are shown in Table 7. Values were significantly greater in the patient group for all of the multiple comparisons between healthy subjects and light cases, healthy subjects and moderate cases, and healthy subjects and severe cases.
9TABLE 7
|
|
C4E HG-U95A statistical analysis results (β-actin-corrected)
Parametric multiple comparisonNon-parametric multiple comparison
Name of geneDunnettp-valueTukeyp-valueDunnettp-valueTukeyp-value
|
L13740TR3AS > Nm0.0533AL > Nm0.0339AM > Nm0.0189
orphanAM > Nm0.01AS > Nm0.0378
receptorAS > Nm0.0204
|
(Nm = normal subject,
AL = Light case of atopic dermatitis,
AM = Moderate case of atopic dermatitis,
AS = Severe case of atopic dermatitis)
[0239] Genes indicative of apoptotic character may be enhanced in the peripheral blood eosinophils of patients with atopic dermatitis conditions due to negative feedback regulation, which acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition.
TR3 Receptor Ligand Search
[0240] Enhanced TR3 function can be used to promote a pathway that specifically leads eosinophils to cell death. It is highly possible that this will lead to therapies for not only asthma, but also for a variety of allergic diseases including atopic dermatitis, which was investigated by the present inventors. Structurally, TR3 is a nuclear receptor; however, it is an orphan receptor and its native ligand and activator are still unknown. If these can be discovered, TR3 can be directly activated in eosinophil cells to promote apoptosis. Therefore, it was thought that the ligand activators were highly likely to be anti-allergic agents, and a high-throughput system for ligand screening was constructed.
[0241] As shown in FIG. 2, a mammalian two hybrid system was slightly modified by inserting the ligand binding domain sequence or full-length TR3 gene (FIG. 3) into pBIND. This was done to facilitate expression of a protein in which the DNA binding domains of TR3 and GAL4 were fused in frame. A plasmid comprising the TR3 ligand binding domain sequence inserted into pBIND, and a luciferase reporter plasmid comprising a GAL4 binding site, were co-transfected into NIH3T3 cells. Luciferase activity was measured automatically. At this time, activity was also measured by adding a retinoic acid X receptor (RXR) α-gene, which is a transcription factor that forms a heterodimer with TR3. By further adding low molecular weight substances to this system, transcriptional enhancement activity can also be used for screening.
[0242] TR3 expression is enhanced in activated eosinophils, such as in the peripheral blood of atopic dermatitis patients. Ligands existing in vivo may exist in sites where nuclear receptors are highly expressed. Therefore, small molecule lipid-soluble mediators considered to be produced under such conditions were added to the assay system, and evaluated based on their ability to enhance luciferase activity. Of these lipid-soluble mediators, the activity of enhancing the transcription-activating ability of TR3 was found in prostaglandins comprising a cyclopentenone structure, such as prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 13,14-dihydro-15-keto prostaglandin A2, 15(R)-15-methyl prostaglandin A2, and delta12-prostaglandin J2 (FIG. 4, Tables 8 to 12). In this manner, the method established by the present inventors paved the way for the high throughput discovery of native and synthetic TR3 ligands. At the same time the present inventors also found that compounds such as prostaglandin A2, prostaglandin A1, and similar metabolites have a high probability of being authentic TR3 native ligands.
10TABLE 8
|
|
Nur77FullNurr1
LBD-ligandlength Nur77-LBD-ligandFull length Nurr1-
activityligand activityactivityligand activity
Name of compoundStructural formulaRXR(+)RXR(−)RXR(+)RXR(−)RXR(+)RXR(−)RXR(+)RXR(−)
|
|
Prostaglandin A21◯10 μMX◯10 μM◯10 μM◯10 μMX◯10 μM◯10 μM
|
Prostaglandin A12◯10 μMX◯10 μM◯10 μM◯10 μMX◯10 μM◯10 μM
|
16,16-dimethyl Prostaglandin A23XXXXXXXX
|
[0243]
11
TABLE 9
|
|
|
|
Prostaglandin A3
4
X
X
X
X
X
X
X
X
|
|
Prostaglandin A1ethyl ester
5
X
X
X
X
X
X
X
X
|
|
15-epi Prostaglandin A1
6
◯10 μM
X
◯10 μM
◯10 μM
◯10 μM
X
X
X
|
|
16,16-dimethyl Prostaglandin A1
7
X
X
X
X
X
X
X
X
|
|
[0244]
12
TABLE 10
|
|
|
|
13,14-dihydro 15-keto Prostaglandin A2
8
X
X
X
X
X
X
X
X
|
|
15(R)-15-methyl Prostaglandin A2
9
◯10 μM
X
◯10 μM
◯10 μM
◯10 μM
X
◯10 μM
X
|
|
15-deoxy-Δ12.14_Prostaglandin A2
10
X
X
X
X
X
X
X
X
|
|
16-phenoxy tetranor Prostaglandin A2
11
X
X
X
◯30 μM
◯10 μM
X
◯30 μM
◯30 μM
|
|
[0245]
13
TABLE 11
|
|
|
|
17-phenyl trinor Prostaglandin A2
12
◯10 μM
X
◯10 μM
X
◯10 μM
X
◯10 μM
X
|
|
17-phenyl trinor- 13,14-dihydro Prostaglandin A2
13
X
X
X
X
X
X
X
X
|
|
19(R)-hydroxy Prostaglandin A2
14
X
X
X
X
X
X
X
X
|
|
15-deoxy-Δ12.14_Prostaglandin A1
15
◯30 μM
X
◯30 μM
X
X
X
X
X
|
|
[0246]
14
TABLE 12
|
|
|
|
Prostaglandin J2
16
X
X
X
X
X
X
X
X
|
|
15-deoxy-Δ12.14_Prostaglandin J2
17
X
X
◯10 μM
◯10 μM
◯10 μM
X
X
X
|
|
Δ12- Prostaglandin J2
18
X
X
X
X
X
X
X
X
|
|
9.10-dihydro-15- deoxy-Δ12.14_Prostaglandin J2(CAY10410)
19
X
X
X
X
X
X
X
X
|
|
8-iso Prostaglandin A1
20
◯10 μM
ND
◯ 3 μM
ND
◯10 μM
ND
◯10 μM
ND
|
|
Expression Analysis of the TINUR Gene
[0247] TINUR, a β-type of the nuclear orphan receptor subfamily, was not selected from expression comparison analysis by DD and GeneChip using clinical peripheral blood samples. This receptor's association with specific diseases, including allergic diseases, has not been very clearly elucidated. However, since TINUR was predicted to have functional similarity with TR3, expression comparison with TINUR was carried out between healthy subjects and patients in the same manner as for TR3, that is, using ABI7700 and the same patient peripheral blood eosinophil panel (Table 5) in which the number of examples in a group amounts to more than ten. The primers and probe used for the TaqMan method were the following:
15|
Primer 1 (5′):AGCACAGGCTACGACGTCAA(SEQ ID
NO:8)
|
Primer 2 (3′):TCTTCTACCTTAATGGAGGACTGC(SEQ ID
NO:9)
|
TaqMan probe:TTGTACCAAATGCCCCTGTCCGGA(SEQ ID
NO:10)
[0248] As shown in Table 13 and FIG. 5, significant enhancement was confirmed in atopic dermatitis patients as compared to normal subjects, regardless of case severity.
16TABLE 13
|
|
C1E-2β-actin (raw)TINUR (raw)For β-correctionTINUR-correction
TINURBloodcopy/ngcopy/5 ngcopy/1 ngraw(/ng)/averageraw/beta-correction
|
13 healthy125312601.011303010
subjects254116681382162762.162094347528
321423900.8559389460
4369621136368272741.47672939318469
571653602.8627419350
616917300.6758875080
7601310203504407012.4023863316942
821306278318156640.85123603618401
9371589121882243761.48459185416420
10646297105612211222.5821198488180
11208737165619331240.83395635239719
1221211400.847449030
13379539112142224281.51635552614791
15 light14508758146688293382.03261852714433
cases1524893700.9945646910
16221813414582829160.88619860493564
17315168275505551011.25917479643760
18141827279290558580.56663676998578
19244028246709493420.97495358450609
20348051332180664361.39055235147777
21387693119505239011.54893123415431
22268468144812289621.07259990727002
23206673216900433800.82570995552537
24136652228928457860.54595903383863
25218963135292270580.87481232930930
26209273198420396840.83609700947463
2713197700.527282360
28121064115898231800.48368079747923
6 moderate2916590100.6628153310
cases in the30134119273684547370.535841346102151
remission318634000.3449490820
stage32472440259151518301.88751907127459
33170914151666303330.68284524444422
3436781871428142861.4695259499721
9 moderate351622585192051038410.648261218160184
cases in the369096900.3634432110
exacerbation37246460338300676600.98467104268713
stage38146805221751443500.58652230175616
39179179240130480260.71586381867088
40138858107895215790.55477136638897
41133317163876327750.53263505161534
42171308333904667810.68441996697573
432852953832176641.1398277536724
10 severe44154902121579243160.61887287639290
cases in the4578948162181324360.315418709102835
remission46231612402817805630.92534690587063
stage47155564149795299590.62151658448203
48385848148392296781.54156178719252
4926474456146112291.0577207810616
50144715194006388010.57817446567110
51205943249286498570.82279501760595
52155395157681315360.6208416950796
5315170300.6060925050
8 severe5439782101.5893959710
cases in the55446400263974527951.78348004529602
exacerbation5628072454818109641.1215648459775
stage57161385102355204710.64477520731749
5813497885303170610.53927162431637
59247404474389490.098841490534
60241793322099644200.96602399166686
6193068135613271230.37183179972943
total15268113
Av.250297
|
TINUR Receptor Ligand Search
[0249] Like TR3, TINUR is an orphan nuclear receptor whose native ligands and activators are still unknown. If discovered, they may directly activate TINUR in eosinophil cells and promote apoptosis. Such ligand activators would therefore be anti-allergic agents, and thus a high-throughput system for ligand screening was constructed, using the same methods as for TR3.
[0250] The TINUR ligand binding domain sequence or full length gene (FIG. 3) was inserted into pBIND as shown in FIG. 2, in order to facilitate expression of a protein in which the DNA binding domains of TINUR and GAL4 are fused in frame. A plasmid comprising the TINUR ligand binding domain sequence inserted into pBIND, and a luciferase reporter plasmid comprising a GAL4 binding site, were co-transfected into NIH3T3 cells. Luciferase activity was measured automatically. At the same time, activity measurement was also carried out by adding retinoic acid X receptor (RXR) α-gene, a transcription factor that forms a heterodimer with TINUR. Low molecular weight substances can also be added to this system to screen using transcriptional enhancement activity.
[0251] Like TR3 expression, TINUR expression is enhanced in activated eosinophils. Ligands existing in vivo may exist in sites where nuclear receptors are highly expressed. The native ligands of TR3 were found to be prostaglandin A2 and prostaglandin A1. Ligands of the nuclear receptor subfamily are assumed to have structural redundancy. Therefore, derivatives similar to TR3 activator compounds were added, and enhancement of transcriptional activity was investigated. Compounds found to comprise the activity of activating TINUR transcription were prostaglandins comprising a cyclopentenone structure, such as prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandinA2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin J2 and 8-isoprostaglandin A1 (FIG. 6, Tables 8 to 12). According to the X-ray crystallography results of Wang et al, the TINUR (Nurr1) ligand pocket is closed, suggesting it may be a nuclear receptor without a native ligand (Z. Wang, G. Benoit, J. Liu, S. Prasad, P. Aarnisalo, X. Liu, H. Xu, N. P. C. Walker, T. Perlmann, “Structure and function of Nurrl identifies a class of ligand-independent nuclear receptors” (Tularik Inc.); Nature 423, 29 May, p555-560 (2003)). However, since the above-mentioned reactions are reproducible, and structure-activity relationships exist in compounds similar to prostaglandin A2, the present inventors revealed that there is a strong possibility that compounds such as prostaglandin A2, prostaglandin A1, and similar metabolites are native ligands of not only TR3, but also of TINUR.
Virtual Compounds
[0252] A pharmacophore model was used to simulate the binding position of the PGA derivatives to the TR3 ligand binding domain (LBS) (FIG. 7). Based on structure-activity relationship information for the PGA derivative reporter system, compounds other than PGA derivatives that matched the binding pocket were selected from the Catalyst database (screened from BioByte Master File 2001 39,383 compounds, 2,198,646 conformations).
[0253] The 158 compounds selected as strongly binding compounds using this simulation are shown in Tables 14 to 45 (including the structural formula). The 117 compounds subsequently selected are shown in Tables 46 to 49.
17TABLE 14
|
|
LUDI_HB_LIPO_
Compoundscorescorescore
|
|
|
21
2DEOXY3FLUORO CYTIDINEN4DIMET HYLAMINOMETHY LENE2040325
|
22
1ACETOMORPHIN E1580254
|
23
BORNYLSALICYL ATE1510272
|
24
NETHYLMORPHIN E1360257
|
25
2HYDROXY42NAP HTHALENYL4OXO 2BUTENOICACIDM ETHYLESTER1260222
|
[0254]
18
TABLE 15
|
|
|
|
26
3ACETYLMORPHI NE
123
0
219
|
|
27
BEREFRINE
112
0
233
|
|
28
DIDEOXYARAA2M ETHYL2FLUORO
112
83
150
|
|
29
STRIAZINE46DIAM INO12H222DIMETH YL13PROPOXYPH ENYL
108
0
254
|
|
[0255]
19
TABLE 16
|
|
|
|
30
DIDEOXYTHIOTHY MIDINE
107
83
145
|
|
31
CINCHONAMINE
103
0
275
|
|
32
STRIAZINE46DIAM INO12DIHYDRO22 DIMETHYL13ETHO XYPHENYL
103
0
224
|
|
33
23DIDEOXYCYTIDI NEN4DIMETHYLA MINOMETHYLENE
101
0
222
|
|
34
DIDEOXYARAA2N6 DIMETHYL2FLUOR O
101
0
222
|
|
[0256]
20
TABLE 17
|
|
|
|
35
DIDEOXYTHIOTHY MIDINE23DEHYDR O
101
72
150
|
|
36
DIDEOXYTHYMIDI NE
101
83
139
|
|
37
ETHYLMORPHINE
101
0
222
|
|
38
MERIBENDAN
96
0
192
|
|
[0257]
21
TABLE 18
|
|
|
|
39
MORPHINE3PROPI ONYL
95
0
216
|
|
40
PHOSPHOROHYD RAZIDICACIDDIPH ENYLESTER
93
0
189
|
|
41
53HYDROXYBENZ OYLH2PYRROLOP YRROLE1CARBOX YLICACID
92
0
213
|
|
42
OXAZEPAM
90
0
236
|
|
[0258]
22
TABLE 19
|
|
|
|
43
TRENBOLONE
90
0
186
|
|
44
STAVUDINE
89
74
136
|
|
45
THYMINE123DIDE OXY2FLUOROPEN TOFURANOSYL
89
83
127
|
|
46
ALOVUDINE
87
63
145
|
|
[0259]
23
TABLE 20
|
|
|
|
47
NAPROXOL
87
0
233
|
|
48
MDL72638
86
0
207
|
|
49
12DIHYDROTRIAZI NE46DIAMINO22DI METHYL13METHO XYPHENYL
84
0
180
|
|
50
4QUINOLINAMINE 2PAMINOSTYRYL
84
0
180
|
|
[0260]
24
TABLE 21
|
|
|
|
51
2AMINO4PHENYL QUINAZOLINE
81
0
177
|
|
52
DIPHENYLACETAL DEHYDEENOL
81
0
177
|
|
53
GUANABENZ
81
0
177
|
|
54
MHYDROXYDIPHE NYLAMINE
81
0
177
|
|
55
PRECLAMOL
81
0
227
|
|
[0261]
25
TABLE 22
|
|
|
|
56
FENISOREX
77
0
198
|
|
57
LY195115
75
0
171
|
|
58
PLATINUMBISCYC LOHEXYLAMMONI ODIAQUADINITRA TE
75
0
171
|
|
59
11DIMETHYL33AM INOPHENYLUREA
72
0
168
|
|
[0262]
26
TABLE 23
|
|
|
|
60
BENZOICACIDHYD RAZIDEO33DIMET RIAZINO
72
0
168
|
|
61
BENZOPHENONE2 4DIHYDROXY
72
0
168
|
|
62
5FLUOROCYTOSI NE123DIDEOXY2F LUOROPENTOFUR ANOSYL
71
0
192
|
|
63
STRIAZINE46DIAM INO12DIHYDRO22 DIMETHYL13ETHY LPHENYL
71
0
192
|
|
64
YM060
71
0
192
|
|
[0263]
27
TABLE 24
|
|
|
|
65
12DIHYDROXYBEN ZENE4HEXEN1YL
70
0
242
|
|
66
BENZAMIDE23MET HYL3ALLYLTRIAZ ENYL
70
0
216
|
|
67
2METHYL4PTOLY LAMINO123BENZO TRIAZINIUMIODID E
69
0
165
|
|
68
BENZENESULFON AMIDE2IBUTYROY LAMINO4METHOX Y
67
0
213
|
|
69
233DIMETHYL1TRI AZINOBENZAMIDE
66
0
162
|
|
70
BENZOPHENONEH YDRAZONE
66
0
162
|
|
[0264]
28
TABLE 25
|
|
|
|
71
BENZOPHENONE OXIME
66
0
162
|
|
72
1HYDROXYMETHY LPENTACHLOROC YCLOHEXANE
65
0
186
|
|
73
CHLOROGUANIDE
65
0
186
|
|
74
ARECAIDINEALPH APHENYLPROPAR GYLESTER
64
0
236
|
|
75
46DIAM22DIME13C YANOPHSTRIAZIN E
63
0
159
|
|
76
BENZENESULFON AMIDE22ETHYLBU TANOYLAMINO4M ETHYL
63
0
260
|
|
[0265]
29
TABLE 26
|
|
|
|
77
CARBAMAZEPINE
63
0
159
|
|
78
N1PHENYLN1BEN ZOYLHYDRAZINE
63
0
159
|
|
79
VIRIDICATIN
63
0
159
|
|
80
CHLORPROGUANI L
62
0
183
|
|
81
DIDEOXYCYTIDIN E2ALPHAFLUORO
62
0
183
|
|
[0266]
30
TABLE 27
|
|
|
|
82
ZALCITABINE
62
0
183
|
|
83
234DIHYDROXYPH ENYLIMINOIMIDAZ OLIDINE
60
0
156
|
|
84
23BENZOOCTAHY DRONAPHTHALEN E34DIOH34DIAX
60
0
156
|
|
85
4AMINOSALICYLI CACID2TOLYLEST ER
60
0
156
|
|
86
5CHLOROCYTIDIN E
60
63
118
|
|
[0267]
31
TABLE 28
|
|
|
|
87
AFURILDIOXIME
60
0
156
|
|
88
BENZOYLPHENYL HYDROXYLAMINE
60
0
156
|
|
89
DOMOXIN
60
0
257
|
|
90
IMIDAZOLE1METH YL2HYDROXYIMIN OMETHYL412DIME THYLPROPOXYME THYL
60
0
257
|
|
[0268]
32
TABLE 29
|
|
|
|
91
46DIAM12HSYMTR IAZINE1MHEXYLP HENYL
59
0
281
|
|
92
BENZENESULFON AMIDE2IBUTYROY LAMINO4METHYL
58
0
204
|
|
93
11DIPHENYLUREA
57
0
153
|
|
94
12DIHYDROTRIAZI NE22DIMETHYL46 DIAMINO13METHY LPHENYL
57
0
153
|
|
95
AFURILMONOXIM E
57
0
153
|
|
[0269]
33
TABLE 30
|
|
|
|
96
DIACETONEGLUC OSE
57
0
153
|
|
97
PYRIDINE2PHENA CYLENOL
57
0
153
|
|
98
DIDEOXYCYTIDIN E5FLUORO
56
0
177
|
|
99
UREA1ETHYL1ME THOXYPHENYL
55
0
201
|
|
100
PYRIDO12APYRIMI DIN4ONE3CONH2 H716DIMEAX
54
0
150
|
|
[0270]
34
TABLE 31
|
|
|
|
101
PHENOL26DIMETH YLOL4METHYL
53
66
133
|
|
102
2CYCLOHEXYLPH ENOL
52
0
148
|
|
103
5AMINO1245TRIC HLOROPHENYLTE TRAZOLE
52
0
148
|
|
104
BENZAMIDE23AZE TIDINYLTRIAZENE
52
0
148
|
|
105
BENZAMIDE23MET HYL3BUTYLTRIAZ ENYL
52
0
224
|
|
[0271]
35
TABLE 32
|
|
|
|
106
BENZENEMETHAN IMINEA3HYDROXY PHENYL
52
0
148
|
|
107
DICHLOROPHENA RSINE
52
0
148
|
|
108
MEDETOMIDINE
52
0
198
|
|
109
NAPHTHALENE2A MINO4METHOXYC ARBONYL
52
0
148
|
|
110
NAPHTHONONE
52
0
148
|
|
111
NNDIMETHYLCAR BAMATEMAMINOB ENZYLESTER
52
0
198
|
|
[0272]
36
TABLE 33
|
|
|
|
112
RO600213
52
0
198
|
|
113
5HYDROXY1METH YL2AMINOTETRAL INNPROPYL
50
0
222
|
|
114
BENZAMIDEODICH LOROACETYLAMI NO
50
0
171
|
|
115
RA131423
50
0
171
|
|
116
1234H4ISOQUINO LINE58DIMETHOX Y
49
0
145
|
|
[0273]
37
TABLE 34
|
|
|
|
117
3HYDROXYCOTINI NE
49
0
145
|
|
118
OBENZYLOXYBEN ZAMIDE
49
0
195
|
|
119
2ETHYL4PTOLYLA MINO123BENZOT RIAZINIUMIODIDE
47
0
168
|
|
120
BENZAMIDEOBUT YLAMINO
47
0
219
|
|
121
OCTAHYDROPHE NANTHREN4AAMI NENMETHYL9HYD ROXY
47
0
168
|
|
122
13BROMOPHENYL 22DIMETHYL46DIA MINOSTRIAZINE
46
0
142
|
|
[0274]
38
TABLE 35
|
|
|
|
123
9ANTHRACENECA RBOXAMIDE
46
0
142
|
|
124
DEBOXAMET
46
0
192
|
|
125
N1PCHLOROPHEN YLN5PROPYLBIG UANIDE
46
0
192
|
|
126
24IBUTYLPHENYL PROPIOHYDROXA MICACID
45
0
242
|
|
127
PAMINOSALICYLI CACID6CHLOROH EXYLESTER
45
0
293
|
|
[0275]
39
TABLE 36
|
|
|
|
128
3METHIO4AMINO6 CYCLOHEXYL124 TRIAZINE5ONE
44
0
165
|
|
129
4PDIMETHYLANILI NOMETHYLPYRID OXOL
44
0
216
|
|
130
BENZOCYCLOHEP TANE58METHENO 10AMINO3TRIFLU OROMETHYL
44
0
165
|
|
131
PYRIMIDINE4AMIN O2DIETHYLAMINO ETHYLAMINO6ME THYL
44
0
266
|
|
132
TERPENE319717
44
45
145
|
|
133
12DIHYDROTRIAZI NE22DIMETHYL46 DIAMINO13HYDRO XYPHENYL
43
0
139
|
|
[0276]
40
TABLE 37
|
|
|
|
134
13DITHIOLANE4O XIMINO2DIMETHY LHYDRAZINO55DI METHYL
43
0
139
|
|
135
245TRIMETHOXYA MPHETAMINE
43
0
189
|
|
136
DARSIDOMINE
43
0
139
|
|
137
OAMINODIPHENYL AMINE
43
0
139
|
|
138
OXAZOLE4ACETO XIME25DIMETHYL
43
0
139
|
|
139
PERHYDROTHIAZI N3ONE2OXIMINON 2FURANYLMETHY L
43
0
189
|
|
[0277]
41
TABLE 38
|
|
|
|
140
PYRIMIDINE24DIA MINO5BENZYL6M ETHYL
43
0
189
|
|
141
5NORBORNEN2YL HYDROXIMICACID METHYLESTER
41
0
162
|
|
142
DIDEOXYTHIOURI DINE23DEHYDRO
41
0
162
|
|
143
ISOPROPYLSALIC YLATE
41
0
162
|
|
144
MDL72145
41
0
213
|
|
[0278]
42
TABLE 39
|
|
|
|
145
QUINAZOLINE2TB UTYL34DIHYDRO4 HYDROXY
41
0
162
|
|
146
1NAPHTHALENEA MINE3METHOXY
40
0
136
|
|
147
BENZOQUINONE2 5BISAZIRIDINYL3 METHYL6HYDROX YETHYL
40
0
186
|
|
148
NAPRODOXIME
40
0
186
|
|
149
OPHENOXYANILIN E
40
0
136
|
|
150
PHENOL2CYCLOP ENTYL
40
0
136
|
|
[0279]
43
TABLE 40
|
|
|
|
151
ST404
40
0
136
|
|
152
UREA1ETHYL1PA NISYL
38
0
159
|
|
153
12DIHYDROPYRAZ OLONE4PROPYL5 PHENYL
37
0
183
|
|
154
24DIMETHOXYAM PHETAMINE
37
0
183
|
|
155
2AMINOBIPHENYL
37
0
133
|
|
156
AMPHENIDONE
37
0
133
|
|
[0280]
44
TABLE 41
|
|
|
|
157
BENZENESULFON AMIDE2IBUTYROY LAMINO
37
0
183
|
|
158
NAPHTHALENE1A MINO6METHOXY
37
0
133
|
|
159
SAUOYLAMIDENN DIMETHYL
37
0
133
|
|
160
BENZAMIDE23MET HYL3ETHYLTRIAZ ENYL
35
0
156
|
|
161
INDOLE3NETHYLC ARBOXAMIDO
35
0
156
|
|
162
NNDIPHENYLPRO PYLENEDIAMINE
35
0
207
|
|
[0281]
45
TABLE 42
|
|
|
|
163
PAMINOSALICYLI CACIDNBUTYLES TER
35
0
207
|
|
164
2OH46BISIPROPY LAMINOSTRIAZIN E
34
0
180
|
|
165
2PHENYLPHENOL
34
0
130
|
|
166
46DIAM22DIME14 METHYLPHSTRIA ZINE
34
0
130
|
|
167
BENZAMIDE233DI ETHYLTRIAZENYL
34
0
180
|
|
[0282]
46
TABLE 43
|
|
|
|
168
CICLOPIROX
34
0
130
|
|
169
INDOLE3THIAZOL 4YL2GUANADYLM ETHYLANALOG
34
0
180
|
|
170
QUINAZOLINE4CA RBAMOYL26DIME THYL
34
0
130
|
|
171
UREA1PROPYL1M TOLYL
34
0
180
|
|
172
BSANTALOL
33
0
230
|
|
[0283]
47
TABLE 44
|
|
|
|
173
12DIHYDROPYRAZ OLONE4ETHYL5P HENYL
32
0
153
|
|
174
2BIPHENYLYLSEL ENIOUSACID
32
0
153
|
|
175
DESETHYLATRAZI NE
32
0
153
|
|
176
DIDEOXYTHIOURI DINE
32
0
153
|
|
177
ETHYLENEGLYCO L12BIS6METHYLP YRID2YL
32
0
204
|
|
[0284]
48
TABLE 45
|
|
|
|
178
ISOLADOL
32
0
204
|
|
[0285]
49
TABLE 46
|
|
|
LUDI—
HB—
LIPO—
Rule of 5
|
Name of compound
MW
score
score
score
Violations
Rotlbonds
|
|
|
13HYDROXYPHENYL3METHOXY3METHYLUREA
196.2054
32
0
153
0
5
|
1HYDROXYPENTACHLOROCYCLOHEXANE
272.3857
28
0
124
0
1
|
1OHYDROXYMEPHENYL33DIMETRIAZENE
179.2212
18
0
139
0
4
|
226DIHYDROXYPHENYLIMINOIMIDAZOLINE
193.2048
16
0
112
0
3
|
24DIAMINO52BR45DIMEOBENZYLPYRIMIDINE
339.1911
19
0
165
0
4
|
24DIAMINOPYRIMIDINE52CL35DIMEOBENZYL
294.7401
22
0
168
0
4
|
26DIMETHYL1NAPHTHOL
172.2262
19
0
115
0
1
|
2ENDOAMINOBENZOBICYCLO222OCTENE
173.2572
25
0
121
0
0
|
2HPYRAZOLO34AQUINOLIZINE1236710BHEXAHYDRO
177.2486
16
0
112
0
0
|
2METHOXY4MEAMINO6IPROPYLAMINOSTRIAZINE
197.2394
18
0
139
0
4
|
2METHYL5IPROPYLPHENOL
150.22
15
0
136
0
2
|
2OH4ETAMINO6DIETAMINOSTRIAZINE
211.2662
23
0
195
0
6
|
2OH4IPROPYLAMINO6DIETAMINOSTRIAZINE
225.293
20
0
192
0
6
|
2PROPYL4PTOLYLAMINO123BENZOTRIAZINIUMIODIDE
279.3639
22
0
168
0
4
|
2PTERIDINAMINE5678TETRAHYDRO4HYDROXY67DIMETHY
195.2236
16
0
112
0
1
|
35DIMETHOXYPHENOL
154.1652
16
0
112
0
3
|
35DITBUTYLPHENOL
206.3272
16
0
162
0
3
|
3AMINOBENZOICACIDETHYLESTER
165.1914
21
0
142
0
3
|
3CYCLOHEXENOL1ISOPROPYL4METHYL
154.2516
18
0
139
0
2
|
3HYDROXY4METHOXYCINNAMICACIDETHYLESTER
222.2402
18
0
139
0
6
|
3OPENTYLMORPHINE
355.476
16
0
213
0
6
|
4HYDROXYETHYLVANILLIN
196.2024
31
0
177
0
6
|
4QUINOLINAMINE2METHYL
158.2024
22
0
118
0
0
|
4QUINOLINAMINE6ETHOXY24PHENYLBUTADIENYL
316.4018
15
0
136
0
5
|
5METHOXY8QUINOLINOL
175.1866
28
0
124
0
2
|
6METHYL5INDANOL
148.2042
16
0
112
0
1
|
8QUINOLINAMINE6METHOXY
174.2018
25
0
121
0
1
|
AAMIDOETHYLCINNAMATE
219.2396
27
0
148
0
5
|
AAMIDOMETHYLCINNAMATE
205.2128
28
0
124
0
4
|
ANILINE35DIMETHOXY
153.1804
22
0
118
0
2
|
ANILINE35DITBUTYL
205.3424
19
0
165
0
2
|
ANTHRALIN102HYDROXYETHIO
302.3442
17
0
189
0
6
|
|
[0286]
50
TABLE 47
|
|
|
ATROMEPINE
303.4004
22
0
219
0
6
|
BENZAMIDENHEXYL34DIHYDROXY
237.298
29
0
251
0
9
|
BENZAMIDEOISOPROPYLAMINO
178.2334
15
0
136
0
3
|
BENZENEMETHANIMINE25DIMETHYLAPHENYL
209.2902
16
0
112
0
2
|
BENZENESULFONAMIDE22ETHYLBUTANOYLAMINO
270.3458
22
0
219
0
6
|
BENZOICACID2AMINOMETHYLESTER
151.1646
19
0
115
0
2
|
BENZOICACIDHYDRAZIDEO33DIMETRIAZINO
207.2346
40
0
136
0
4
|
BENZOIN
212.2476
16
0
162
0
4
|
BENZOINOXIME
227.2622
28
0
174
0
5
|
BENZYLALCOHOL35DIMETHOXY4HYDROXY
184.1914
21
0
142
0
5
|
CARVEOL
152.2358
18
0
139
0
2
|
CINAMETICACID
238.2396
16
0
162
0
8
|
CYPENAMINE
161.2462
25
0
121
0
1
|
CYTIDINE23DIDEHYDRO23DIDEOXY
209.2042
15
0
136
0
3
|
CYTIDINEDIDEOXY3FLUORO
229.2105
18
0
139
0
3
|
CYTOSINE2BUTOXY
167.2102
17
0
189
0
4
|
DMDC
239.2304
27
0
148
0
4
|
ECGONINEMETHYLESTER
199.2492
27
0
148
0
3
|
ETHYCHLOZATE
238.6731
20
0
192
0
4
|
ETHYLENEGLYCOLMONO24DICHLOROPHENYLETHER
207.056
25
0
171
0
4
|
ETHYLMETHYLGLYOXIME
130.1462
27
83
65
0
4
|
F11105
203.2432
15
0
136
0
2
|
FLOVERINE
198.2182
22
0
168
0
6
|
GUANIDINE1METHYL14CHLOROPHENYL
183.6401
19
0
115
0
2
|
GUANIDINEN43AMINOPHENYLTHIAZOL2YL
233.2904
22
0
118
1
2
|
HEXAHYDROFLUOREN9AAMINE
187.284
28
0
124
0
0
|
ILEPRO
228.2906
16
83
130
0
6
|
IMIDAZOLINE22HYDROXYPHENYL
162.1908
19
0
115
0
2
|
INDOLE3CARBOXYLICACIDETHYLESTER
189.2134
18
0
139
0
3
|
INDOLE3IMIDAZOL1YLMETHYL
197.239
16
0
162
0
2
|
INDOLE3NMETHYLCARBOXAMIDO
174.2018
28
0
124
0
2
|
LAMIVUDINE
229.2532
18
0
139
0
3
|
METHYLBENZOATE2AMINO5CHLORO
185.6097
16
0
112
0
2
|
METHYLSALICYLATE
152.1494
16
0
112
0
3
|
|
[0287]
51
TABLE 48
|
|
|
MORPHINE3HEXANOYL
383.4864
25
0
222
0
7
|
MPENTOXYPHENOL
180.2462
16
0
213
0
6
|
N1PCHLOROPHENYLN5METHYLBIGUANIDE
225.6803
43
0
139
0
5
|
N2N4N6TRIMETHYLNNNHYDROXYMETHYLMELAMINE
258.2796
17
3
186
0
9
|
NAPHTHALENE1AMINO3CHLORO
177.6329
25
0
121
0
0
|
NAPHTHALENE1AMINO3METHYL
157.2146
25
0
121
0
0
|
NAPHTHALENE1AMINO6CHLORO
177.6329
28
0
124
0
0
|
NBUTYLSALICYLIDENEIMINE
177.2456
20
0
192
0
5
|
NCYCLOPENTYLCINNAMAMIDE
215.2944
27
0
148
0
4
|
NETHYLMORPHINE
299.3688
136
0
257
0
3
|
NHYDROXYETHYLPTP
203.2834
31
0
177
0
4
|
NITRAFUDAM
231.2104
25
0
121
0
2
|
NNDIMETHYLTRYPTAMINE6METHOXY
218.298
29
0
201
0
4
|
OMETHOXYBENZAMIDE
151.1646
16
0
112
0
2
|
OMETHYLCINNAMAMIDE
161.203
22
0
118
0
2
|
OMETHYLTYROSINEETHYLESTER
223.2712
30
0
227
0
6
|
PAMINOSALICYLICACID4CHLOROBUTYLESTER
243.6895
22
0
219
0
7
|
PAMINOSALICYLICACIDNAMYLESTER
223.2712
25
0
222
0
7
|
PENTA24DIENYLAMINE23455PENTACHLORO
255.3583
18
0
139
0
2
|
PENTALAMIDE
207.2718
19
0
216
0
6
|
PHENOL2HEPTYL
192.3004
21
0
142
0
7
|
PHENYLBORONICACIDMETHOXYACETAMIDO
209.0081
19
0
165
0
7
|
PICOLINHYDROXAMICACID
166.1792
19
0
115
0
3
|
PROTOCATECHUICACIDETHYLESTER
182.1756
15
0
136
0
5
|
PYRAZINE2AMIDINO56DIMETHYL3METHYLAMINO
179.2242
25
0
121
0
2
|
PYRAZOLE23DIHYDRO3IMINO15DIMETHYL2PHENYL
187.2438
25
0
121
0
1
|
PYRAZOLE24DIMETHYL5PHENYL
172.2292
16
0
112
0
1
|
PYRAZOLE426DIMETHYLPHENYLMETHYL
186.256
16
0
162
0
2
|
PYRAZOLE4METHYL5PHENYL
158.2024
16
0
112
0
1
|
PYRIDINE22HYDROXYPHENYL
171.1982
25
0
121
0
2
|
PYRIDINE4HYDROXY26BISMETHOXYCARBONYL
211.1738
22
0
118
0
5
|
PYRIMIDINE24DIAMIO6METHYL5PHENYL
200.2426
22
0
118
0
1
|
PYRIMIDINE2AMINO4DIETHYLAMINOETHYLAMINO56DIMETHYL
237.3472
26
0
248
0
6
|
|
[0288]
52
TABLE 49
|
|
|
PYRIMIDINE2DIMETHYLAMINO4METHYLAMINO
152.1986
19
0
115
0
2
|
PYRIMIDINE2HYDRAZINO4METHOXY6METHYL
154.1712
19
0
115
0
2
|
PYRIMIDINE4AMINO2DIMETHYLAMINO
138.1718
28
0
124
0
1
|
QUINOLINE4AMINO7CHLORO
178.6207
22
0
118
0
0
|
RA161045
371.484
19
0
216
0
5
|
SYMTRIAZINE2ETHYLAMINO4TBUTYLAMINO6HYDROXY
211.2662
22
0
168
0
5
|
TERPENE319712
268.3954
25
0
171
0
4
|
TETRAHYDROPYRAN24DIONE31ETHOXYIMINOBUTYL66SPIRO35
323.4314
23
0
195
0
6
|
DIMETHYLCYCLOHEXYL
|
TIMIRDINE
227.7111
16
0
112
0
1
|
TIZOLEMIDE
335.8229
27
0
148
0
3
|
UREA1BUTYL1PTOLYL
206.287
17
0
189
0
5
|
UREA1ETHYL1MTOLYL
178.2334
29
0
150
0
3
|
UREA1ETHYL1OANISYL
194.2328
29
0
150
0
4
|
UREA1ETHYL1OETHOXYPHENYL
208.2596
31
0
177
0
5
|
UREA1METHYL1MTOLYL
164.2066
22
0
118
0
2
|
VERBENOL
152.2358
19
0
115
0
1
|
VESTITOL
272.3
22
0
118
0
4
|
|
Decrease of Activity by LBD Deletion Mutant
[0289] Prostaglandin A2 transcriptional activity was suppressed in a Mammalian Two Hybrid reporter system that used a TR3 or TINUR gene completely lacking an LBD region (FIG. 8). Thus, it was implied that prostaglandin A2 functions by acting on the LBD region of the nuclear receptor.
Demonstration of the Binding of PGA Derivatives to TR3 or TINUR Using BIAcor
[0290] To conclusively demonstrate PGA derivative ligand binding activity to TR3 or TINUR, revealed using the Mammalian Two Hybrid reporter system, TR3 GST-LBD and TINUR GST-LBD were respectively expressed in E. coli, and then purified. PGA1 and PGA2 binding to the LBD of TR3 or TINUR was detected by BIAcor S51, using comparison with GST as a base (FIG. 9). The negative control compound, 13,14-dihydro-15-keto-PGA2, did not demonstrate any activity in the reporter system, and did not bind to the LBD.
EXAMPLE 8
[0291] Genes such as TR3 or TINUR, which comprise apoptotic character, may be enhanced in the peripheral blood eosinophils of atopic dermatitis conditions due to negative feedback regulation that acts to reduce the increase in peripheral blood eosinophils that occurs in association with a pathologic condition. Therefore, the present inventers investigated in vitro the type of stimulation that causes expression of this kind of gene in eosinophils.
[0292] A large number of peripheral blood eosinophils were collected from healthy subjects and cultured, while suppressing their activation, in suspension in petri dishes. Eosinophil activation by stimulation with cytokines such as IL-5 and IL-4 did not lead to TR3 induction. In contrast, induction of cell apoptosis using anti-CD30 antibody resulted in dramatic induction of TR3 and TINUR in cultured peripheral blood eosinophils over a one to three-hour treatment (Table 50, FIGS. 10 and 11). This anti-CD30 antibody comprises agonist activity towards eosinophil CD30, and has recently received attention due to possible use as a therapeutic agent for asthma or the like, by inducing apoptosis in eosinophils by a specific intracellular pathway. Table 50 below summarizes the apoptosis induction of human peripheral blood eosinophils.
53TABLE 50
|
|
Annexin
V-positive cells (%)
|
|
Fresh4.0
Control 1 hr2.30
Anti-CD30 antibody9.20
Anti-Fas antibody5.20
Control 3 hr4.50
Anti-CD30 antibody20.00
Anti-Fas antibody13.80
Control24 hr11.70
Anti-CD30 antibody63.00
Anti-Fas antibody31.20
|
[0293] Although the anti-Fas antibody induced apoptosis, albeit more slowly than the anti-CD30 antibody, it did not induce TR3 and TINUR. Thus, apoptosis induction by the anti-CD30 antibody, accompanied by TR3 and TINUR induction, may occur through an eosinophil-specific apoptosis pathway that is different from conventional pathways. These phenomena (apoptosis induction and expression induction of TR3 or TINUR) were similarly observed when AML14.3D10, an eosinophil-specific cell line, was treated with anti-CD30 antibody (FIGS. 12, 13 and 14).
[0294] It is very likely that promotion of a pathway that specifically leads eosinophils to cell death through the enhancement of TR3 or TINUR function will lead to the treatment of not only asthma, but also of various allergic diseases including atopic dermatitis, which was investigated by the present inventors. An example of the therapeutic strategy intended by the present inventors is shown in FIG. 15.
Claims
- 1. A method of testing for an allergic disease, said method comprising the steps of:
a) measuring the expression level of a TR3 or TINUR receptor protein, or a gene encoding the TR3 or TINUR receptor protein, in eosinophil cells of a test subject; and b) comparing the expression level of the protein or gene in the eosinophil cells of the test subject with an expression level in eosinophil cells of a healthy subject.
- 2. The testing method of claim 1, wherein the gene expression level is measured by cDNA PCR.
- 3. The testing method of claim 1, wherein the allergic disease is atopic dermatitis.
- 4. A reagent for testing for an allergic disease, said reagent comprising an oligonucleotide of at least 15 nucleotides in length that comprises a nucleotide sequence complementary to a polynucleotide encoding a TR3 or TINUR receptor protein, or to its complementary strand.
- 5. A method of detecting the influence of a candidate compound on the expression level of a polynucleotide of (a) or (b) below, wherein said method comprises the steps of:
(1) contacting the candidate compound with a cell that expresses a polynucleotide of (a) or (b):
(a) a polynucleotide encoding a TR3 or TINUR receptor protein; and (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein; and (2) measuring the expression level of the polynucleotide of (a) or (b).
- 6. The method of claim 5, wherein the cell is from a leukocyte cell line.
- 7. A method of detecting the influence of a candidate compound on the expression level of a polynucleotide of (a) or (b) below, wherein said method comprises the steps of:
(1) administering the candidate compound to a test animal; and (2) measuring the expression intensity of a polynucleotide in the eosinophil cells of the test animal, wherein the polynucleotide is selected from (a) or (b):
(a) a polynucleotide encoding a TR3 or TINUR receptor protein; and (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein.
- 8. A method of screening for a compound that increases the expression level of the polynucleotide (a) or (b), wherein said method comprises the steps of detecting the influence on expression level by the method of claim 5, and selecting a compound that increases that expression level as compared to a control.
- 9. A method of detecting the influence of a candidate compound on the expression level of a polynucleotide encoding a TR3 or TINUR receptor protein, wherein said method comprises the steps of:
(1) contacting a candidate compound with a cell or cell extract containing a DNA comprising a structure such that a reporter gene and the transcription regulatory region of a gene encoding a TR3 or TINUR receptor protein are operably linked; and (2) measuring the activity of the reporter gene.
- 10. A method of screening for a candidate compound that increases the expression level of a gene encoding a TR3 or TINUR receptor protein, wherein said method comprises the steps of detecting the influence of a compound on the activity of the reporter gene by the method of claim 9, and selecting a compound that increases the activity compared to a control.
- 11. A method of screening candidate compounds for a therapeutic agent for an allergic disease, wherein said method comprises the steps of:
1) contacting a test compound with a TR3 or TINUR receptor protein; 2) measuring the binding activity between the test compound and the TR3 or TINUR receptor protein; and 3) selecting the compound that binds to the TR3 or TINUR receptor protein.
- 12. A method of screening candidate compounds for a therapeutic agent for an allergic disease, wherein said method comprises the steps of:
1) providing cells transfected with (a) a DNA that can express a fusion protein of a TR3 or TINUR receptor protein or its ligand binding domain and a transcription regulatory region binding protein, and (b) a DNA having a reporter gene is operably linked downstream of a DNA sequence to which the transcription regulatory region binding protein binds; 2) contacting the cell with the test compound; 3) measuring the activity of the reporter gene; and 4) selecting the compound that changes this activity.
- 13. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a compound obtainable by the screening method of claim 10.
- 14. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 10.
- 15. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a ligand of a TR3 or TINUR receptor.
- 16. The therapeutic agent for an allergic disease of claim 15, wherein the ligand of a TR3 or TINUR receptor is a prostaglandin comprising a cyclopentenone structure.
- 17. The therapeutic agent for an allergic disease of claim 16, wherein the prostaglandin having a cyclopentenone structure is selected from the group consisting of prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, and 8-isoprostaglandin A1.
- 18. The therapeutic agent for an allergic disease of claim 15, wherein the ligand of a TR3 receptor is any one of the compounds listed in Tables 14 to 49.
- 19. The therapeutic agent for an allergic disease of claim 13, wherein the allergic disease is atopic dermatitis.
- 20. An animal model for an allergic disease, wherein the animal is a transgenic non-human vertebrate in which the expression intensity of polynucleotide (a) or (b) below is decreased in eosinophil cells:
(a) a polynucleotide encoding a TR3 or TINUR receptor protein; and (b) a polynucleotide encoding a protein whose expression in the eosinophils of an atopic dermatitis patient is increased, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide encoding a TR3 or TINUR receptor protein.
- 21. The animal model of claim 20, wherein the transgenic animal is a knockout animal.
- 22. A method of inducing cell apoptosis, said method comprising activation of a TR3 or TINUR receptor protein in the cell.
- 23. A method of inducing cell apoptosis, said method comprising activation of a TR3 or TINUR receptor protein in the cell, which comprises the step of contacting a cell with a compound that is obtainable by the screening method of claim 10, or a prostaglandin comprising a cyclopentenone structure.
- 24. The apoptosis induction method of claim 22, wherein said cell is an eosinophil cell.
- 25. An apoptosis-inducing agent, which comprises a compound or a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 10.
- 26. An apoptosis-inducing agent comprising a ligand of a TR3 or TINUR receptor as an active ingredient.
- 27. The apoptosis-inducing agent of claim 26, wherein the ligand of the TR3 or TINUR receptor is. a prostaglandin comprising a cyclopentenone structure.
- 28. The apoptosis-inducing agent of claim 27, wherein the prostaglandin comprising a cyclopentenone structure is selected from the group consisting of prostaglandin A2, prostaglandin A1, 15-epi prostaglandin A1, 15(R)-15-methyl prostaglandin A2, 16-phenoxy tetranor prostaglandin A2, 17-phenyl trinor prostaglandin A2, 15-deoxy-delta 12,14-prostaglandin A1, 15-deoxy-delta 12,14-prostaglandin J2, and 8-isoprostaglandin A1.
- 29. The apoptosis-inducing agent of claim 26, wherein the ligand of the TR3 receptor is any one of the compounds listed in Tables 14 to 49.
- 30. A TR3 or TINUR gene expression-inducing agent, which comprises a ligand of an eosinophil CD30 receptor.
- 31. The testing method of claim 2, wherein the allergic disease is atopic dermatitis.
- 32. A method of screening for a compound that increases the expression level of the polynucleotide (a) or (b), wherein said method comprises the steps of detecting the influence on expression level by the method of claim 6, and selecting a compound that increases that expression level as compared to a control.
- 33. A method of screening for a compound that increases the expression level of the polynucleotide (a) or (b), wherein said method comprises the steps of detecting the influence on expression level by the method of claim 7, and selecting a compound that increases that expression level as compared to a control.
- 34. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a compound obtainable by the screening method of claim 11.
- 35. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a compound obtainable by the screening method of claim 12.
- 36. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 11.
- 37. A therapeutic agent for an allergic disease, said agent comprising, as an active ingredient, a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 12.
- 38. The therapeutic agent for an allergic disease of claim 14, wherein the allergic disease is atopic dermatitis.
- 39. The therapeutic agent for an allergic disease of claim 15, wherein the allergic disease is atopic dermatitis.
- 40. The therapeutic agent for an allergic disease of claim 16, wherein the allergic disease is atopic dermatitis.
- 41. The therapeutic agent for an allergic disease of claim 17, wherein the allergic disease is atopic dermatitis.
- 42. The therapeutic agent for an allergic disease of claim 18, wherein the allergic disease is atopic dermatitis.
- 43. The therapeutic agent for an allergic disease of claim 34, wherein the allergic disease is atopic dermatitis.
- 44. The therapeutic agent for an allergic disease of claim 35, wherein the allergic disease is atopic dermatitis.
- 45. The therapeutic agent for an allergic disease of claim 36, wherein the allergic disease is atopic dermatitis.
- 46. The therapeutic agent for an allergic disease of claim 37, wherein the allergic disease is atopic dermatitis.
- 47. A method of inducing cell apoptosis, said method comprising activation of a TR3 or TINUR receptor protein in the cell, which comprises the step of contacting a cell with a compound that is obtainable by the screening method of claim 11.
- 48. A method of inducing cell apoptosis, said method comprising activation of a TR3 or TINUR receptor protein in the cell, which comprises the step of contacting a cell with a compound that is obtainable by the screening method of claim 12.
- 49. The apoptosis induction method of claim 23, wherein said cell is an eosinophil cell.
- 50. The apoptosis induction method of claim 47, wherein said cell is an eosinophil cell.
- 51. The apoptosis induction method of claim 48, wherein said cell is an eosinophil cell.
- 52. An apoptosis-inducing agent, which comprises a compound or a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 11.
- 53. An apoptosis-inducing agent, which comprises a compound or a prostaglandin comprising a cyclopentenone structure and that is obtainable by the screening method of claim 12.
Priority Claims (1)
Number |
Date |
Country |
Kind |
2002-193841 |
Jul 2002 |
JP |
|