JP2001128695A - Screening method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a compound for promoting or inhibiting function of orphan receptor protein or its salt. SOLUTION: This method for screening a compound for promoting or inhibiting the function of the orphan receptor protein or its salt comprises (i) measuring a cell stimulation activity in the case of bringing a test compound into contact with an orphan receptor protein expression cell or its cell membrane fraction and a cell stimulation activity in the case of bringing the test compound into contact with an orphan receptor nonexpression cell or its cell membrane fraction, comparing the cell stimulation activities of the test compounds to give a compound having agonist activity and using this compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for screening high-concentration test compounds using activation of orphan receptor protein-expressing cells as an index, and utilizing the common structure of test compounds having agonist activity to orphans. The present invention relates to a method for efficiently screening for a compound that promotes or inhibits the function of a receptor protein, and a method for determining an (endogenous) ligand of an orphan receptor protein by using the common structure.

[0002]

2. Description of the Related Art Many physiologically active substances such as hormones and neurotransmitters exert their actions by binding to receptor molecules present on the cell surface, and regulate various life phenomena.
Searching for a substance that supplements, enhances or inhibits the action of these physiologically active substances is one of the main means for the research and development of new drugs. It is extremely important to understand the properties of body molecules. Recent developments in molecular biological techniques have made it possible to analyze receptors for many physiologically active substances at the molecular level. A group of such receptor molecules having a common structural feature that penetrates the cell membrane seven times is known and is called a seven-transmembrane receptor. Further, since they are coupled to the intracellular signal transduction system via GTP-binding proteins (G proteins), they are also called G protein-coupled receptors. Ligands of the seven-transmembrane receptor include proteins, peptides, amines, amino acids,
There are a wide variety of nucleotides, nucleosides, eicosanoids, phospholipids, odorants, light, and more. With recent advances in genomic or cDNA analysis techniques, many genes encoding receptors have been reported. Some of them are putatively belonging to a family of seven transmembrane receptors from sequence characteristics, but are called orphan receptors because their corresponding ligands are not known. If such orphan receptor ligands could be identified, it is expected that new physiological functions or disease states regulated by the receptors and ligands will be revealed. for that reason,
The search for ligands for orphan receptors has attracted attention as a means to discover new targets for drug development. For orphan receptors that have shown some remarkable similarity to known ligands so far, we examined known ligands or their analogs as candidate ligands and actually identified endogenous ligands. FQ / nocice
Although there are examples such as ptin (Meunier, J.-C. Nature 393: 211-212, 1998), there is a limit in estimating the structure of a ligand from only the structural similarity of a known ligand or its analog. There is. In many cases, endogenous ligands are identified by purification using activation of the signal transduction system of orphan receptor protein-expressing cells as an index (Sakura
i, T. et al. Cell 92: 573-585, 1998; Hinuma, S. et
al. Nature 393: 272-276, 1998; Tatemoto, K. et al.
Biochem. Biophys. Res. Commun. 251: 471-476, 199
8). However, the signal transduction system through the seven-transmembrane receptor is not unique, and it is necessary to screen several assay systems in parallel in order to detect the activity derived from the endogenous ligand. In addition, ligands for the seven-transmembrane receptor are diversified, and it has been difficult to rationally select a sample as a ligand candidate to be used in an assay.

[0003]

PROBLEM TO BE SOLVED BY THE INVENTION A method for efficiently and reliably screening a compound or a salt thereof which promotes or inhibits the function of an orphan receptor protein and a method for determining an (endogenous) ligand of an orphan receptor protein Is an issue.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, it has been found that (i) orphan receptor protein-expressing cells or their cell membrane fractions (preferably high The cell stimulating activity of each of the test compound was measured when the test compound was brought into contact with the test compound (at a concentration of) or when the test compound was brought into contact with a cell not expressing the orphan receptor protein or its cell membrane fraction. Determining the common structure of the test compound having agonist activity by comparing the cell stimulating activities of the compounds, (iii)
When the orphan receptor protein-expressing cell or its cell membrane fraction is brought into contact with a candidate ligand having the common structure, and when the orphan receptor protein-expressing cell or its cell membrane fraction functions as the orphan receptor protein Comparing the cell stimulating activity in the case of contacting with a candidate substance of a compound that promotes or inhibits, and identifying the orphan receptor protein and a candidate substance of a compound that promotes or inhibits the function of the orphan receptor protein By measuring the amount of the binding, it is found that a compound or a salt thereof that promotes or inhibits the function of the orphan receptor protein can be efficiently screened,
Further, based on these findings, as a result of repeated studies, the present invention has been completed.

That is, the present invention relates to (1) (i) a method in which a test compound is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and a method in which an orphan receptor protein is not expressed or a cell membrane fraction thereof. When a test compound is brought into contact with the test compound, the respective cell stimulating activities are measured, and (ii) the common structure of the test compound having agonist activity is determined by comparing the cell stimulating activities of each test compound, iii) contacting the orphan receptor protein-expressing cell or its cell membrane fraction with a candidate ligand having the common structure, and contacting the orphan receptor protein-expressing cell or its cell membrane fraction with the orphan receptor protein Cell stimulating activity when contacted with a candidate compound that promotes or inhibits the function of Promoting or inhibiting the function of the orphan receptor protein, which comprises measuring the amount of specific binding between the orphan receptor protein and a candidate compound that promotes or inhibits the function of the orphan receptor protein. A method for screening a compound or a salt thereof, (2) a compound or a salt thereof obtainable by the screening method described in the above (1), and (3)
(I) When a test compound was brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and when a test compound was brought into contact with a cell that does not express an orphan receptor protein or its cell membrane fraction, (Ii) comparing the cell stimulating activities of each test compound to determine the common structure of the test compound having agonist activity, and (iii) determining the common structure of the ligand candidate substance having the common structure. The present invention relates to a method for determining a ligand of the orphan receptor protein or a subtype thereof by measuring a specific binding amount to the orphan receptor protein. More specifically, (4) (i)
When the test compound (a) is brought into contact with an orphan receptor protein-expressing cell or its cell membrane fraction, and when the test compound (a) is brought into contact with a cell that does not express an orphan receptor protein or its cell membrane fraction. In (2), a compound having an agonist activity is obtained by measuring the cell stimulating activity of each, and (ii) comparing the cell stimulating activity of each test compound (a), and (iii) a compound having the agonist activity. When the ligand candidate deduced from the structure is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and when the test compound (b) is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction. Comparing the cell stimulating activity and measuring the specific binding amount between the orphan receptor protein and the test compound (b). Method of screening a compound or its salt that promotes or inhibits the function of the orphan receptor proteins, (5) above (4)
A compound or a salt thereof obtainable by the screening method described above, and (6) (i) a test compound (a) on an orphan receptor protein-expressing cell or a cell membrane fraction thereof.
And contacting the test compound (a) with a cell that does not express an orphan receptor protein or a cell membrane fraction thereof, the respective cell stimulating activities were measured, and (ii) each test compound ( By comparing the cell stimulating activity of a), a compound having an agonist activity is obtained, and (ii)
i) measuring the amount of specific binding of the ligand candidate substance to the orphan receptor protein deduced from the common structure possessed by the compound having agonist activity to determine the ligand of the orphan receptor protein or a subtype thereof And how to determine.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION (A) Orphan receptor protein: As used herein, the term "orphan receptor protein" means a protein whose ligand is not known. And unknown. Specific examples of the orphan receptor protein include, for example, the FM-3 receptor protein (Tan, C. P et al, Genomics 52, 223-22) used in Example 1 described later.
9) or mas receptor protein (Young D. et al., Pro
Natl. Acad. Sci. USA, 85, 5339-5342, 1988), and those described in the orphan receptor database of Swiss-plot. The orphan receptor protein used in the present invention may form a salt, and a salt of the “orphan receptor protein” may be a physiologically acceptable base (eg, an alkali metal) or an acid (organic acid). , An inorganic acid), but a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Salts with tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. The DNA encoding the orphan receptor protein used in the present invention may be any DNA containing a DNA encoding the orphan receptor protein. Genome DN
A, genomic DNA library, human or warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit,
Cells of pigs, sheep, cattle, monkeys, etc. (eg, retinal cells, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β)
Cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Or precursors of these cells, stem cells or cancer cells, etc.) or any tissue in which those cells are present, such as the brain, each part of the brain (eg, retina, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus,
Hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary,
Stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate , Testicles, ovaries, placenta, uterus, bones, joints, skeletal muscle, etc.
Or cells of blood cell lineage or cultured cells thereof (for example, M
EL, M1, CTLL-2, HT-2, WEHI-3,
HL-60, JOSK-1, K562, ML-1, MO
LT-3, MOLT-4, MOLT-10, CCRF-
CEM, TALL-1, Jurkat, CCRT-HS
B-2, KE-37, SKW-3, HUT-78, HU
T-102, H9, U937, THP-1, HEL, J
K-1, CMK, KO-812, MEG-01), a cDNA library derived from the tissue / cell, or a synthetic DNA. The vectors used for the library are bacteriophage, plasmid,
Any of cosmid, phagemid and the like may be used. In addition, Reverse Transcriptase Polymerase Chain was directly used by using an RNA fraction prepared from the above-described tissue / cell.
It can also be amplified by Reaction (hereinafter abbreviated as RT-PCR method).

[0007] DNA encoding the orphan receptor protein used in the present invention can also be produced by the following genetic engineering techniques. As a means for cloning DNA that completely encodes the orphan receptor protein, a known PC primer using a synthetic DNA primer having a partial nucleotide sequence of the orphan receptor protein is used.
The target DNA is amplified from the DNA library or the like by the R method, or the DNA incorporated in an appropriate vector is labeled, for example, using a DNA fragment or a synthetic DNA having a partial or entire region of the orphan receptor protein. Selection can be carried out by hybridization with those obtained. Hybridization methods are described, for example, in Molecular Cloning (2nd ed .; J. Sambr).
ook et al., Cold Spring Harbor Lab. Press, 1989)
And the like. When a commercially available library is used, the procedure is performed according to the method described in the attached instruction manual. The DNA encoding the cloned orphan receptor protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker if desired. The DNA is at its 5 '
It has ATG as a translation initiation codon on the terminal side, and TAA and TGA as translation termination codons on the 3 ′ terminal side.
Alternatively, it may have a TAG. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.

(B) Orphan receptor protein-expressing cells or cell membrane fraction thereof or cells not expressing orphan receptor protein or cell membrane fraction thereof: In the present specification, “orphan receptor protein-expressing cells” Refers to a host cell that expressed the orphan receptor protein of (A) above. As a method for expressing an orphan receptor protein in a host cell, for example, the DNA encoding the orphan receptor protein of the above (A) can be expressed by the following method. That is, an expression vector for an orphan receptor protein can be obtained by, for example, (a) cutting out a target DNA fragment from DNA encoding the orphan receptor protein, and (ii) converting the DNA fragment into a promoter of an appropriate expression vector. It can be manufactured by connecting downstream. As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pU
C13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as phage λ, and animal viruses such as retrovirus, vaccinia virus, and baculovirus. The promoter to be used may be any promoter as long as it is appropriate for the host used for gene expression.

When the host used for the transformation is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter and the like can be used. When the host is Escherichia, Trp promoter,
T7 promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, etc. When the host is yeast penP promoter, PHO5 promoter, PGK Promoter, GAP promoter, ADH
One promoter, GAL promoter and the like are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In the present invention, the host is preferably an animal cell or an insect cell in order to measure the cell stimulating activity mediated by the orphan receptor protein. In addition to those described above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used as the expression vector. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (Hereinafter sometimes abbreviated as Neo,
G418 resistance). In particular, CHO (dhf
When the DHFR gene is used as a selection marker using r ⁻ ) cells, selection can also be made using a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the polypeptide or its partial peptide. When the host is a genus Escherichia,
When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like, and when the host is yeast, a mating factor α (MFα )
・ Signal sequence, invertase signal sequence, etc.
When the host is an animal cell, for example, insulin
Signal sequence, α-interferon signal sequence,
Antibody molecules, signal sequences, etc. can be used. Using the vector containing the DNA encoding the orphan receptor protein thus constructed, a transformant can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect or insect cells, animal cells and the like are used. As described above, insect cells and animal cells are preferably used. Examples of Escherichia bacteria include Escherichia coli K12.
・ DH1 [Procedings of the National
Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Volume 60,
160 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Research), Vol. 9,
309 (1981)], JA 221 [Journal of
Molecular biology (Journal of Molecular B
iology)], 120, 517 (1978)], HB10
1 [Journal of Molecular Biology,
41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus genus bacteria include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry.
emistry), Vol. 95, 87 (1984)].

Examples of the yeast include Saccharomyces cerevisiae AH22,
^{AH22R -, NA87-11A, DKD-5D} , 20
B-12 or the like is used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Natu
re), 315, 592 (1985)]. As insect cells, for example, when the virus is AcNPV, Spodoptera frugiperda cell; Sf
Cells), MG1 cells from Trichoplusia ni midgut, Tri
High Five ^TM cells from choplusia ni eggs, Mamestra br
Cells derived from assicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (AT
CC CRL1711), Sf21 cells [Vaughn, JL et al.
In Vitro, Volume 13, 213-217
Page (1977)]. Examples of animal cells include monkey COS-7 cells, Vero cells, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO cells), mouse L cells, mouse 3T3 cells, mouse myeloma cells, human HEK293 cells, Human FL cells, C1
27 cells, BALB3T3 cells, Sp-2 / O cells and the like are used. To transform Escherichia,
For example, Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2
110 (1972) and Gene, 17, 107
(1982). To transform Bacillus sp., For example,
And General Genetics (Molecular &
General Genetics), Vol. 168, 111 (1979). To transform yeast, for example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 75
Vol., 1929 (1978).

To transform insect cells or insects,
For example, Bio / Technology, 6
Volume, pp. 47-55 (1988). Transformation of animal cells is described, for example, in Virology, 52, 456 (197).
This is performed according to the method described in 3). As a method for introducing an orphan receptor protein expression vector into cells, for example, a lipofection method [Felgner, PL et a
l. Proceedings of The National Academy of Sciences
of The United States of America), 84, 7413
(1987)], calcium phosphate method [Graham, F.
L. and van der Eb, AJ virology (Virolog
y), 52, 456-467 (1973)], electroporation [Nuemann, E. et al. Embo Journal (E
MBO J.), 1, 841-845 (1982)]. Thus, cells expressing the orphan receptor protein are obtained. In addition, using animal cells,
As a method for stably expressing an orphan receptor protein, there is a method in which cells in which an expression vector introduced into an animal cell is integrated into a chromosome are selected by clonal selection. Specifically, a transformant is selected using the above-mentioned selection marker as an index. Furthermore, a stable animal cell line having high ability to express an orphan receptor protein can be obtained by repeatedly performing clonal selection on the animal cells obtained using the selectable marker. When the dhfr gene was used as a selection marker, the DNA encoding the orphan receptor protein was amplified in the cell together with the dhfr gene by culturing the MTX concentration gradually and selecting a resistant strain, together with the dhfr gene. In addition, an animal cell line with higher expression can be obtained. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are contained. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Also, yeast extract,
Vitamins and growth promoting factors may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing Escherichia bacteria, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, as a medium, for example, Burkholder minimal medium [Bostian, KL et al.
"Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 450
5 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., "Processings of the National Academy of Sciences of the USA (Proc. Acad. Sci. US
A), 81, 5330 (1984)].
Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.

When culturing a transformant whose host is an insect cell, Grace's Insect Medium (Grace,
TCC, Nature, 195,788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or stirring are added. When culturing a transformant whose animal host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], D
MEM medium [Virology, 8, 396
(1959)], RPMI 1640 medium [Journal
The Journal of The American Medical Association
n) Volume 199, 519 (1967)], 199 Medium [Proceeding of the Society for the Biological Medicine (Proceeding of the Society)]
for The Biological Medicine), 73, 1 (195
0)]. Preferably, the pH is between about 6-8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added. Especially CHO
When (dhfr ⁻ ) cells and the dhfr gene are used as selection markers, it is preferable to use a DMEM medium containing dialyzed fetal bovine serum containing almost no thymidine.

As the cell membrane fraction, after crushing the cells,
It refers to a fraction containing a large amount of cell membrane obtained by a method known per se. As a method for disrupting cells, Potter-El
Examples include a method of crushing cells with a vehjem-type homogenizer, crushing with a Waring blender or Polytron (manufactured by Kinematica), crushing with ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure by a French press, and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is fed at a low speed (500 r
pm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes).
Min) and centrifuged, and the supernatant
(0000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 ³ to 10 ⁸ molecules per cell, more preferably 10 ⁵ to 10 ⁷ molecules per cell. It is suitable. The higher the expression level, the higher the agonist (ligand) binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system, but also enables the measurement of a large number of samples in the same lot. Become like The cells that do not express the orphan receptor protein or the cell membrane fraction thereof are the cells listed as the above host cells and do not express the orphan receptor protein. As the cell membrane fraction, those similar to the above can be used.

(C) Test compound or test compound (a): As used herein, the term “test compound” or “test compound (a)” refers to, for example, a natural or unnatural peptide,
Examples include natural and non-natural proteins, natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, and the like. These test compounds or test compounds (a)
The peptides, proteins, compounds or fermentation products used for the above may form salts, and these salts include physiologically acceptable bases (eg, alkali metals) and acids (organic acids, inorganic acids) And particularly preferably a physiologically acceptable acid addition salt.
Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

(D) Cell stimulating activity: A test compound (specifically, test compound (a)) is brought into contact with an orphan receptor protein-expressing cell or its cell membrane fraction, A test compound (specifically, a test compound)
When (a)) is contacted, the respective cell stimulating activities include, for example, (a) fluctuation of extracellular pH, (b) arachidonic acid release, (c) acetylcholine release, (d) intracellular Ca ²⁺ release (E) fluctuation of intracellular cAMP, (f) fluctuation of intracellular cGMP, (g) inositol phosphate production, (h) fluctuation of cell membrane potential,
(i) phosphorylation of intracellular proteins, (j) activation of c-fos,
Using (k) GTPγS binding, (l) reporter gene expression, and the like as indices, it can be measured according to a known method or using a commercially available measurement kit. As the measurement of the cell stimulating activity performed in the screening method or the ligand determination method of the present invention, among the above, a measurement method using a change in extracellular pH as an index is preferably used. Specifically, first, orphan receptor protein-expressing cells or their cell membrane fractions, and cells that do not express orphan receptor proteins or their cell membrane fractions are separately cultured in a multiwell plate or the like. Before measuring the cell stimulating activity, the cells are replaced with a fresh medium or an appropriate buffer that is not toxic to cells in advance, and the test compound (specifically, test compound (a)) is added to each cell.
After adding for a certain period of time, the cells or their cell membrane fractions are extracted or the supernatant is recovered, and the generated products are quantified according to the respective methods. If the production of a substance as an indicator of the cell stimulating activity is difficult to be assayed by a degrading enzyme contained in the cell, the assay may be performed by adding an inhibitor against the degrading enzyme. Also, cAM
The activity such as P production suppression can be detected as a production suppression effect on cells whose basal production has been increased by forskolin or the like or on cell membrane fractions thereof. In order to perform screening by measuring cell stimulating activity, a cell expressing an appropriate orphan receptor protein or a cell membrane fraction thereof and a cell not expressing an orphan receptor protein or a cell membrane fraction thereof are required. The transformant-expressing orphan receptor protein-expressing cell may be a stable expression strain or a transient expression strain.
In order to measure the cell stimulating activity, it is necessary to measure the cell stimulating activity at a concentration as high as possible within a range where a specific reaction of the cell can be identified so that a weak agonist activity can be detected. preferable. The high concentration in this case is generally 10 ⁻⁸ M to 1 M, preferably 10 ⁻⁶ M to 10 M.
^-2 say M. Test compound (specifically, test compound
Examples of (a)) include those similar to those described in (C) above.

The above cell stimulating activity measuring system will be described more specifically below. The following (1) to (7)
In the description, the term "test compound"
(a) ". (1) Cell stimulating activity measuring system characterized by measuring fluctuation of extracellular pH (acidification rate) Changes in response to a test compound having an agonistic activity in an orphan receptor protein-expressing cell or a cell membrane fraction thereof. Extracellular pH is measured by cytosensor (Cytosens
or) cell stimulating activity can be measured by using an apparatus (such as Molecular Devices). A specific method for measuring the cell stimulating activity of a test compound by measuring a change in extracellular pH using a site sensor device is described below. After culturing the orphan receptor protein-expressing cell or the cell membrane fraction thereof for about 2 hours to about 48 hours, preferably for about 5 hours to about 24 hours (for example, in a capsule for a cytosensor device), the pH of the medium is adjusted. To stabilize. Until the pH of the medium is stabilized, it is preferable to perfuse, for example, an RPMI1640 medium (manufactured by Molecular Devices) containing 0.1% bovine serum albumin. Next, the test compound is brought into contact with an orphan receptor protein expressed in an orphan receptor protein-expressing cell or a cell membrane fraction thereof. As a method for contacting, a method of perfusing a medium containing a test compound into orphan receptor protein-expressing cells or a cell membrane fraction thereof is generally used. Next, the pH change of the medium caused by contacting the medium containing the test compound with the orphan receptor protein expressed in the orphan receptor protein-expressing cells or its cell membrane fraction is measured. The above methods (1) to (4) are carried out using cells that do not express the orphan receptor protein or cell membrane fractions thereof.

(2) Cell stimulating activity measuring system characterized by measuring the radioactivity of GTPγS When an orphan receptor protein-expressing cell is stimulated by a test compound having an agonistic activity, the intracellular G protein is activated. And GTP binds. This phenomenon is also observed in the membrane fraction of orphan receptor protein-expressing cells. Normally, GTP is hydrolyzed to change to GDP. At this time, if GTPγS is added to the reaction solution, GTPγS binds to G protein similarly to GTP, but contains G protein without hydrolysis. The state of binding to the cell membrane is maintained. When labeled GTPγS is used, the activity of stimulating the receptor-expressing cells of the test compound can be measured by measuring the radioactivity remaining on the cell membrane. Using this reaction, the stimulating activity of the test compound on orphan receptor protein-expressing cells and cells that do not express the orphan receptor protein can be measured and compared. This method is an assay method using an orphan receptor protein and a membrane fraction containing cells that do not express the orphan receptor protein, and measures cell stimulating activity. Substances that exhibit GTPγS binding promoting activity to the protein membrane fraction and do not exhibit GTPγS binding promoting activity to the membrane fraction that does not express the orphan receptor protein are ligand candidate substances. A specific method for measuring the cell stimulating activity according to this method is described below. Cell membrane fraction containing orphan receptor protein,
Membrane dilution buffer (eg, 50 mM Tris, 5 mM MgCl ₂ , 150
Dilute with mM NaCl, 1 μM GDP, 0.1% BSA pH 7.4). The dilution ratio depends on the expression level of the receptor protein. Next, transfer the obtained liquid to a suitable container (for example, Falcon
2053), the test compound is added, and [ ³⁵ S] GTPγS is further added to a final concentration of about 200 pM. Next, after keeping the medium obtained in the above at about 25 ° C. for about 1 hour, a washing buffer (for example, ice-cold 50 mM Tris, 5
mM MgCl ₂ , 150 mM NaCl, 0.1% BSA, 0.05% CHAPS pH
7.4 1.5 ml) and add (eg glass fiber filter paper)
(Using GF / F etc.). After the filter paper is kept warm (for example, at about 65 ° C. for about 30 minutes) and dried, the radioactivity of [ ³⁵ S] GTPγS bound to the membrane fraction remaining on the filter paper is measured by a liquid scintillation counter. The above methods (1) to (4) are carried out using a cell membrane fraction containing no orphan receptor protein.

(3) Cell stimulating activity measuring system characterized by measuring the fluctuation of intracellular cAMP The orphan receptor protein-expressing cells are stimulated by an agonistic stimulation of a test compound having an agonistic activity.
MP amount fluctuates. Using this reaction, the cell stimulating activity of the test compound on cells expressing the orphan receptor protein can be measured. The amount of cAMP produced by various animal cells expressing orphan receptor protein was determined by immunizing mice, rats, rabbits, goats, cows, etc.
By using MP antibody and ¹²⁵ I-labeled cAMP (both are commercially available), it can be measured by RIA or other EIA systems combining anti-cAMP antibody and labeled cAMP. Also anti-cAM
It is also possible to quantify the P antibody by SPA method using beads containing scintillant and ¹²⁵ I-labeled cAMP, which are immobilized using an antibody against protein A or an IgG of an animal used for production of anti-cAMP antibody (for example, Use a kit from Amersham Pharmacia Biotech). In this measurement system, the activity of promoting cAMP production can be measured.
Alternatively, the intracellular cAMP level is increased by a substance that increases the intracellular cAMP level, such as forskolin, and the change in the intracellular cAMP level is observed by adding a test compound. Production inhibitory activity) can be measured. A specific method for measuring the cell stimulating activity according to this method is described below. Animal cells (eg, CHO cells) expressing the orphan receptor protein are seeded at an appropriate concentration (eg, 5 × 10 ⁴ cells / well) in a 24-well plate and cultured for about 48 hours. Next, the animal cells in which the orphan receptor protein was expressed were buffered (for example, Hanks buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES (hereinafter referred to as 0.2 mM 3 -Hank's buffer (pH 7.4) containing isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES is called a reaction buffer)). Thereafter, an appropriate amount (for example, about 0.5 ml) of a reaction buffer is added to the cells, and the mixture is incubated in an incubator for about 30 minutes. Next, after removing the reaction buffer, an appropriate amount (eg, about 0.25 ml) of the reaction buffer is added to the cells, and then an appropriate amount (eg, about 0.25 ml) of the reaction buffer (cAMP production inhibitory activity is determined). When measuring, an appropriate amount (for example, about 2 μM forskolin is preferable)
1 nM) of the test compound was added to the cells, and about 3
Incubate at 7 ° C for about 24 minutes. Next, an appropriate amount (for example, about 100 μl) of 20% perchloric acid is added to stop the reaction, and then the cells are placed on ice for about 1 hour to extract intracellular cAMP. The amount of cAMP in the extract is measured using a cAMP EIA kit (Amersham Pharmacia Biotech) or the like. The above methods (1) to (4) are performed using cells that do not express the orphan receptor protein.

(4) Cell stimulating activity measuring system characterized by introducing a CRE-reporter gene DNA containing a CRE (cAMP response element) is picked up by Picker Gene Basic Vector or Picker Gene Enhancer Vector (Toyo Ink Manufacturing Co., Ltd.) or the like. Into a multiple cloning site upstream of the luciferase gene, and this is used as a CRE-reporter gene vector. CRE
-In cells transfected with the reporter gene vector, stimulation with elevated cAMP induces CRE-mediated luciferase gene expression and subsequent luciferase protein production. That is, by measuring the luciferase activity, a change in the amount of cAMP in the cells into which the CRE-reporter gene vector has been introduced can be detected. Cell stimulating activity can be measured using cells transfected with a CRE-reporter gene vector into orphan receptor protein-expressing cells. A specific method for measuring the cell stimulating activity according to this method is described below. Orphan receptor protein-expressing cells into which the CRE-reporter gene has been introduced are seeded at an appropriate concentration (for example, 5 × 10 ³ cells / well) on a 24-well plate, and cultured for about 48 hours. Cells are diluted with an appropriate amount (eg, 0.2 mM) of a buffer (eg, 3-isobutyl-methylxanthine and 0.05% BSA).
Hanks buffer (pH 7.4) containing 20 mM HEPES or less, 0.
2 mM 3-isobutyl-methylxanthine and 0.05% BSA
And Hank's buffer (pH 7.4) containing 20 mM HEPES are called a reaction buffer)). Thereafter, an appropriate amount (for example, 0.5 ml) of a reaction buffer is added to the cells, and the mixture is incubated in an incubator for about 30 minutes. Next, after removing the reaction buffer, a new appropriate amount (eg, 0.25 ml) of the reaction buffer is added to the cells.
An appropriate amount (eg, 1 nM) of a test compound and an appropriate amount (eg, 0.25 ml) of a reaction buffer (preferably containing 2 μM forskolin when measuring cAMP production inhibitory activity) are added to the cells. Incubate at about 37 ° C. for about 24 hours. The cells are lysed with a cell lysing agent for Picker Gene (Toyo Ink Mfg. Co., Ltd.) or the like, and a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) is added to the lysate. Luminescence by luciferase is measured with a luminometer, liquid scintillation counter, top counter, or the like. The above methods (1) to (4) are carried out using cells that do not express the orphan receptor protein or cell membrane fractions thereof. As a reporter gene, other than luciferase, for example, alkaline phosphatase, chloramphenicol acetyltransferase, or β-galactosidase can also be used. The enzymatic activities of the gene products of these reporter genes can be easily measured using a commercially available measurement kit as follows. That is, alkaline phosphatase activity, for example, Lumi-P manufactured by Wako Pure Chemical
chloramphenicol acetyltransferase by hos 530
e) The activity is measured, for example, by FAST CAT chrolamphenico manufactured by Wako Pure Chemical.
l Acetyltransferase Assay K
β-galactosidase activity can be measured by iT using, for example, Aurora Gal-XE manufactured by Wako Pure Chemical Industries.

(5) Cell stimulating activity measuring system characterized by measuring arachidonic acid release Orphan receptor protein-expressing cells release arachidonic acid metabolites extracellularly as a result of stimulation by an agonist.
By previously introducing arachidonic acid having radioactivity into cells, the cell-stimulating activity can be measured by measuring the radioactivity released outside the cells. At this time, the cell stimulating activity can be measured by adding the test compound and examining the arachidonic acid metabolite releasing activity of the test compound. A specific method for measuring the cell stimulating activity according to this method is described below. Orphan receptor protein-expressing cells (eg, orphan receptor protein-expressing CHO cells, etc.) are seeded in a 24-well plate at an appropriate concentration (eg, 5 × 10 ⁴ cells / well) and cultured for 24 hours. After culturing, [ ³ H] arachidonic acid is added in an appropriate amount (eg, 0.25
μCi / well). About 16 hours after the addition of [ ³ H] arachidonic acid, the cells are washed (eg, 0.05% BSA and 20%
Wash with Hank's buffer (pH 7.4) containing mM HEPES) and add buffer (e.g., 0.05% BSA and 20 mM HEPES) to each well.
Hanks buffer (pH 7.4) containing: 0.05% BSA
And a Hanks buffer (pH 7.4) containing 20 mM HEPES are referred to as a reaction buffer. 3.) Add the test compound dissolved in). After incubating at about 37 ° C. for about 60 minutes, the reaction buffer is added to an appropriate amount (for example, 400 μl) of a scintillator, and the amount of [ ³ H] arachidonic acid metabolite released in the reaction solution is measured by a scintillation counter. The above methods (1) to (4) are performed using cells that do not express the orphan receptor protein.

[0023] (6) Ca ² in cells by the cell stimulating activity measurement system orphan receptor protein-expressing cells, characterized by measuring the release of intracellular Ca ²⁺ stimulated by the test compounds with agonist activity ⁺ Increases concentration. By utilizing this, the cell stimulating activity can be measured. A specific method for measuring the cell stimulating activity according to this method is described below. The orphan receptor protein-expressing cells are seeded on a sterilized cover glass for a microscope, and after about 2 days, an appropriate amount (for example, 4 mM) of Fura-2 AM (Dojindo Laboratories) is used.
Is replaced with suspended HBSS and left at room temperature for about 2 hours and 30 minutes. After washing with HBSS, a cover glass is set on the cuvette, and the increase in the ratio of the fluorescence intensity at 505 nm at the excitation wavelengths of 340 nm and 380 nm when the test compound is added is measured with a fluorimeter. FLIPR (Molecular Devices) can also be used as follows. That is, after adding Fluo-3 AM (manufactured by Dojindo Laboratories) to the cell suspension and allowing it to be taken up into the cells, the supernatant was washed several times by centrifugation.
Seed cells in 6-well plate. The sample is set in a FLIPR apparatus, a test compound is added in the same manner as in the case of Fura-2, and a change in the fluorescence intensity observed by the addition of the test compound is measured. The orphan receptor protein-expressing cells are co-expressed with a protein gene such as aequorin, which emits light by increasing intracellular Ca ions, and ae concentration is increased by increasing intracellular Ca ion concentration.
Utilizing the fact that quorin becomes Ca-binding and emits light, it is also possible to measure the cell stimulating activity by measuring the change in the emission intensity observed by the addition of the test compound. The method in this case is the same as described above except that the fluorescent substance is not incorporated. In addition, in order to facilitate the measurement of the change in intracellular Ca ion concentration, a cell in which a modified G protein such as a chimeric G protein is simultaneously expressed can be used. Substitution and is the chimeric G protein, Gi, Go, the G protein that does not use the Ca ²⁺ etc. Gs as signaling system in a functional domain of G proteins using Ca ^2+, such as G _4, G ₁₁ as a signal transduction system G protein. By using the chimeric protein, the signal transduction system of any G protein can be monitored by changes in Ca ²⁺ . The above methods (1) to (4) are performed using cells that do not express the orphan receptor protein.

(7) Cell stimulating activity measuring system characterized by measuring inositol phosphate production When a test compound having an agonist activity is added to orphan receptor protein-expressing cells, the intracellular inositol triphosphate concentration increases. I do. By observing this response in orphan receptor protein expressing cells produced by the test compound, the cell stimulating activity can be measured. A specific method for measuring the cell stimulating activity according to this method is described below. Orphan receptor protein expression was cultivated for 1 day in a medium in which myo- [2- ³ H] inositol (2.5 microCi / well) was added to orphan receptor protein-expressing cells on day 1 after seeding on a 24-well plate. After thoroughly washing the cells, the test compound is added, and 10% perchloric acid is added to stop the reaction. Neutralize with an appropriate amount (for example, 1.5 M) of KOH and an appropriate amount (for example, 60 mM) of HEPES solution, and use AG1x8 resin (Bio-Ra
After d) is passed through a packed column and washed, an appropriate amount (eg, 1 M) of HCOONH ₄ and an appropriate amount (eg, 0.1 M) H
The radioactivity eluted with COOH is measured with a liquid scintillation counter. The above methods (1) to (4) are performed using cells that do not express the orphan receptor protein.

(E) Regarding (test) compound having agonist activity: In the present specification, the “(test) compound having agonist activity” refers to the test compound described in the above (C) (for example, a natural or non-natural compound). Cell stimulation as described in (D) above, among peptide, natural / non-natural protein, natural / non-natural non-peptidic compound, synthetic compound, natural / non-natural fermentation product, etc.) or test compound (a) Any of the activity measurement systems (preferably, extracellular pH (acidificat
cell stimulatory activity is measured using an orphan receptor protein-expressing cell or a cell membrane fraction thereof by an orphan receptor protein. Test cells that do not show cell stimulating activity when using cells that do not express the same or cell membrane fractions thereof (for example, natural / non-natural peptides, natural / non-natural proteins, natural / non-natural non-peptidic compounds, Synthetic compounds, natural and non-natural fermentation products, etc.). More specifically, for example,
The orphan receptor protein is FM-3 (Tan, CP et
al., Genomics 52, 223-229, 1998), the (test) compounds having agonist activity include an R—X—NH ₂ structure at the C-terminus (X is Gly, Ala, Val, L
eu, Ile, Ser, Thr, Cys, Met, Gl
u, Asp, Lys, Arg, His, Phe, Ty
Indicates any amino acid residue such as r, Trp, Pro, Asn, Gln. ), And more specifically, peptides containing the amino acid sequences represented by SEQ ID NOs: 2, 6, and 20. Peptides, proteins, compounds or fermentation products which are these (test) compounds having agonist activity may also form salts, and these salts may include physiologically acceptable bases (eg, alkali metals) and the like. Examples thereof include salts with acids (organic acids and inorganic acids), and particularly preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like. Specific examples of criteria for determining whether or not the test compound (or test compound (a)) is a (test) compound having agonist activity in the cell stimulating activity measurement system described in (D) above are shown below. The following criteria are merely examples, and whether or not a test compound (or test compound (a)) has an agonist activity is not limited to the following criteria.

Extracellular pH as described in (D)-(1) above
(Acidification rate), when a cell stimulating activity measuring system characterized by using a cell stimulating activity measuring system, when the extracellular pH before contacting the test compound with the cells is 100%, the test compound A test in which the extracellular pH at the peak of the reaction when contacted with a cell exceeds 105% and no cell stimulating activity is observed when cells that do not express the orphan receptor protein or their cell membrane fractions are used The compound is selected as a (test) compound having agonist activity. The labeled G according to (D)-(2) above
When a cell stimulating activity measuring system characterized by measuring the radioactivity of TPγS was used, the radioactivity of the test group without the test compound was taken as 100%, and the radioactivity of the test group with the test compound was added. Is greater than 105%, and a test compound which does not show cell stimulating activity when cells that do not express orphan receptor protein or its cell membrane fraction is used is selected as a compound having agonist activity (test). . When a cell stimulating activity measuring system characterized by measuring the fluctuation of intracellular cAMP described in the above (D)-(3) is used, when the cAMP production inhibitory action is used as an index, false When the amount of cAMP produced by choline stimulation is 100% and the amount of cAMP produced by the addition of the test compound is 95% or less, and cells that do not express the orphan receptor protein or a cell membrane fraction thereof are used. The test compound having no cell stimulating activity is selected as a (test) compound having an agonist activity. On the other hand, when the cAMP production promoting action is used as an index, the amount of cAMP when no test compound is added is 10%.
0%, and the amount of cAMP produced by the addition of the test compound is
For test compounds that are not less than 105% and do not show cell stimulating activity when cells that do not express orphan receptor protein or their cell membrane fractions are used,
It is selected as a (test) compound having agonist activity. When a cell stimulating activity measuring system characterized by introducing the CRE-reporter gene described in the above (D)-(4) is used, when cAMP production inhibitory action is used as an index, stimulation with forskolin 100% of generated light
The test compound, which emits no more than 95% of the luminescence generated by the addition of the test compound and does not show cell stimulating activity when cells that do not express the orphan receptor protein or their cell membrane fractions are used Is selected as a (test) compound having agonist activity. on the other hand,
When the cAMP production promoting action is used as an index, the amount of luminescence when no test compound is added is defined as 100%, and the amount of luminescence generated by addition of the test compound is 105% or more. A test compound that does not show cell stimulating activity when using a cell that does not express a protein or a cell membrane fraction thereof is selected as a compound having an agonist activity (test). The above (D)-(5)
When using a cell stimulating activity measuring system characterized by measuring arachidonic acid release described in, the amount of [ ³ H] arachidonic acid metabolite in the medium with the reaction buffer without the test compound was set to 100%. When the amount of the [ ³ H] arachidonic acid metabolite in the medium when the test compound is added is 105% or more and cells that do not express the orphan receptor protein or a cell membrane fraction thereof are used, A test compound having no cell stimulating activity is selected as a (test) compound having an agonist activity. The above (D)-(6)
When using a cell stimulating activity measurement system characterized by measuring the release of intracellular Ca ²⁺ described in, the fluorescence intensity observed when no test compound is added is set to 100%, and the test compound is A test compound that has a fluorescence intensity of 105% or more when added and has no cell-stimulating activity when cells that do not express an orphan receptor protein or a cell membrane fraction thereof have agonist activity. (Test) compound to be selected. The above (D)-(7)
When using a cell stimulating activity measurement system characterized by measuring inositol phosphate production described in the above, the radioactivity in the medium by the reaction buffer without the test compound was 100
%, And the radioactivity in the medium when the test compound is added is 105% or more. When cells that do not express the orphan receptor protein or their cell membrane fractions are used, cell stimulating activity is observed. For test compounds not available,
It is selected as a (test) compound having agonist activity.

(F) Structure comparison of (test) compound having agonist activity: After selecting (test) compound having agonist activity according to (E) above, the structure of each (test) compound having each agonist activity was determined. By comparison, the common structure of the compound having the agonist activity (test) is estimated (or determined), and a ligand candidate substance having the common structure is prepared or obtained. The ligand candidate substance has a structure common to the (test) compound having the agonist activity, and each test compound (specifically, a test compound)
Compared with (a)), it refers to the substance (D) having a stronger cell stimulating activity (for example, a natural peptide, a natural protein, a natural non-peptidic compound, etc.). When the compound having the agonist activity (test) is a natural / non-natural peptide, a natural / non-natural protein, etc., the amino acid sequences encoding those peptides or proteins are compared, and their partial sequences having high homology are compared. Or a portion having a similar three-dimensional structure can be said to be a common structure. Specifically, for example, the orphan receptor protein is FM-3
(Tan, CP et al., Genomics 52, 223-229, 1998, etc.), the common structure is SEQ ID NO: 2, 6
Comparison of the amino acid sequences represented by and
Terminus R-X-NH ₂ structure (X is. To indicate any amino acid residue) can be derived common structure of having "a. From the common structure, in the screening method or the ligand determination method of the present invention, the orphan receptor protein is F
If a M-3, the ligand candidate substance having a common structure, believed to turn the FM-3 (endogenous) ligand is a peptide having an R-X-NH ₂ structure at its C-terminus. Peptides having an R—X—NH ₂ structure at the C-terminal include A-18-F-NH ₂ and F-8-F-NH ₂ (Perry,
SJ et al. FEBS Lett. 409: 426-430, 1997), pro
lactin-releasing peptide (Hinuma, S. et al. Natur
e 393: 272-276, 1998). In lower animals, it is universally present as a group of RF amide peptide families. Furthermore, unknown R
It believed -X-NH ₂ peptide is present in variously as with subhuman animals, in a novel R-X-NH ₂ peptide in mammalian FM-3 of (endogenous) believed ligand is present. When the (test) compound having agonist activity is a natural or non-natural non-peptidic compound, a synthetic compound, or the like, the chemical structures of those compounds are compared, and a common basic skeleton (for example, a specific ring) Structure, e.g., saturated or unsaturated alicyclic hydrocarbon such as cycloalkyl, cycloalkenyl, cycloalkanedienyl as "alicyclic hydrocarbon", aromatic heterocyclic ring as "heterocycle", saturated or unsaturated Saturated non-aromatic heterocycle (aliphatic heterocycle)
Etc.) can be said to be a common structure. Further, in the method for determining a ligand of the present invention, a ligand is determined using the above-mentioned common structure as an index. Therefore, obtaining a subtype of a ligand having a high structural similarity to the ligand is also significantly different from the conventional method. Easy and reliable.

(G) Steps after preparation or acquisition of a candidate ligand substance deduced (or determined) from a common structure possessed by a (test) compound having agonist activity:
Hereinafter, a method for preparing or obtaining a ligand candidate substance estimated (or determined) from the common structure possessed by the (test) compound having the agonist activity according to the above (F) will be specifically described. (1) Searching for natural peptides, natural proteins, and natural non-peptidic compounds having a common structure, selecting candidate ligands having a common structure, and preparing or obtaining candidate ligands having the common structure how to. Based on the common structure of the test compound described in (F) above (specifically, the test compound (a)), searching for a natural peptide, a natural protein, or a natural non-peptidic compound having a common structure Can deduce the ligand or its subtype. There are various known databases that can be used, but typical ones include, for example, the Beilstein Handbook of Organi
c Chemistry (Beilstein), CROPR (Serwent Crop Pro
tection Registry) file (Derwent), Derwent Dr
ug file (Derwent), The Merck Index (Merck
Company). Next, using the cell stimulating activity measurement system described in (D) above for the putative ligand,
By measuring the cell stimulating activity and comparing it with the cell stimulating activity with the ligand candidate substance, it can be determined whether or not the ligand is a ligand or a subtype thereof.

(2) (When the (test) compound having agonist activity is a peptide, protein or a salt thereof) A primer or probe containing a base sequence encoding a common structure is prepared to prepare a ligand or a ligand thereof. A method for obtaining a candidate ligand substance by cloning a cDNA or a gene encoding a subtype. First, it contains the common structure of the (test) compound having agonist activity described in (F) above, that is, a base sequence encoding a highly homologous sequence portion between the amino acid sequences encoding the (test) compound having agonist activity. Make primers or probes to be used. Next, using the primers, PCR, which is known per se, can be performed on any tissue (eg, pituitary gland) such as human, warm-blooded animal (eg, guinea pig, rat, mouse, pig, sheep, cow, monkey, etc.) and fish , Pancreas, brain, kidney, liver, gonads,
DNA encoding the ligand candidate substance of interest is amplified from thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, etc.) or cell-derived genomic DNA library, cDNA library, or the like. The cloned cDNA containing the nucleotide sequence encoding the ligand candidate substance can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA has ATG at the 5 'end as a translation initiation codon and TAA, TGA or TAG at the 3' end as a translation stop codon.
May be provided. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. Next, a transformant containing a DNA encoding a ligand candidate substance is cultured according to the method for producing an orphan receptor protein-expressing cell described in (A) and (B) above, and from the culture, for example, The ligand candidate substance can be separated and purified by the following method. That is, after culturing, the cells or cells are collected by a method known per se, and this is suspended in an appropriate buffer,
After the cells or cells are disrupted by lysozyme and / or freeze-thawing, a method of obtaining a presumed extract of the ligand or its subtype, which is estimated by centrifugation or filtration, is used as appropriate. The buffer may contain a protein denaturing agent such as urea or guadinine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, sometimes abbreviated as TM hereinafter). Purification of the ligand candidate substance contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation / purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation,
Methods that mainly use differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, methods that use differences in charge, such as ion exchange chromatography, and affinity chromatography. Methods that utilize the specific affinity of DNA, methods that use differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods that use differences in isoelectric points such as isoelectric focusing or chromatofocusing. Can be When the thus obtained ligand candidate substance is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto. The compound can be converted into a free form or another salt by an analogous method. Further, it can be produced from the amino acid sequence encoding the ligand candidate substance according to a protein synthesis method known per se, or by cleaving a protein containing the ligand candidate substance with an appropriate peptidase. As a method for synthesizing a protein, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that can constitute a ligand candidate substance is condensed with the remaining part, and when the product has a protecting group, the protecting group is eliminated to produce the target ligand candidate substance. Known condensation methods and elimination of protecting groups include, for example,
The following methods are described. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Chemistry of Protein IV, 205, (1977) Supervision of Haruaki Yajima, Development of Continuing Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten , For example, a combination of solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc., to purify and isolate the candidate ligand substance. When the ligand candidate substance obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when obtained as a salt, it can be converted to a free form by a known method. be able to. The cell stimulating activity of the ligand candidate substance is measured using the cell stimulating activity measuring system described in the above (D), and is compared with the cell stimulating activity of a test compound (specifically, test compound (a)). The presence or absence of agonist (ligand) activity can be confirmed. Agonist (ligand)
Whether or not the ligand candidate substance having activity is the (endogenous) ligand of the orphan receptor protein or a subtype thereof can be confirmed by the ligand determination method described in (J) below. Also, the mRN of the target gene can be obtained using the above-mentioned probe like a gene trapper.
A was purified, and the cD
The NA can also be obtained. Further, according to the method for producing an orphan receptor protein described in the above (A),
A candidate ligand substance can be obtained by culturing a transformant containing DNA encoding the candidate ligand substance.

(3) (has agonist activity (test)
A method of searching for a ligand candidate substance by searching a sequence database for a peptide or protein having a common structure (when the compound is a peptide, protein or a salt thereof). A peptide having a common structure of the (test) compound having an agonist activity described in the above (F), that is, a peptide containing a base sequence encoding a sequence part having high homology between amino acid sequences encoding the (test) compound having an agonist activity Alternatively, a candidate protein can be determined by searching a sequence database for proteins. As a sequence database, for example, GenBank
(Registered trademark) file (National Institute of Healt
h) and VTS (Virtual Transcribed Sequence). Once the nucleotide sequence encoding the ligand candidate substance has been determined, the method described in (G) -2 above can be used.
It is possible to obtain a ligand candidate substance. Also,
If the database search reveals a sequence presumed to encode part of the ligand candidate substance,
Create a primer or probe based on the sequence,
According to the method described in the above (G) -2, a ligand candidate substance can be obtained.

(4) A method of searching for a ligand candidate substance by preparing an antibody recognizing a common structure. The peptide represented by the common structure of the (test) compound having agonist activity (test) described in (F) above, that is, a peptide represented by a sequence portion having high homology between amino acid sequences encoding the (test) compound having agonist activity, (Protein) Created using a synthetic method. Next, an antibody against the peptide is prepared according to the following method. Incidentally, if the antibody is an antibody capable of recognizing the peptide, a polyclonal antibody,
Any of monoclonal antibodies may be used. An antibody against the peptide can be produced by using the peptide as an antigen according to a method for producing an antibody or antiserum known per se. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The peptide is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where the antibody can be produced by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens and the like. When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in the spleen or lymph node. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled peptide described below with the antiserum and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 256, 49
5 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
G is used.

Examples of myeloma cells include NS-1,
Myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1 can be mentioned, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%,
Preferably, cell fusion can be carried out efficiently by incubating at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a peptide antigen is directly or adsorbed together with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A
A method for detecting a monoclonal antibody bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a peptide labeled with a radioactive substance, an enzyme, or the like, A method for detecting a monoclonal antibody bound to a phase is exemplified. The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Normal HA
It can be performed in a medium for animal cells to which T (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, 1
R containing 20%, preferably 10-20% fetal calf serum
PMI 1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. . The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, a separation and purification method of immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis Method, adsorption and desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or an active adsorbent such as protein A or protein G to collect only antibodies and dissociate the bond. Specific Purification Method for Obtaining Antibody].

[Preparation of Polyclonal Antibody] The above-mentioned peptide antibody can be produced by a method known per se or a method analogous thereto. For example, an immune antigen (peptide antigen) itself or a complex thereof with a carrier peptide is formed, and a warm-blooded animal is immunized in the same manner as in the above-described monoclonal antibody production method, and an antibody-containing substance for the peptide is collected from the immunized animal. Then, the antibody can be produced by separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier peptide and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0.1 to 2 with respect to 1 hapten.
A method of coupling at a rate of 0, preferably about 1 to 5 is used. In addition, various coupling agents can be used for coupling the hapten and the carrier, but glutaraldehyde, carbodiimide, maleimide active ester,
An active ester reagent containing a thiol group or a dithioviridyl group is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually about 2
It is performed once every 6 weeks, about 3-10 times in total.
The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. Measurement of polyclonal antibody titer in antiserum
The measurement can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody. By using the cross-reactivity of the antibody recognizing the common structure of the (test) compound having agonist activity thus obtained, a ligand candidate substance is detected, and the immunological activity is used as an index to combine various extraction methods and chromatography. And a ligand candidate substance can be obtained.

(H) Screening method for compounds that promote or inhibit the function of orphan receptor protein: Compounds that promote the function of orphan receptor protein using the candidate ligands obtained in (G) above (Highly active agonists) or compounds that inhibit their function (antagonists) can be screened. A compound that promotes the function of an orphan receptor protein is referred to as a “highly active agonist”,
“High activity” means more cell stimulating activity (specifically, the cell stimulating activity described in (D) above) than the “(test) compound having agonist activity” described in (E) above.
Etc. means strong. Hereinafter, the screening method will be described in detail. The ligand obtained in the above (G) can be obtained by using an orphan receptor protein or by constructing an expression system for a recombinant orphan receptor protein and using a receptor binding assay system using the expression system. Compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof that alter the binding between the candidate substance and the orphan receptor protein can be efficiently screened.
That is, first, the ligand candidate substance was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction and the test compound (b) (that is, the orphan receptor protein-expressing cell or its cell membrane fraction was exposed to the orphan receptor protein-expressing cell or its cell membrane fraction). (A) fluctuations in extracellular pH, (b) arachidonic acid release, (c) described in (D) above when contacted with a candidate compound that promotes or inhibits the function of the fan receptor protein.
Acetylcholine release, (d) intracellular Ca ²⁺ release, (e) intracellular cAMP fluctuation, (f) intracellular cGMP fluctuation, (g) inositol phosphate production, (h) cell membrane potential fluctuation, (i) cell Phosphorylation of internal proteins, (j) activation of c-fos, (k) GTP
The cell stimulating activity is compared using γS binding, (l) reporter gene expression, and the like as indicators. Compared with the cell stimulating activity when the candidate ligand substance is brought into contact with the orphan receptor protein-expressing cells or the cell membrane fraction thereof, the test compound
(b) strong cell stimulating activity when the orphan receptor protein-expressing cell or its cell membrane fraction is contacted with a candidate compound for promoting or inhibiting the function of the orphan receptor protein Contains the test compound
(b) (or candidate substance) is likely to be a compound that promotes the function of the orphan receptor protein (a so-called agonist). Whether or not the test compound (b) (or candidate substance) is a compound (so-called agonist) that promotes (selectively, selectively) the function of the orphan receptor protein depends on whether the orphan receptor protein and the orphan receptor protein function. What is necessary is just to measure the amount of specific binding to the test compound (b) (or the candidate substance). As a method for measuring the specific binding amount,
For example, when a labeled test compound (b) (or candidate substance) is brought into contact with an orphan receptor protein, the amount of binding of the labeled test compound (b) (or candidate substance) to the orphan receptor protein is determined. There is a method of measuring. As a result of the measurement, a sufficient binding amount (an increase in binding amount of 1% or more with respect to non-specific binding, preferably
% Of the test compound)
(b) (or candidate substance) is recognized as a compound that promotes the function of the orphan receptor protein (a so-called agonist). The specific description of the measuring method will be described below. First, the orphan receptor protein used in the measurement method may be any as long as it contains the above-mentioned orphan receptor protein. In order to obtain a large amount of an orphan receptor protein used for screening, an orphan receptor protein or the like which is expressed in a large amount using a recombinant is suitable. The above-mentioned method is used for producing the orphan receptor protein, but it is preferably carried out by expressing the DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells. A complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, gene fragments or synthetic DNA
May be used. In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and express them efficiently, the DNA fragment must be transformed into a nuclear polyhedrosis virus (nu
clear polyhedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter,
It is preferable to incorporate into downstream such as human heat shock promoter, cytomegalovirus promoter and SRα promoter. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-
19559, 1992]. Therefore, in the above measurement method, the one containing the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing the orphan receptor protein may be used. Alternatively, a membrane fraction of cells containing an orphan receptor protein may be used. The cell containing the orphan receptor protein refers to a host cell that has expressed the receptor protein, and the host cell is preferably an insect cell, an animal cell, or the like. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing by ultrasonic waves, or squirting the cells from a thin nozzle while applying pressure with a French press. Crushing and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is fed at a low speed (500 rpm to 3000 rpm).
m) for a short time (generally about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15,000 to 30000 rpm) for usually 30 minutes to 2 hours to obtain a precipitate as a membrane fraction. The membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of orphan receptor protein in cells or membrane fractions containing the orphan receptor protein,
It is preferably 10 ³ to 10 ⁸ molecules per cell,
0 that ⁵ a 10 ⁷ molecules are preferred. Examples of the labeled test compound (b) (or candidate substance) include [ ³ H],
A test compound (b) (or a candidate substance) labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used. In order to carry out the measurement method, first, a receptor protein preparation is prepared by suspending a cell or a membrane fraction of the cell containing the orphan receptor protein in a buffer suitable for screening. PH 4-10
Any buffer may be used as long as it does not inhibit the binding of the candidate substance to the receptor protein, such as a phosphate buffer (preferably pH 6 to 8) or a Tris-HCl buffer. In addition, CHAP was used to reduce non-specific binding.
Surfactants such as S, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate can also be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), pepstatin and the like can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. A fixed amount (5000 cpm to 500,000 cpm) of the labeled test compound (b) (or candidate substance) is added to 0.01 ml to 10 ml of the receptor solution. The reaction is carried out at about 0 ° C to 50 ° C, preferably at about 4 ° C to 37 ° C,
It is carried out for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. On the other hand, as compared with the cell stimulating activity when the candidate ligand substance is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, the test compound (b) ( That is, when the cell stimulating activity upon contact with the orphan receptor protein promoting or inhibiting the function of the orphan receptor protein) is weak or not observed,
The test compound (b) (or candidate substance) may be a compound that inhibits the function of the orphan receptor protein (a so-called antagonist). In addition, compared to the cell stimulating activity when a candidate ligand substance is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof,
Test compound (b) (or a cell obtained by contacting the orphan receptor protein-expressing cell or a cell membrane fraction thereof with a candidate substance for a compound that promotes or inhibits the function of the orphan receptor protein) and a ligand candidate substance When the stimulating activity is weak or not observed, the test compound (b) (or candidate substance) may be a compound that inhibits the function of the orphan receptor protein (a so-called antagonist). Whether or not the test compound (b) (or candidate substance) is a compound (so-called agonist) that inhibits (selectively, specifically) the function of the orphan receptor protein depends on whether the orphan receptor protein is May be measured. As a method for measuring the specific binding amount, for example, when the labeled test compound (b) (or candidate substance) is brought into contact with the orphan receptor protein, the labeled test compound (b) (or candidate substance) A method for measuring the amount of binding to the orphan receptor protein is exemplified. As a result of the measurement, sufficient binding amount (increase of binding amount of 1% or more with respect to non-specific binding,
If the increase in binding amount is preferably 10% or more, the test compound (b) (or candidate substance) is recognized as a compound that inhibits the function of the orphan receptor protein (a so-called antagonist). As the measuring method,
The same method as the above-described measurement method for screening a compound that promotes the function of an orphan receptor protein (a so-called highly active agonist) is used. Also,
When the labeled ligand candidate substance is brought into contact with the orphan receptor protein, the labeled ligand candidate substance and the test compound (b) (ie, the candidate substance for the compound that inhibits the function of the orphan receptor protein) are compared with the labeled ligand candidate substance. Screening the compound that inhibits the function of the orphan receptor protein by measuring and comparing the amount of binding of the labeled ligand candidate substance to the orphan receptor protein when brought into contact with the fan receptor protein; Is also possible. The specific description of the screening method is as follows. First, the orphan receptor protein used in the screening method of the present invention may be any as long as it contains the above-mentioned orphan receptor protein. In order to obtain a large amount of an orphan receptor protein used for screening, an orphan receptor protein or the like which is expressed in a large amount using a recombinant is suitable. The above-mentioned method is used for producing the orphan receptor protein, but it is preferably carried out by expressing the DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells.
A complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. DNA encoding orphan receptor protein
In order to introduce the fragments into host animal cells and express them efficiently, the DNA fragments must be transformed into nuclear polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using insects as a host).
It is preferable to incorporate the gene into the downstream of a polyhedrin promoter of hedrosis virus (NPV), a promoter derived from SV40, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, an SRα promoter, or the like. The amount and quality of the expressed receptor can be examined by a method known per se. For example, literature [Namb
i, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559,
1992]. Therefore, in the above screening method, the one containing an orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing an orphan receptor protein may be used. Alternatively, a membrane fraction of cells containing an orphan receptor protein may be used. The cell containing the orphan receptor protein refers to a host cell that has expressed the receptor protein or the like, and the host cell is preferably an insect cell, an animal cell, or the like. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing by ultrasonic waves, or squirting the cells from a thin nozzle while applying pressure with a French press. Crushing and the like.
For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is fed at a low speed (500 rpm to 3000 rpm).
m) for a short time (generally about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15,000 to 30000 rpm) for usually 30 minutes to 2 hours to obtain a precipitate as a membrane fraction. The membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of orphan receptor protein in cells or membrane fractions containing the orphan receptor protein,
It is preferably 10 ³ to 10 ⁸ molecules per cell,
0 that ⁵ a 10 ⁷ molecules are preferred. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . In order to carry out a method for screening a compound that changes the binding property between a ligand candidate substance and an orphan receptor protein, for example, an appropriate orphan receptor protein fraction and a labeled ligand or a subtype thereof are required. It is. Examples of labeled ligand candidate substances include [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], and [ ³⁵
S] and the like. Specifically, to screen for a compound that inhibits the function of an orphan receptor protein, first suspend cells or a membrane fraction of the cell containing the orphan receptor protein in a buffer suitable for screening. Thus, a receptor protein standard is prepared. The buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer, which does not inhibit the binding between the ligand and the receptor protein. In addition, in order to reduce non-specific binding, CHAPS, Tween-80 ^™ (Kao-
Surfactants such as Atlas, digitonin and deoxycholate can also be added to the buffer. further,
A protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can also be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. A certain amount (5,000 cpm to 50,000) is added to 0.01 to 10 ml of the receptor solution.
0 cpm) of the labeled candidate ligand is added, and at the same time, 10 ^-4 M to ^{10 -10} M of the test compound (b) (that is, a candidate compound for inhibiting the function of the orphan receptor protein) is allowed to coexist. A reaction tube containing a large excess of an unlabeled ligand candidate substance is also prepared to determine the non-specific binding amount (NSB). The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Nonspecific binding amount from the count (B ₀₎ where any antagonizing substance is absent (N
When count minus SB) and (B _0-NSB) as 100%, the specific binding amount (B-NSB) is, for example, 50
% Or less of the test compound (b) (ie, a candidate compound that inhibits the function of the orphan receptor protein) can be selected as a compound that inhibits the function of the orphan receptor protein (a so-called antagonist).

(I) Test compound (b) described in (H) above
(Ie, a candidate compound for promoting or inhibiting the function of the orphan receptor protein), for a compound that promotes or inhibiting the function of the orphan receptor protein: test compound (b) (ie, Compounds that promote or inhibit function)
Natural and non-natural peptides, natural and non-natural proteins,
It is selected from natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, and the like. As the compound that promotes or inhibits the function of the orphan receptor protein, the compound that promotes the function of the orphan receptor protein or the compound that inhibits the function of the orphan receptor protein in the screening method described in (H) above. And the compound may be in the form of a salt. Examples of the salt of the compound include salts with physiologically acceptable bases (eg, alkali metals) and acids (organic acids, inorganic acids), and particularly preferred are physiologically acceptable acid addition salts. . Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like. Further, the compound that promotes (highly active agonist) or inhibits (antagonist) the function of the orphan receptor protein obtainable by the method (H) includes:
Since it has an action similar to the physiological activity of the (endogenous) ligand or a subtype thereof described below, it is useful as a safe and low-toxic drug according to the ligand activity. Antagonists to orphan receptor proteins include
Since the physiological activity of the ligand for the orphan receptor protein or its subtype can be suppressed, it is useful as a safe and low-toxic drug for suppressing the ligand activity. A highly active agonist for an orphan receptor protein is useful as a safe and low toxic drug for enhancing the physiological activity of a ligand for an orphan receptor protein.

When the antagonist or highly active agonist obtainable by the method of the present invention is used as a pharmaceutical composition, it can be administered according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.
The preparations obtained in this way are safe and have low toxicity. For example, they should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be. The dose of the ligand or a subtype thereof, and the antagonist or agonist may vary depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example,
About 0.1 to 100 mg per day, preferably about 1.0 mg
５０50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. For example, in the case of an injection, it is usually, for example, about 0.01 to 30 mg per day. Degree, preferably about 0.1-20m
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(J) Method for Determining Orphan Receptor Protein Ligand or Subtype Thereof: The present invention provides a method for screening a compound that promotes or inhibits the function of an orphan receptor protein described in (H) above. In addition to the method, the present invention also provides a method for more efficiently and reliably determining the ligand of the orphan receptor protein or its subtype using the common structure of the (test) compound having agonist activity as an index. That is,
(I) contacting a test compound with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, measuring the cell stimulating activity mediated by the orphan receptor protein, and (ii) comparing the cell stimulating activity of each test compound By determining the common structure of the test compound having agonist activity, (i
ii) A method for determining the ligand of the orphan receptor protein or a subtype thereof by measuring the amount of specific binding of the ligand candidate substance having the common structure to the orphan receptor protein. is there.
More specifically, (i) the test compound (a) is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and the test is carried out on a cell that does not express an orphan receptor protein or a cell membrane fraction thereof. When the compound (a) was brought into contact, the respective cell stimulating activities were measured, and (i
i) a compound having agonist activity is obtained by comparing the cell stimulating activity of each test compound (a), and (iii) the orphan of a ligand candidate substance deduced from the common structure of the compound having agonist activity. A method for determining the ligand of the orphan receptor protein or a subtype thereof by measuring the amount of specific binding to the receptor protein is provided. In the determination method, (i) a test compound (specifically, test compound (a)) is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and a cell stimulating activity mediated by the orphan receptor protein is determined. (Ii) Each test compound (specifically, test compound (a))
By comparing the cell stimulating activities of the above, a (test) compound having agonist activity is obtained, and the common structure of the compound having agonist activity is estimated (or determined) to obtain a ligand candidate substance. Is used. Next, a method for determining whether the ligand candidate substance is an (endogenous) ligand or a subtype thereof using the ligand candidate substance having the common structure (described above) will be described in detail below. Whether or not the ligand candidate substance is a specific ligand of the orphan receptor protein can be determined by measuring the specific binding amount of the ligand candidate substance to the orphan receptor protein. That is, when the labeled ligand candidate substance is brought into contact with the orphan receptor protein,
A method of measuring the binding amount of the labeled ligand candidate substance to the orphan receptor protein and the like can be mentioned.
As a result of the measurement, if a sufficient binding amount (an increase in the binding amount of 1% or more relative to the non-specific binding, preferably an increase in the binding amount of 10% or more) is recognized, the candidate substance is identified as (Endogenous) ligand.
Conversely, when a sufficient binding amount is not recognized, the ligand candidate substance has a cell stimulating activity, but has a low specific binding ability to the orphan receptor protein,
That is, it is highly possible that the substance is a non-specific agonist-like substance. A specific description of the determination method will be given below.
First, the orphan receptor protein used in the determination method may be any as long as it contains the above-mentioned orphan receptor protein. In order to obtain a large amount of an orphan receptor protein used for screening, an orphan receptor protein or the like which is expressed in a large amount using a recombinant is suitable. To produce an orphan receptor protein, the method described above is used,
It is preferable to carry out the expression by expressing the DNA encoding the orphan receptor protein in the aforementioned animal cells or insect cells. DNA encoding the protein portion of interest
Complementary DNA is used for the fragment, but is not necessarily limited thereto. For example, gene fragments or synthetic DN
A may be used. In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and express them efficiently, the DNA fragment must be expressed in a nuclear polyhedrosis virus belonging to baculovirus using an insect as a host. (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 1
9555-19559, 1992]. Therefore, in the above measurement method, the one containing the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing the orphan receptor protein may be used. Alternatively, a membrane fraction of cells containing an orphan receptor protein may be used. The cell containing the orphan receptor protein refers to a host cell that has expressed the receptor protein, and the host cell is preferably an insect cell, an animal cell, or the like. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing by ultrasonic waves, or squirting the cells from a thin nozzle while applying pressure with a French press. Crushing and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, a cell lysate is supplied at a low speed (500 rpm to 300 rpm).
0 rpm) for a short time (usually about 1 minute to 10 minutes),
The supernatant is further accelerated (15,000 rpm to 30,000 rpm).
m) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed orphan receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the orphan receptor protein in the cell or membrane fraction containing the orphan receptor protein is preferably 10 ³ to 10 ⁸ molecules per cell, more preferably 10 ⁵ to 10 ⁷ molecules per cell. It is suitable. Examples of labeled ligand candidate substances include [ ³ H],
A ligand candidate substance labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used. In order to carry out the measurement method, first, a receptor protein preparation is prepared by suspending a cell or a membrane fraction of the cell containing the orphan receptor protein in a buffer suitable for screening. The buffer may have a pH of 4 to 10 (preferably pH 6
Any buffer may be used as long as it does not inhibit the binding of the candidate protein to the receptor protein, such as the phosphate buffer and Tris-HCl buffer of (8). In addition, in order to reduce non-specific binding, CHAPS, Tween-80 are used.
Surfactants such as ^TM (Kao-Atlas), digitonin, deoxycholate and the like can also be added to the buffer. In addition, PMSF, leupeptin, E-6
4 (manufactured by Peptide Research Laboratories) and protease inhibitors such as pepstatin can also be added. 0.01ml ~ 1
A fixed amount (5000 cpm) is added to 0 ml of the receptor solution.
５００500,000 cpm) of labeled candidate substance. The reaction is carried out at about 0 ° C. to 50 ° C., preferably at about 4 ° C. to 3 ° C.
The reaction is carried out at 7 ° C. for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter.

In the present specification and drawings, when bases, amino acids, and the like are represented by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in the relevant field. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lysine arginine arginine : Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid HEPES: N- [2-hydroxyethyl] piperazine
-N '-[2-ethanesulfonic acid] HBSS: Hank's Balanced Salt Solution

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 2] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 3] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 4] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 5] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 6] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 7] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 8] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 9] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 10] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 11] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 12] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 13] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 14] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 15] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 16] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 17] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 18] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 19] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 20] This shows the amino acid sequence of the sample used in Example 1 described later (see Table 1). [SEQ ID NO: 21] DNA encoding human FM-3
Is shown (Reference Example 1 described later). [SEQ ID NO: 22] FM3 used in Reference Example 1 described later
2 shows the nucleotide sequence of F2. [SEQ ID NO: 23] FM3 used in Reference Example 1 described later
2 shows the base sequence of R2.

[0042]

The present invention will be described in more detail with reference to the following Reference Examples and Examples, which do not limit the scope of the present invention. Reference Example 1 Human FM-3 expressing CH
Preparation of O cells (see Example 1 of Japanese Patent Application No. 2000-52251) Human FM-3 was obtained as follows. Genomics
52,223-229 (1998), the following two types of synthetic DNAs were synthesized based on the sequence of human FM-3. FM3F2: 5'-GTCGACCATGGCTTGCAATGGCAGTGCGGCCAGG-3 '(SEQ ID NO: 22) FM3R2: 5'-GCTAGCTCAGGATGGATCGGTCTCTTGCTG-3' (SEQ ID NO: 23) Using these synthetic DNAs, human fetal brain cDNA was obtained by PCR. PCR reaction solution: 1 μl rat hypothalamus cDNA solution
(From 0.2 ng poly (A) ⁺ RNA), 1 μFM3F2 (10 μM),
1 μl FM3R2 (10 μM), 5 μl 10 x reaction mixture, 5 μl
dNTP (10 mM), 1 μl Ex Taq (Takara) and 36 μl distilled water were added to make a total of 50 μl. The reaction solution is ThermalCycler96
00 was used for the PCR reaction. The PCR conditions were: denaturation at 95 ° C for 2 minutes, and a cycle of 98 ° C for 10 seconds, 65 ° C for 10 seconds, and 72 ° C for 90 seconds was repeated 28 times. After confirming the amplification of a PCR product of about 1,2 kb by electrophoresis using a part of the PCR product, the PCR product was subcloned into E. coli using a TA cloning kit (Invitrogen). A plasmid was extracted from Escherichia coli obtained by subcloning using a plasmid extractor (Kurabo), the base sequence of the inserted fragment was determined, and the human FM-3 cDNA whose sequence was the same as that reported in the above literature (SEQ ID NO: 21). Next, the restriction enzymes SalI and N
After digestion with heI, a human FM-3 cDNA fragment of about 1.2 kb was obtained. Furthermore, p, which is an expression vector for animal cells,
AKKO-111H was digested with restriction enzyme sites SalI and NheI at the multiple cloning site and then electrophoresed to recover the vector. The human FM-3 cDNA fragment and the expression vector prepared by the above procedure were ligated by ligation, and
M109 was transformed into E. coli JM109 / pAKK
OFM3 was obtained. Transformant E. coli JM109 / pA
KKOFM3 was cultured to prepare a large amount of plasmid pAKKOFM3 DNA. After dissolving 20 μg of the plasmid DNA in 1 ml of physiological saline (PBS),
Inject into a vial of Gene Transfer (Wako Pure Chemical Industries), and vigorously stir using a vortex mixer.
Liposomes containing A were formed. CHOdhfr
^- to cells 1 were seeded 2 × 10 ⁶ cells into a cell culture dish with a diameter of 35 mm, the culture solution after 20 hours of culture was replaced with fresh. 0.5 μg DNA for each dish
The liposome solution in an amount (25 μl) corresponding to
After 6 hours of incubation, plasmid DNA was introduced. After further replacing the medium with fresh medium and culturing for 1 day, replacing the medium with selection medium and continuing culturing for 3 days. The cells were seeded in minimum essential medium and alpha medium plus 10% dialyzed bovine serum), and transformants were selected. Only the transformants were able to grow in the selection medium, and the selection was repeated by repeating the subcultures to establish CHO-FM3 cells.

Example 1 Detection of Stimulation Activity for FM-3 Expressing CHO Cells by Cytosensor Assay Using Various Peptide Samples FM-3 expressing CHO cells were seeded at a density of 2.7 × 10 ⁵ cells / capsule in a cytosensor capsule. After culturing overnight, the cells were mounted on the workstation of the cytosensor. Assay medium (0.1
Low buffered RPMI1 containing 5% bovine serum albumin
640 medium), pump ON (80 seconds), pump OFF
(40 seconds) to the cells in each cycle, and change the extracellular pH change rate for 30 seconds from 8 seconds after the pump is stopped in each cycle.
Calculated as acidification rate. acidification ra
The time-dependent change of te was monitored, and when a stable value was reached, the peptides shown in the table were exposed to the cells for 7 minutes and 2 seconds by switching the flow path. Acidific for each well
The values of the ation Rate were normalized to the value of the three cycles immediately before the exposure of the peptide as 100%, and the reaction of the cells was compared. The concentration of the exposed samples was 1-10 μM. Table 1 shows the results of detection of the stimulating activity of the peptide sample on FM-3 expressing CHO cells by cytosensor assay.
If the acidification rate value at the peak of the reaction exceeds 120% (）), it is 120% or less but the reaction is clearly recognized (△), and the reaction is not recognized (認 め). , Items that cannot be said either are indicated by (?). As a result, FM-3 expressing CHO cells were H-2980, F-
Peptide S from 8-F-NH2 and Aplysia
A reaction was detected in mall Cardioactive Peptide B (SCPB). Of these, the response to SCPB was the strongest.

[0044]

[Table 1]

The peptides (H-2980, F-8-
Compares the F-NH2, SCPB structure), it found RX-NH ₂ structure C- terminal part a common structure to all. FIG. 1 shows a comparison of the amino acid sequences in common. Because of the strongest response to SCPB, it was expected that the endogenous ligand of FM-3 would adopt a structure similar to SCPB in other parts in addition to the C-terminal RX-NH ₂ structure. Also, H-2980 and
It is considered that F-8-F-NH2 also has a partially similar structure. As a result of searching for a known peptide based on such prediction, neuromedin U (NMU-8) was found.

[0046]

According to the method of the present invention, a test compound is brought into contact with an orphan receptor protein-expressing cell or its cell membrane fraction or an orphan receptor protein-expressing cell or its cell membrane fraction-expressed orphan receptor protein. By measuring the cell stimulating activity mediated by the orphan receptor protein, comparing the cell stimulating activity of each test compound, selecting an agonist, and then comparing the structure of each agonist, the orphan receptor protein A ligand or a subtype thereof, an antagonist, a highly active agonist, and the like can be obtained efficiently and reliably.

[0047]

[Sequence listing] [Sequence Listing] <110> Takeda Chemical Industries, Ltd. <120> Screening Method <130> B00227 <150> JP 11-236597 <151> 1999-08-24 <160> 23 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 1 Phe Met Arg Phe 1 4 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 2 Tyr Phe Met Arg Phe 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 3 Tyr Gly Gly Ply Met Arg Phe 1 5 7 <210> 4 <211 > 7 <212> PRT <213> Artificial Sequence <220><223><400> 4 Tyr Gly Gly Ply Met Arg Phe 1 5 7 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220 ><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 5 Pro Gln Arg Phe 1 4 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 6 Phe Leu Phe Gln Pro Gln Arg Phe 1 5 8 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (- CONH ₂ ) form <233> Xaa means pGlu <400> 7 Xaa Asp Pro Phe Leu Arg Phe 1 5 7 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C- terminus of the polypeptide is amide (-CONH ₂ ) form <400> 8 Asp Arg Asn Phe Leu Arg Phe 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C -terminus of the polypeptide is amide (-CONH ₂ ) form <400> 9 Asn Arg Asn Phe Leu Arg Phe 1 5 7 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 10 Thr Asn Arg Asn Phe Leu Arg Phe 1 5 8 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 11 Pro Asp Val Asp His Val Phe Leu Arg Phe 1 5 10 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 12 Lys Asn Glu Phe Ile Arg Phe 1 5 7 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C- terminus of the polypeptide is amide (-CONH ₂ ) form <400> 13 Lys His Glu Tyr Leu Arg Phe 1 5 7 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 14 Leu Pro Leu Arg Phe 1 5 <210> 15 <211> 31 <212> PRT <213> Artificial Sequence <220><223> the C -terminus of the polypeptide is amide (-CONH ₂ ) form <400> 15 Ser Arg Ala His Gln His Ser Met Glu Ile Arg Thr Pro Asp Ile Asn 1 5 10 15 Pro Thr Trp Tyr Thr Gly Arg Gly Ile Arg Pro Val Gly Arg Phe 20 25 30 31 <210> 16 <211> 20 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 16 Ser Pro Glu Ile Asp Pro Phe Trp Val Tyr Gly Arg Gly Val Arg Pro 1 5 10 15 Ile Gly Arg Phe 20 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 17 Ser Gly Gln Ser Trp Arg Pro Gln Gly Arg Phe 1 5 10 11 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 18 Leu Ser Ser Phe Val Arg Ile 1 5 7 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220 ><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 19 Ala Arg Pro Gly Tyr Leu Ala Phe Pro Arg Met 1 5 10 11 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220><223> the C-terminus of the polypeptide is amide (-CONH ₂ ) form <400> 20 Met Asn Tyr Leu Ala Phe Pro Arg Met 1 5 9 <210> 21 <211> 1209 <212> DNA <213> Human <400> 21 ATGGCTTGCA ATGGCAGTGC GGCCAGGGGG CACTTTGACC CTGAGGACTT GAACCTGACT 60 GACGAGGCAC TGAGACTCAA GTACCTGGGG CCCCAGCAGA CAGAGCTGTT CATGCCCATC 120 TGTGCCACAT ACCTGCTGAT CTTCGTGGTG GGCGCTGTGG GCAATGGGCT GACCTGTCTG 180 GTCATCCTGC GCCACAAGGC CATGCGCACG CCTACCAACT ACTACCTCTT CAGCCTGGCC 240 GTGTCGGACC TGCTGGTGCT GCTGGTGGGC CTG CCCCTGG AGCTCTATGA GATGTGGCAC 300 AACTACCCCT TCCTGCTGGG CGTTGGTGGC TGCTATTTCC GCACGCTACT GTTTGAGATG 360 GTCTGCCTGG CCTCAGTGCT CAACGTCACT GCCCTGAGCG TGGAACGCTA TGTGGCCGTG 420 GTGCACCCAC TCCAGGCCAG GTCCATGGTG ACGCGGGCCC ATGTGCGCCG AGTGCTTGGG 480 GCCGTCTGGG GTCTTGCCAT GCTCTGCTCC CTGCCCAACA CCAGCCTGCA CGGCATCCGG 540 CAGCTGCACG TGCCCTGCCG GGGCCCAGTG CCAGACTCAG CTGTTTGCAT GCTGGTCCGC 600 CCACGGGCCC TCTACAACAT GGTAGTGCAG ACCACCGCGC TGCTCTTCTT CTGCCTGCCC 660 ATGGCCATCA TGAGCGTGCT CTACCTGCTC ATTGGGCTGC GACTGCGGCG GGAGAGGCTG 720 CTGCTCATGC AGGAGGCCAA GGGCAGGGGC TCTGCAGCAG CCAGGTCCAG ATACACCTGC 780 AGGCTCCAGC AGCACGATCG GGGCCGGAGA CAAGTGACCA AGATGCTGTT TGTCCTGGTC 840 GTGGTGTTTG GCATCTGCTG GGCCCCGTTC CACGCCGACC GCGTCATGTG GAGCGTCGTG 900 TCACAGTGGA CAGATGGCCT GCACCTGGCC TTCCAGCACG TGCACGTCAT CTCCGGCATC 960 TTCTTCTACC TGGGCTCGGC GGCCAACCCC GTGCTCTATA GCCTCATGTC CAGCCGCTTC 1020 CGAGAGACCT TCCAGGAGGC CCTGTGCCTC GGGGCCTGCT GCCATCGCCT CAGACCCCGC 1080 CACAGCTCCC ACAGCCTCAG CAGGATGACC ACAGGCAGCA CCCTGTGTGA TGTGGGCTCC 1140 CTGGGCAGCT GGGTCCACCC CCTGGCTGGG AACGATGGCC CAGAGGCGCA GCAAGAGACC 1200 GATCCATCC 1209 <210> 22 <211> 34 <212> DNA <213> Artificial Sequence <220><223><400> 22 GTCGACCATG GCTTGCAATG GCAGTGCGCAGC <210 30 <212> DNA <213> Artificial Sequence <220><223><400> 23 GCTAGCTCAG GATGGATCGG TCTCTTGCTG 30

[0048]

[Brief description of the drawings]

FIG. 1 shows a comparison diagram of the commonality of amino acid sequences in Example 1. Common amino acid residues are boxed. Phenylalanine (F) and tyrosine (Y) show very similar structures in terms of their steric structure, and are therefore similarly boxed.

Claims (3)

[Claims] (I) contacting a test compound (a) with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and adding a test compound to a cell not expressing an orphan receptor protein or a cell membrane fraction thereof. When (a) was brought into contact, the respective cell stimulating activities were measured, and (ii)
By comparing the cell stimulating activity of each test compound (a), a compound having agonist activity is obtained, and (iii) a ligand candidate substance deduced from a common structure of the compound having agonist activity is converted to the orphan receptor. Comparing the cell stimulating activity when the test compound (b) is brought into contact with the orphan receptor protein-expressing cells or the cell membrane fraction thereof when the test compound (b) is brought into contact with the protein-expressing cells or the cell membrane fraction thereof; and A method for screening a compound or a salt thereof that promotes or inhibits the function of said orphan receptor protein, comprising measuring the amount of specific binding between the receptor protein and the test compound (b). 2. A compound or a salt thereof obtainable by the screening method according to claim 1. (I) contacting a test compound (a) with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, and adding a test compound to a cell not expressing an orphan receptor protein or a cell membrane fraction thereof. When (a) was brought into contact, the respective cell stimulating activities were measured, and (ii)
By comparing the cell stimulating activity of each test compound (a), a compound having an agonist activity is obtained, and (iii) the orphan receptor of a ligand candidate substance deduced from the common structure of the compound having the agonist activity A method for determining the ligand of the orphan receptor protein or its subtype by measuring the amount of specific binding to the protein.