WO2001014883A1

WO2001014883A1 - Screening method

Info

Publication number: WO2001014883A1
Application number: PCT/JP2000/005639
Authority: WO
Inventors: Shuji Hinuma; Masaki Hosoya
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 1999-08-24
Filing date: 2000-08-23
Publication date: 2001-03-01
Also published as: AU6726400A

Abstract

A ligand of an orphan receptor protein or its subtype, antagonist, highly active agonist, etc. can be efficiently and surely obtained by bringing test compounds into contact with cells showing the expression of the orphan receptor protein or a cell membrane fraction thereof, or an expression product of the orphan receptor protein expressed in cells showing the expression of the orphan receptor protein or a cell membrane fraction thereof; measuring the cell stimulating activity of each test compound mediated by the orphan receptor protein; comparing the cell stimulating activities of the test compounds with each other; thereby selecting agonists; and then comparing the structures of these agonists.

Description

Description Screening method Technical field

The present invention screens a high-concentration test compound using the activation of orphan receptor protein-expressing cells as an index, and utilizes the common structure of the test compound having agonist activity to produce an orphan receptor protein. The present invention relates to a method for efficiently screening a compound that promotes or inhibits the function of a protein, and a method for determining a (endogenous) ligand of an orphan receptor protein using the common structure. Background art

Many physiologically active substances such as hormones and neurotransmitters exert their effects 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 pharmaceuticals. It is extremely important to understand the properties of body molecules. Recent advances in molecular biological techniques have made it possible to analyze the receptors of many physiologically active substances at the molecular level. A group of such receptor molecules with a common structural feature that penetrates the cell membrane seven times is known and is called a seven-transmembrane receptor. They are also called G protein-coupled receptors because they are coupled to intracellular signaling systems via GTP-binding proteins (G proteins). Ligands of the seven-transmembrane receptor are diverse, including proteins, peptides, amines, amino acids, nucleotides, nucleosides, eicosanoids, phospholipids, odorants, and light.

Recent advances in genomic or cDNA analysis techniques have led to the reporting of many genes encoding receptors. 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 ligands for orphan receptors could be identified, it would be expected to reveal new physiological functions or pathologies regulated by the receptors and ligands. For this reason, the search for orphan receptor ligands is attracting attention as a means to uncover new drug development targets. For orphan receptors that have shown some remarkable similarity to known ligands to date, orphanin FQ, which has studied the known ligands or their analogs as candidate ligands, has actually identified the endogenous ligand. / noc i cept in (Meunier, J. -C. Nature 393: 211-212, 1998), but the structure of the ligand is estimated only from the structural similarity of the known ligand or its analog. There is a limit to In many cases, endogenous ligands are identified by purification using the activation of the signal transduction system of orphan receptor protein-expressing cells as an index (Sakurai, T. et al. Cell 92: 573- 585, 1998; Hinuma, S. et al. Nature 393: 272-276, 1998; Tatemoto, K. et al. Biochem. Biopys. Res. Co., Ltd. 251: 471-476, 1998). However, the signal transduction system via the seven-transmembrane receptor is not unique, and it is necessary to screen several Atsui systems in parallel to detect the activity derived from endogenous ligands. Was. 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 for the assay.

Establishment of efficient and reliable screening method for compounds or salts thereof that promote or inhibit the function of ophan receptor protein and method for determining (endogenous) ligand of ophan receptor protein Is an issue. Disclosure of the invention

The present inventors have conducted intensive studies in order to solve the above problems,

(i) contacting a test compound (preferably at a high concentration) with an orphan receptor protein-expressing cell or a cell membrane fraction thereof, or a cell or a cell membrane fraction that does not express orphan receptor protein; When the test compound is brought into contact, the cell stimulating activity of each is measured, and

(ii) By comparing the cell stimulating activity of each test compound, Determine the common structure of the test compound

(iii) (1) When the ligand-containing substance having the common structure is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction and the orphan receptor protein-expressing cell or its cell membrane fraction Compare the cell stimulating activity when contacting a candidate substance for a compound that promotes or inhibits the function of the orphan receptor protein, and ② promote or function the functions of the orphan receptor protein and the orphan receptor protein. By measuring the specific binding amount of a compound to be inhibited to a candidate substance, it was found that a compound or a salt thereof that promotes or inhibits the function of an orphan receptor protein can be efficiently screened. As a result of repeated studies based on the knowledge, the present invention has been completed.

That is, the present invention

(1)

(1) When the test compound is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and when the test compound is brought into contact with the cell that does not express the orphan receptor protein or its cell membrane fraction, Measure the stimulating activity,

(ii) determining the common structure of test compounds having agonist activity by comparing the cell stimulating activity of each test compound,

(iii) (1) When the ligand-containing substance having the common structure is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction and the orphan receptor protein-expressing cell or its cell membrane fraction The cell stimulating activity when contacting a candidate substance for a compound that promotes or inhibits the function of the orphan receptor protein is compared, and the function of the orphan receptor protein and the function of the orphan receptor protein are compared. A method for screening for a compound or a salt thereof which promotes or inhibits the function of the orphan receptor protein, which comprises measuring the amount of specific binding of a promoting or inhibiting compound to a candidate substance;

(2) a compound or a salt thereof obtainable by the screening method described in the above (1), and (i) When the test compound was brought into contact with the orphan receptor protein-expressing cells or its cell membrane fraction, and when the test compound was brought into contact with cells that do not express the orphan receptor protein or its cell membrane fraction, respectively. The cell stimulating activity of

(ii) determining the common structure of test compounds having agonist activity by comparing the cell stimulating activities of each test compound,

(iii) a method of determining the ligand of the orphan receptor protein or a subtype thereof by measuring the specific binding amount of the ligand candidate substance having the common structure to the orphan receptor protein, etc. About.

More specifically

( Four )

(0) The test compound (a) was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and the test compound (a) was brought into contact with a cell that does not express the orphan receptor protein or its cell membrane fraction. The cell stimulating activity of each

(i i) By comparing the cell stimulating activity of each test compound (a), a compound having agonist activity is obtained,

(iii) (i) When the candidate ligand substance deduced from the common structure possessed by the compound having agonist activity is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and when the test compound (b) is Comparing the cell stimulating activity when contacting the orphan receptor protein-expressing cell or its cell membrane fraction, and (2) measuring the specific binding amount between the orphan receptor protein and the test compound (b). A method for screening a compound or a salt thereof which promotes or inhibits the function of the orphan receptor protein,

(5) a compound or a salt thereof obtainable by the screening method described in (4) above,

and

(6)

(i) Test compound is added to orphan receptor protein-expressing cells or its cell membrane fraction. When the test compound (a) was brought into contact with (a) and when the test compound (a) was brought into contact with cells not expressing the orphan receptor protein or its cell membrane fraction, the respective cell stimulating activities were measured.

(iii) measuring the amount of specific binding of the candidate ligand substance to the orphan receptor protein deduced from the common structure of the compound having agonist activity, whereby the orphan receptor protein is determined. And a method for determining the ligand or its subtype. BRIEF DESCRIPTION OF THE FIGURES

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) have very similar steric structures, so they are similarly boxed. BEST MODE FOR CARRYING OUT THE INVENTION

(A) About o-fan receptor protein:

As used herein, the term “orphan receptor protein” means a protein whose ligand is unknown, and includes unknown proteins in addition to known proteins.

-Specific examples of orphan receptor proteins include, for example, the FM-3 receptor protein (Tan, C. P et al, Genomi cs 52, 223-229) or the mas receptor used in Example 1 described below. Protein (Young D. et al., Proc. Natl. Acad. Sci. USA, 85, 5339-5342, 1988), etc., as well as those listed in the orphan receptor database of Swiss- plot And so on.

The orphan receptor protein used in the present invention may form a salt. Examples of the salt of the “orphan receptor protein” include physiologically acceptable bases (eg, alkali metals) and acids (organic compounds). Acid, inorganic acid) Physiologically acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or 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, and benzenesulfonic acid).

The DNA encoding the orphan receptor protein used in the present invention may be any DNA containing the DNA encoding the orphan receptor protein. In addition, genomic DNA, genomic DNA libraries, human warm-blooded animals (eg, guinea pigs, rats, mice, chicks, egrets, bushes, sheep, swords, monkeys, etc.) (eg, retinal cells, hepatocytes, Spleen cells, neuronal cells, glial cells, kidney / 3 cells, bone marrow cells, mesangial cells, Langerens cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, Immune cells (eg, macrophage, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells Osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any of those cells Tissue, for example, brain, parts of the brain (eg, retina, olfactory bulb, amygdala, cerebral basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, stomach, Kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, Ovary, placenta, uterus, bone, joints, skeletal muscle, etc., or blood cells or their cultured cells (eg, MEL, Ml, CTLL-2, HT-2, WEH I-3, HL-60, J OSK) -1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT HSB-2, KE-37, S KW_3, HUT-78, CDNA derived from HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.), cDNA library derived from the tissue / cell, synthetic DNA Any The vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Also, It can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RTPCR method) using an RNA fraction prepared from the above-mentioned tissues and cells.

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, the DNA library was synthesized by a PCR method known per se using a synthetic DNA primer having a partial nucleotide sequence of orphan receptor protein. Amplification of the target DNA from the first place, or the DNA incorporated into an appropriate vector, for example, labeled using a DNA fragment or a synthetic DNA having a partial or entire region of the orphan receptor protein. Sorting can be performed by hybridization. The hybridization is performed according to, for example, the method described in Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, follow the method described in the attached instruction manual.

The DNA encoding the cloned orphan receptor protein can be used as it is, or as desired, after digestion with a restriction enzyme or addition of a linker. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation start codon and translation stop codon can be added using an appropriate synthetic DNA adapter.

(B) For orphan receptor protein-expressing cell or its cell membrane fraction or cell that does not express orphan receptor protein or its cell membrane fraction: In the present specification, the “orphan receptor protein-expressing cell” is as defined above. (A) A host cell that has expressed the protein receptor for ophan.

As a method for expressing the orphan receptor protein in host cells, for example, the DNA encoding the orphan receptor protein (A) can be expressed by the following method.

That is, the expression vector of the orphan receptor protein is, for example, It can be produced by cutting out a DNA fragment of interest from DNA encoding an an receptor protein, and ligating the DNA fragment downstream of a promoter in an appropriate expression vector.

Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pCl94), yeast-derived plasmids (eg, pSHl) 9, pSHl 5), bacteriophage such as λ phage, and animal viruses such as retrovirus, vaccinia virus, and baculovirus are used. The promoter used may be any promoter suitable for the host used for gene expression.

When the host used for the transformation is an animal cell, promoters derived from SV40, a retrovirus promoter, a metamouth thionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRa promoter, etc. Available. If the host is Escherichia, Trp promoter, T7 promoter overnight, lac promoter, recA promoter, APL promoter, 1 pp promoter, etc.If the host is Bacillus, SPO When the host is an enzyme such as 1 promoter, SP〇2 promoter, pen P promoter, etc., preferred are PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter and the like. 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 via the orphan receptor protein. In addition to the above, the expression vector may further include 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, if desired. Can be used. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance] and ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^) And a neomycin-resistant gene (hereinafter sometimes abbreviated as Neo, G418-resistant). Special In the case where the DHFR gene is used as a selection marker using CH〇 (dhfr ~) cells, selection can also be performed 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. If the host is a bacterium belonging to the genus Escherichia, the phoA-signal sequence and the O-immediate A / signal sequence are included. In the case of yeast, the mating factor a (M Fa) signal sequence, invertase, signal sequence, etc. When the host is an animal cell, for example, insulin signal sequence, a-in yellow ferron Signal sequence, antibody molecule, signal sequence, etc. can be used respectively.

A transformant can be produced using the vector containing DNA encoding the orphan receptor protein thus constructed.

As the host, for example, a bacterium belonging to the genus Escherichia, a bacterium belonging to the genus Bacillus, a yeast, an insect or an insect cell, an animal cell, or the like is used. As described above, the insect cell or the animal cell is preferably used.

Examples of the genus Escherichia include Escherichia coli (Escherichia col 0 K12 • DH1 [Prosessing's of the National Academy of Sciences] of the United States (Pro Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal-Robb 'Molecular' Noology 1] (Journal of Molecular Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39] , 440 (1954)].

Examples of Bacillus bacteria include, for example, Bacillus subtilis M1114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95 , 87 (198 4)].

Examples of yeast include Saccharomyces cerevisiae) AH22, AH22R—, NA87-11A, DKD—5D, 20B-12 and the like are used.

As insects, for example, silkworm larvae are used [Maeda et al., Neycha (

Nature), 315, 592 (1 985)].

As insect cells, for example, when the virus is Ac NPV, a cell line derived from a larva of night rob moth (Spodopter.a frugiperda cell; S f cell), and the midgut of Trichoplusia ni

ΤΜ

For example, MG1 cells derived therefrom, High Five cells derived from Trichoplusia ni eggs, cells derived from Mamestra brasskae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyxmori N; BmN cell) is used. Examples of the Sf cells include, for example, Sf9 cells (ATCC CRL1711), Sf21 cells [Vaughn, J. L et al., In Vitro, Vol.

13-217 (1977)].

Examples of animal cells include monkey COS-7 cells, Vero cells, Chinese nose, muster cells CH〇, DHFR gene deficient Chinese hamster cells CHO (dh fr-CH〇 cells), mouse L cells, mouse 3T3 cells, mouse Myeloma cells, human HEK293 cells, human FL cells, C127 cells, BALB 3T3 cells

, Sp-2 cells and the like are used.

To transform Escherichia, for example,

• National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene (Gene

), Vol. 17, 107 (1 982).

For transformation of Bacillus sp., For example, Molecular & General Genetics, Vol. 168, 1 1 1 (

1 979).

To transform yeast, for example, Prossings of the National

• Academy 'ob' 'Sciences'' ob 'the' U.S.A. (Pro atl. Acad.

Sci. USA), 75, 1929 (1978). To transform insect cells or insects, for example, biotechnology (

Bio / Technology), 6, 47-55 (1988), etc. Done.

Transformation of animal cells is performed, for example, according to the method described in Virology, 52, 456 (1973).

Methods for introducing orphan receptor protein expression vectors into cells include, for example, the lipofection method (Felgner, PL et al. Processing's 'the' National Academy of Sciences, Obs. U.S.A. (Proceedings of The National Academy of Sciences of The United States of America), 84, 7413 (1987)), calcium phosphate method [Graham, FL and van der Eb, AJ Virology, 52, 456- 46 p. 7 (1973)], electroporation [Nuemann, E. et al., Empo J. (EMBO J.), 1, 841-845 (1982)], and the like.

In this manner, cells expressing the protein receptor for orphan are obtained.

As a method for stably expressing an orphan receptor protein using animal cells, there is a method of selecting, by clonal selection, cells in which the expression vector introduced into the animal cell is integrated into the chromosome. . Specifically, a transformant is selected using the above selection abilities as an index. Furthermore, by repeatedly performing clonal selection on the animal cells obtained using the selectable marker in this manner, a stable animal cell line having high expression ability of orphan receptor protein can be obtained. You. When the dh fr gene or the dh fr gene is used as a selection marker, the MTX concentration is gradually increased, and the cells are cultured. By selecting resistant strains, the DNA encoding the orphan receptor protein together with the dh fr gene can be transferred to the cells. It can also be amplified in situ to obtain higher expressing animal cell lines.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. As a carbon source

Nitrogen sources such as glucose, dextrin, soluble starch, sucrose, etc.Inorganic or organic substances such as ammonium salts, nitrates, corn chip liqueur, peptone, casein, meat extract, soybean meal, potato extract, etc. Inorganic substances such as calcium chloride, sodium dihydrogen phosphate, and magnesium chloride And the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

Examples of a medium for culturing Escherichia sp. Include, for example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, Journal of Experiments in Molecular Genetics, 431). -433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3) 3-indolylacrylic acid can be added in order to make the promoter work efficiently.

When the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.

When the host is a bacterium belonging to the genus Bacillus, culturing is usually performed at about 30 to 40 "C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.

When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., "Processing's the National Academy of Sciences" Natl. Acad. Sci. USA, 77, 4505 (1980)] and an SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. The National Academy's Academy of Sciences, Inc., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH is preferably adjusted to about 5 to 8. Culture is usually performed at about 20 to 35 for about 24 to 72 hours, and aeration and stirring are added as necessary.

When culturing a transformant whose host is an insect cell, the medium used is Grace's Insect.

Medium (Grace, T.C., Immobilized on Nature, 195, 788 (1962)) 1

A solution to which an additive such as 0% serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3-5 days.

Add aeration and agitation as needed.

When culturing a transformant in which the host is an animal cell, for example, about 5

MEM medium containing ~ 20% fetal bovine serum [Science, 122, 5

01 (1952)], DMEM medium [Virology, 8, 396 ( 1959)], RPMI 1640 medium [The Journal of The American Medical Association, Vol. 199, 519 (1967)], 199 medium · The · Society 'For' the 'Biological' Medicine (Proceeding of The Society for The Biological Medicine), 73, 1 (1950)]. The pH is preferably about 6-8. Culture is usually performed at about 30 to 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.

In particular, when CHO (dhfr-) cells and the dhfr gene are used as selection markers, it is preferable to use a DMEM medium containing dialysed fetal serum containing almost no thymidine. .

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. Methods for crushing cells include crushing cells with a Potter-Elvehjem homogenizer, crushing with a Pelling Blender and Polytron (Kinematica), crushing with ultrasonic waves, and narrowing the cells while pressing with a French press. Crushing by ejecting from For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 min. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the 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 to 10 molecules per cell, and more preferably 10 to 10 molecules per cell. The higher the expression level, the higher the agonist (ligand) binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to use a large amount of sample in the same lot. Be able to measure.

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. Means As the cell membrane fraction, those similar to the above can be used.

(C) Test compound or test compound (a):

In the present specification, “test compound” or “test compound (a)” refers to, for example, natural / non-natural peptide, natural / non-natural protein, natural / non-natural non-peptide compound, synthetic Compounds, natural and non-natural fermented products.

The test compound or the peptide, protein, compound or fermentation product used in the test compound (a) may form a salt, and these salts include a physiologically acceptable base (eg, an alkali metal). And the like, and salts with acids (organic acids and inorganic acids), and especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or 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:

When a test compound (specifically, test compound (a)) is brought into contact with an orphan receptor protein-expressing cell or its cell membrane fraction, the test compound is added to a cell that does not express an orphan receptor protein or its cell membrane fraction. (Specifically, when the test compound (a)) is brought into contact, the respective cell stimulating activities include, for example, (a) fluctuation of extracellular pH, (b) release of arachidonic acid, (c) release of acetylcholine, ( d) intracellular Ca release, (e) intracellular cAMP fluctuation, (f) intracellular cGMP fluctuation, (g) inositol phosphate production, (h) cell membrane potential fluctuation, (i) cell phosphorylation of the inner proteins (] ') Gee ^1: 0 5 activation,

Using (k) GTPTS binding, (1) 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 the 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 The cultures are separately cultured in a multiwell plate or the like. Before measuring cell stimulating activity, replace the cells with fresh medium or an appropriate buffer that is not toxic to cells, and add the test compound (specifically, test compound (a)) to each cell. After incubation for a certain period of time, the cells or their cell membrane fractions are extracted or the supernatant is collected, and the resulting products are quantified according to the respective methods.

If the production of a substance as an indicator of cell stimulating activity is difficult to be detected by a degrading enzyme contained in the cells, an inhibitor for the degrading enzyme may be added to perform the assay. In addition, activities such as inhibition of cAMP production can be detected as an effect of inhibiting production of cells whose basal production has been increased by forskolin or the like or a cell membrane fraction thereof.

To perform screening by measuring cell stimulating activity, cells expressing the appropriate orphan receptor protein or their cell membrane fractions and cells not expressing the orphan receptor protein or their cell membrane fractions are used. is necessary. The transformant orphan receptor protein-expressing cell may be a stable expression cell or a transient expression cell.

In order to measure the cell stimulating activity, the cell stimulating activity should be measured at the highest concentration as far as the specific reaction of the cell can be identified so that the weak agonist activity can be detected. Is preferred. The high concentration of this case, usually 1 0 one ⁸ M～1M, preferably refers to 10- ⁶ M~10- ² M. Examples of the test compound (specifically, test compound (a)) include those similar to those described in the above (C).

The above-mentioned cell stimulating activity measuring system will be described more specifically below.

In the following description of (1) to (7), “test compound” specifically indicates “test compound (a)”.

(1) Cell stimulation activity measurement system characterized by measuring the fluctuation of extracellular pH (acidification rate)

The extracellular pH at which orphan receptor protein-expressing cells or their cell membrane fractions change in response to a test compound having agonist activity is measured using a cytosensor (Cy to sensor) device (Molecular Devices, etc.) To do Therefore, the cell stimulating activity can be measured. A specific method for measuring the cell stimulating activity of a test compound by measuring the extracellular pH change using a site sensor device is described below.

① After culturing the orphan receptor protein-expressing cell or its cell membrane fraction for about 2 hours to about 48 hours, preferably for about 5 hours to about 24 hours (for example, in a capsule for a site sensor device), the culture medium Stabilize pH.

Until the pH of the medium is stabilized, it is preferable to perfuse, for example, an RPMI 1640 medium (manufactured by Molecular Devices) containing 0.1 serum albumin.

(2) Next, the test compound is brought into contact with the orphan receptor protein expressed in orphan receptor protein-expressing cells or its cell membrane fraction.

As a method of 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.

(3) Next, measure the pH change of the medium caused by contacting the medium containing the test compound with the orphan receptor protein-expressing cells or the orphan receptor protein expressed in the cell membrane fraction thereof.

(4) Perform steps (1) to (3) above using cells that do not express orphan receptor protein or their cell membrane fractions.

(2) Cell stimulating activity measurement system characterized by measuring the radioactivity of GT P aS Intracellular G protein is activated when orphan receptor protein-expressing cells are stimulated by a test compound having agonist activity GTP binds. This phenomenon is also observed in the membrane fraction of cells expressing orphan receptor protein. Normally, GTP is hydrolyzed and changes to GDP.At this time, if GTPaS is added to the reaction solution, GTPTS binds to G protein like GTP, but does not undergo carohydrolysis. The state of binding to the cell membrane containing the G protein is maintained. When labeled GT PrS 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 orphan receptor protein can be measured and compared. This method expresses the orphan receptor protein and the orphan receptor protein. This assay uses cell membrane fractions containing no cells, but measures cell stimulating activity.In this assay, it shows GT PrS binding promoting activity to orphan receptor protein membrane fraction, Substances that do not show GTP r S binding promoting activity to the membrane fraction that does not express protein receptor protein are candidate ligand substances.

A specific method for measuring the cell stimulating activity according to this method is described below.

① The cell membrane fraction containing the orphan receptor protein is transferred to a membrane dilution buffer (for example,

Dilute with 50 mM Tris, 5 mM MgCl ₂ , 150 mM NaCl, 1 GDP, 0. \% BSA pH 7.4).

The dilution ratio depends on the expression level of the receptor protein.

② Then a suitable container and the resulting liquid by ① (e.g., Fal etc. Con2053) dispensed into a test compound was added, the ^[35 S] GTP _T S as further final concentration of approximately 200 pM Add.

③ Next, after keeping the medium obtained in ① at about 25 ° C for about 1 hour, wash buffer (for example, ice-cold 50 mM Tris, 5 mM MgCl ₂ , 150 mM NaCl, 0. Add 1% BSA, 0.05¾ CHAPS pH 7.4 1.5 ml) and filter (eg, using glass fiber filter paper GF / F).

④ filter paper insulation (e.g., about 65 ° C, about 30 minutes) to measure to dried, the radioactivity of ^[35 S] GTP § S bound to the remaining membrane fraction on filter paper in the liquid scintillation counter You.

(4) 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. In an orphan receptor protein-expressing cell, the intracellular cAMP level fluctuates by agonist stimulation of a test compound having agonist activity. Using this reaction, the cell stimulating activity of the test compound on orphan receptor protein-expressing cells can be measured. - Ofan amount of cAMP produced various animal cells receptor evening to express protein in mice, rats, Usagi, catcher formic, © and anti-cAMP antibody obtained by immunizing such ^'25 1-labeled cAMP ( Both can be measured by using TRIAS or other EIA system combining anti-cAMP antibody and labeled cAMP. In addition, anti-cAMP antibody Is a quantitative possible by SPA method using a beads and iota ²⁵ iota labeled cAMP containing scintillant was solid boss using such antibodies to such animal IgG used in A or anti-CAMP antibody production (e.g., Amashamufu Use Almatia Biotech kits

) o

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. AMP production inhibitory activity) can be measured.

① Ofan receptor protein animal cells expressing the (e.g. CH0 cells) were plated at the appropriate concentration in 24-well plates (e.g., 5 X〗 ^{0 4 cel l / we ll)} , and cultured for about 48 hours.

(2) Next, the animal cells expressing the orphan receptor protein are buffered (eg, a Hanks buffer (PH7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES). (Hereinafter, Hanks buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES is called a reaction buffer).

(3) Then, add an appropriate amount (for example, about 0.5 ml) of the reaction buffer to the cells, and incubate in the incubator for about 30 minutes.

④ Next, after removing the reaction buffer, add an appropriate amount (eg, about 0.25 ml) of the reaction buffer to the cells, and then add an appropriate amount (eg, about 0.25 ml) of the reaction buffer (eg, about 0.25 ml). c When the AMP production inhibitory activity is measured, a cell containing 2 ^ {forskolin is preferable) and an appropriate amount (for example, about 1ηΜ) of a test compound are added to the cells, and the mixture is added to the cells. Incubate at 24 ° C for approximately 24 minutes.

⑤ Next, add an appropriate amount (for example, about 100 I) of 20% perchloric acid to stop the reaction, and then place on ice for about 1 hour to extract intracellular cAMP.

C The amount of cAMP in the extract can be determined using the cAMP EIA kit

). 。 Perform the above methods ① to ⑥ using cells that do not express the orphan receptor protein.

(4) CRE—DNA that contains a cell stimulating activity measurement system CRE (cAMP response element) characterized by introducing a reporter gene, can be used to prepare a Pitka Gene Basic Vector or a Pitka Gene Enhancer Vector (Toyo Ink Manufacturing) And insert it into the multiple cloning site upstream of the luciferase gene, and use this as the CRE-reporter gene vector.

In cells transfected with the CRE-repo overnight gene, stimulation with elevated cAMP induces CRE-mediated luciferase gene expression and subsequent luciferase protein production. In other words, by measuring the luciferase activity, it is possible to detect a change in the amount of cAMP in the CRE-repo overnight-transfected cells.

Cell stimulating activity can be measured using cells in which the CRE-reporter gene vector has been transfected into orphan receptor protein-expressing cells.

(1) Inoculate the CRE-repo overnight gene-introduced orphan receptor protein-expressing cells into a 24-well plate at an appropriate concentration (for example, 5 × 10 ³ cel l / wel l) and culture for about 48 hours.

(2) Transfer cells to an appropriate volume (eg, 0.2 mM) in a buffer (eg, Hanks buffer (pH 7.4) containing 3-isobutyl-methylxanthine, 0.05% BSA, and 20 mM HEPES) at a concentration of 0.2 mM or less. Wash with Hanks' buffer (PH7.4) containing sobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES).

③ Then, add an appropriate amount (for example, 0.5 ml) of the reaction buffer to the cells, and incubate in the incubator for about 30 minutes.

④ Next, except for the reaction buffer, add an appropriate amount (eg, 0.25 ml) of the reaction buffer to the cells, and add an appropriate amount (eg, 1 nM) of the test compound and an appropriate amount (eg, 0.25 ml) of the reaction buffer (preferably containing forskolin when measuring cAMP production inhibitory activity) to the cells, and at about 37 ° C for about 24 hours. To react for a while.

溶 Lyse the cells with a cell lysing agent for Pitka Gene (Toyo Ink Mfg. Co., Ltd.) and add the luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the lysate.

発光 Luminescence from luciferase is measured with a luminometer, liquid scintillation counter or top counter.

(4) The above methods (1) to (4) are carried out using cells that do not express orphan receptor protein or cell membrane fractions thereof.

As the repo overnight gene, other than luciferase, for example, alkaline phosphatase, chloramphenicol acetyltransferase or 0-galactosidase can also be used. The enzymatic activity of these repo overnight gene products can be easily measured using a commercially available assay kit as follows. That is, alkaline phosphatase activity can be measured, for example, by Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicol acetyltransferase activity can be measured, for example, by FAST CAT chrola manufactured by Wako Pure Chemical immediately henicol Acetyltransferase Assay KiT ; 3-galactosidase activity can be measured, for example, by Aurora Gat XE (manufactured by Hako Pure Chemical Industries, Ltd.).

(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 agonist. By introducing arachidonic acid having radioactivity into cells in advance, 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.

(1) Inoculate orphan receptor protein-expressing cells (for example, orphan receptor protein-expressing CH0 cells) in a 24-well plate at an appropriate concentration (for example, 5 × 10 ⁴ cells / well) and culture for 24 hours.

② After culturing, add [¾] arachidonic acid to an appropriate amount (eg, 0.25 Ci / well). [¾] Approximately 16 hours after the addition of arachidonic acid, the cells are washed (eg, 0.05 BSA). T / JP00 / 05639

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Wash with a Hanks buffer containing 20 mM HEPES (pΗ7.4)) and add a buffer (eg, Hanks buffer (PH7.4) containing 0.05% BSA and 20 mM HEPES: PH 7.4). Add the test compound dissolved in Hanks buffer (PH7.4) containing BSA and 20D1M HEPES (referred to as reaction buffer).

(3) After incubating at about 37 for about 60 minutes, add an appropriate amount of reaction buffer (eg, 400 l) to Scintille overnight, and determine the amount of [¾] arachidonic acid metabolite released in the reaction mixture. It is measured by Yuichi Shiyonukaun.

(4) Perform steps (1) to (3) above using cells that do not express the protein receptor receptor.

(6) Ca ² in by connexion cells to Yotsute stimulate cell stimulating activity measurement system Ofan receptor protein-expressing cells, characterized by measuring the release of intracellular Ca ^{2 +} in the test compound having a Agonisuto activity ⁺ Increases concentration. By utilizing this, cell stimulating activity can be measured.

(1) The orphan receptor protein-expressing cells were seeded on a sterile microscope cover glass. After about 2 days, the culture solution was suspended in an appropriate amount (for example, 4 mM) of Fura-2 AM (Dojindo Laboratories). Replace with HBSS and leave at room temperature for about 2 hours and 30 minutes.

(2) After washing with HBSS, set a glass cover on the cuvette, and measure the increase in the ratio of the fluorescence intensity at 505 nm at excitation wavelengths of 340 nm and 380 nm when the test compound is added using a fluorimeter.

You can also use FLIPR (Molecular Devices) as follows. (1) Add Fluo-3 AM (manufactured by Dojindo Laboratories) to the cell suspension, allow the cells to take up, wash the supernatant several times by centrifugation, and seed the cells on a 96-well plate. . (2) Set on the FLIPR device, add the test compound as in the case of Fura-2, and measure the change in the observed fluorescence intensity due to the addition of the test compound.

The orphan receptor protein-expressing cells are co-expressed with a gene for a protein such as aequor in, which emits light due to an increase in intracellular Ca ions, and aequor in is converted to Ca-binding by increasing intracellular Ca ion concentration. By measuring the change in luminescence intensity observed with the addition of a test compound, Thus, the cell stimulating activity can be measured. The method in this case is the same as 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, cells that simultaneously express a modified G protein such as a chimeric G protein can be used. The chimeric G protein is a functional domain of a G protein that uses Ca ²⁺ as a signal transduction system, such as G ₄ , a G protein that does not use Ca ²⁺ as a signal transduction system, such as Gi, Go, and Gs. Refers to the G protein replaced by. By using the chimeric protein, the signaling system of any G protein can be monitored by changes in Ca ²⁺ .

(3) Perform steps (1) to (4) above 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 agonist activity is added to a cell expressing a receptor receptor protein, the intracellular inositol triphosphate concentration increases. By observing this response in orphan receptor protein expressing cells produced by the test compound, the cell stimulating activity can be measured.

(1) Seed a 24-well plate and cultured for 1 day in a medium containing myo- [2-¾] inositol (2.5 micro Ci / wel 1) added to the orphan receptor protein-expressing cells on day 1 After washing well the cells expressing the receptor protein, add the test compound and add 10% perchloric acid to stop the reaction.

② Neutralize with an appropriate amount (eg, 1.5 M) of K0H and an appropriate amount (eg, 60 Y) of HEPES solution, pass through a column packed with AGlx8 resin (Bio-Rad), wash, and (e.g., 1 M) HC00NH ₄ and an appropriate amount (e.g., 0. 1M) eluted radioactivity HC00H measured by liquid scintillation counter evening one.

(3) Perform steps (1) to (4) above using cells that do not express the receptor protein.

(E) For (test) compounds having agonist activity:

As used herein, the “(test) compound having agonist activity” refers to the above ( Test compounds described in C) (eg, natural 'non-natural peptides, natural' non-natural proteins, natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, etc.) or test Among the compounds (a), any one of the cell stimulating activity measuring systems described in the above (D) (preferably, a cell stimulating activity measuring system characterized by measuring fluctuation of extracellular pH (acidification rate), etc.) ), Cell stimulating activity was observed when using orphan receptor protein-expressing cells or their cell membrane fractions, and cells that did not express orphan receptor protein or cell membrane fractions when using orphan receptor proteins were used. Test compounds with no stimulatory activity (eg, natural and non-natural peptides, natural and non-natural proteins, natural and non-natural non-peptidic compounds, synthetic compounds, natural and non-natural sources) It refers to such products).

More specifically, for example, a compound having an agonist activity when the orphan receptor protein is FM-3 (Tan, CP et al., Genomics 52, 223-229, 1998, etc.) is a C-terminal In the structure of R—X—NH ₂ (X is Gly, Ala, Va1, Leu, Ile, Ser, Thr, Cys, Met ;, Glu, Asp, Lys, Ar g, His, Phe, Tyr, Trp, Pro, Asn, Gin, etc.), and more specifically, a peptide having SEQ ID NOs: 2, 6, and A peptide containing the amino acid sequence represented by 20; Peptides, proteins, compounds or fermentation products which are (test) compounds having these agonist activities may also form salts, and these salts may include physiologically acceptable bases (eg, alkali metals). And salts with an acid (organic acid or inorganic acid), and especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or 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).

In the cell stimulating activity measurement system described in (D) above, specific examples of criteria for determining whether a test compound (or test compound (a)) is a (test) compound having agonist activity are shown below. However, the following criteria are merely examples, and whether the test compound (or test compound (a)) has agonist activity is determined by the following criteria. It is not to be construed as limiting.

When a cell stimulating activity measurement system characterized by measuring the fluctuation of extracellular pH (acidifi cat ion rate) described in (D) — (1) above is used, the test compound is If the extracellular pH at the peak of the reaction when the test compound is brought into contact with the cells exceeds 105%, assuming that the extracellular pH before contacting with the cells is 100%, the orphan receptor protein A test compound that does not show cell stimulating activity when cells that do not express it or its cell membrane fraction is used is selected as a (test) compound having agonist activity.

When a cell stimulating activity measuring system characterized by measuring the radioactivity of the labeled GTPaS described in (D)-(2) above was used, the radioactivity of the test group to which no test compound was added was measured. Assuming that the activity is 100%, the radioactivity of the experimental group containing the test compound exceeds 105%, and cell stimulating activity is observed when cells that do not express the orphan receptor protein or their cell membrane fractions are used. Test compounds that do not have any agonist activity are selected as (test) compounds.

When a cell stimulating activity measurement system characterized by measuring the intracellular cAMP fluctuation described in (D) to (3) above is used, a method using the cAMP production inhibitory effect as an index is used. In this case, the amount of cAMP produced by forskolin stimulation is assumed to be 100, the amount of cAMP produced by the addition of the test compound is 95% or less, and cells that do not express orphan receptor protein or their cell membrane fractions The test compound that does not show cell stimulating activity when using the same is selected as a (test) compound having agonist activity. On the other hand, when the cAMP production promoting effect is used as an index, the amount of cAMP when the test compound is not added is 100%, and the amount of cAMP produced by the addition of the test compound is 105% or more. A test compound that does not show cell stimulating activity when using cells that do not express a somatic protein or cell membrane fraction thereof is selected as a (test) compound having agonist activity.

When using the cell stimulating activity measuring system characterized by introducing the CRE-repo overnight gene described in (D)-(4) above, when the cAMP production inhibitory effect is used as an index, The amount of luminescence generated by forskolin stimulation is 100%, and the luminescence generated by the addition of the test compound is 95% or less. A test compound that does not show cell stimulating activity when cells that do not appear or its cell membrane fraction is used is selected as a (test) compound having agonist activity. On the other hand, when the activity of promoting the production of CAMP is used as an index, the amount of luminescence without the addition of the test compound is 100%, and the amount of luminescence produced by the addition of the test compound is 105% or more. A test compound that does not show cell stimulating activity when using cells that do not express the monofan receptor protein or a cell membrane fraction thereof is selected as a (test) compound having agonist activity.

When the cell stimulating activity measurement system characterized in that arachidonic acid release described in (D)-(5) above is used, [¾] arachidone in the medium with the reaction buffer containing no test compound was used. A cell that does not express the orphan receptor protein or its cell membrane fraction, with the amount of [] arachidonic acid metabolite in the culture medium being 105 or more when the test compound is added, with the amount of the acid metabolite being 100%. If the test compound does not show cell stimulating activity when used, select it as a (test) compound having agonist activity.

When a cell stimulating activity measuring system characterized by measuring intracellular Ca ²⁺ release described in (D) to (6) above is used, the fluorescence observed when no test compound is added is observed. The fluorescence intensity when the test compound is added is 105% or more with the intensity set to 100%, 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 test compound is selected as a (test) compound having agonist activity.

When a cell stimulating activity measuring system characterized by measuring inositol phosphate production described in (D)-(7) above was used, the radioactivity in the medium with the test compound-free reaction buffer was reduced to 100%. %, The radioactivity in the medium when the test compound was added was 105% or more, and cell stimulating activity was observed when cells that did not express the orphan receptor protein or their cell membrane fractions were used. Test compounds that cannot be tested are selected as (test) compounds having agonist activity.

(F) Structure comparison of (test) compounds having agonist activity:

After selecting (test) compounds having agonist activity according to the above (E), comparing the structures of (test) compounds having each agonist activity, (Test) Estimate (or determine) the common structure of a compound having a common activity, and prepare or obtain a ligand candidate substance having the common structure.

The ligand candidate substance has a structure common to the (test) compound having the agonist activity. Compared with each test compound (specifically, test compound (a)), Substances with strong cell stimulating activity (eg, natural peptides, natural proteins, natural non-peptide compounds, etc.).

When the compound having agonist activity (test) is a natural or non-natural peptide, natural-non-natural protein, etc., the amino acid sequences encoding those peptides or proteins are compared, and their partial sequences with high homology are compared. Or, a portion having a similar three-dimensional structure can be said to be a common structure.

Specifically, for example, when the orphan receptor protein is FM-3 (Tan, CP et al., Genomics 52, 223-229, 1998, etc.), the common structure includes SEQ ID NO: 2, 6, and By comparing the amino acid sequences represented by 20, a common structure can be derived that has an R—X —NH ₂ structure (X represents an arbitrary amino acid residue) at the C-terminus. From the common structure, in the screening method or the ligand determination method of the present invention, when the orphan receptor protein is FM-3, the ligand candidate substance having the common structure, and further, the (endogenous) ligand of FM-3 is the C-terminal thereof. It is considered that the peptide has an R—X_NH ₂ structure.

Peptides having an R—X—NH ₂ structure at the C-terminus include A-18-F-NH, and F-8-F-NH ₂ in mammals (Perry, SJ et al. FEBS Lett. 409: 426-430). , 1997) and prolactin-releasing peptide (Hinuma, S. et al. Nature 393: 272-276, 1998). In lower animals, it is ubiquitous as a family of RF amide peptide families. Furthermore, it is thought that unknown R—X—NH ₂ぺ peptides are present in mammals as diverse as lower animals, and a new R _X—NH ₂ peptide in mammals contains FM-3 (endogenous) ligand. Is considered to exist.

When the (test) compound having agonist activity is a natural or non-natural non-peptidic compound or a synthetic compound, the chemical structures of those compounds are compared, and a common basic skeleton (for example, specific Ring structures, eg, cyclo as “cycloaliphatic hydrocarbon” Saturated or unsaturated alicyclic hydrocarbons such as alkyl, cycloalkenyl, and cycloalkenyl, aromatic heterocycles as "heterocycles", saturated or unsaturated non-aromatic heterocycles (aliphatic heterocycles) Etc.) can be said to be a common structure.

In addition, the ligand determination method of the present invention determines ligands using the above-mentioned common structure as an index, so that obtaining a subtype of a ligand having a high structural similarity to the ligand can also be compared with the conventional method. It becomes much easier and more reliable.

(G) Having agonist activity (test) Regarding the steps after the creation or acquisition of the ligand candidate deduced (or determined) from the common structure possessed by the compound: The following have the agonist activity according to (F) above (test) A specific description will be given of a method of creating or obtaining a candidate ligand substance estimated (or determined) from the common structure of the compound. ,

(1) Search for natural peptides, natural proteins and natural non-peptide compounds having a common structure, select candidate ligands having a common structure, and create candidate ligands having the common structure Or how to get.

Based on the common structure of the test compound described in (F) above (specifically, test compound (a)), search for natural peptides, natural proteins, and natural non-peptidic compounds having a common structure. Can be used to estimate the ligand or its subtype.

There are a variety of known databases that can be used. File (Derwent) and The Merck Index (Merck).

Next, the cell stimulating activity of the putative ligand is measured using the cell stimulating activity measuring system described in (D) above, and is compared with the cell stimulating activity with the ligand candidate substance. Can be determined.

(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. And encodes the ligand or its subtype c A method for obtaining candidate ligand substances by cloning DNA or gene.

First, the common structure of the (test) compound having the agonist activity described in (F) above, that is, the base sequence encoding the highly homologous sequence portion between the amino acid sequences encoding the (test) compound having the 7 gonist activity, Create primers or probes to contain.

Next, using the primers, PCR is carried out in a manner known per se, and all tissues (eg, humans, warm-blooded animals (eg, guinea pigs, rats, mice, pigs, sheep, wedges, monkeys, etc.) and fish) are obtained. For example, pituitary gland, brain, brain, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, etc.) or genomic DNA library derived from cells, c Amplify DNA encoding the target ligand candidate substance from a DNA library or the like.

The cDNA containing the nucleotide sequence encoding the cloned ligand candidate substance can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and may have TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.

Next, a transformant containing DNA encoding a ligand candidate substance is cultured according to the method for producing cells expressing an orphan receptor protein described in (A) and (B) above, and the culture is performed. The candidate ligand substance can be separated and purified from the product by, for example, the following method.

That is, after culturing, cells or cells are collected by a method known per se, suspended in a suitable buffer, disrupted by ultrasonication, lysozyme and / or freeze-thawing, and then centrifuged. A method of obtaining a liquid extract of a ligand or a subtype thereof estimated by separation or filtration is appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, sometimes abbreviated as TM hereinafter).

Ligand candidates contained in the culture supernatant or extract obtained in this way The substance can be purified by appropriately combining known separation and purification methods. These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in molecular weight, Method using difference in charge such as ion exchange chromatography, Method using specific affinity such as affinity mouth chromatography, Hydrophobicity such as reverse phase high performance liquid chromatography A method using the difference between isoelectric points, such as isoelectric focusing and chromatofocusing, is used.

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, and conversely, when it is obtained as a salt, a method known per se or The compound can be converted into a free form or another salt by an analogous method.

Further, it can be produced from an amino acid sequence encoding a ligand candidate substance according to a known method for synthesizing a protein, or by cleaving a protein containing a 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, the target ligand candidate substance can be produced by condensing a partial peptide or amino acid that can constitute the ligand candidate substance with the remaining part, and if the product has a protective group, removing the protective group. . Known condensation methods and elimination of protecting groups include, for example, the methods described in the following ① to ⑤.

① M. Bodanszky and M. A. Ondet ti, Peptide synthesis, Interscience Publisher, New York (1966)

② Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(3) Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemical Experiments 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14

After the reaction, use standard purification methods such as solvent extraction, distillation, and column chromatography. The candidate ligand can be purified and isolated by a combination of laffy, liquid chromatography, and recrystallization. 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. Conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.

Measure the cell stimulating activity of the candidate ligand using the cell stimulating activity measuring system described in (D) above, and compare it with the test compound (specifically, test compound (a)). Thus, the presence or absence of agonist (ligand) activity can be confirmed. Whether or not the candidate ligand for which agonist (ligand) activity is recognized is the (endogenous) ligand of the orphan receptor protein or its subtype should be confirmed by the ligand determination method described in (J) below. Can be.

In addition, it is also possible to purify mRNA of the target gene by using the above-mentioned probe like a gene trapper, and to obtain cDNA as a ligand candidate substance from the mRNA. Furthermore, a candidate ligand substance can be obtained by culturing a transformant containing DNA encoding the candidate ligand substance according to the method for producing an orphan receptor protein described in (A) above.

(3) When the (test) compound having agonist activity is a peptide, protein or a salt thereof, a peptide or protein having a common structure is sequenced to form a ligand by searching the database. How to search for candidate substances.

The compound having the agonist activity described in (F) above (test) should have a common structure, that is, a base sequence encoding a sequence with high homology between the amino acid sequences encoding the compounds having agonist activity (test). Peptides or proteins can be searched in sequence databases to determine candidate ligands.

Examples of the sequence database include GenBank (registered trademark) file (National Institute of Health), VTS (Virtual Transcribed Sequence) and the like.

Once the nucleotide sequence encoding the candidate ligand substance is determined, the candidate ligand substance can be obtained according to the method described in (G) ^ 2 above.

When a database search reveals a sequence presumed to encode a part of a candidate ligand substance, primers or pro- It is possible to obtain ligand candidates according to the method described in (G) -2 above.

(4) A method for searching for a candidate ligand by creating an antibody that recognizes a common structure.

A peptide represented by a sequence having high homology between amino acid sequences encoding a compound having the agonist activity (test) having the agonist activity described in the above (F), that is, a compound having the agonist activity (test) (Protein) Created using a synthetic method.

Next, an antibody against the peptide is prepared according to the following method. The antibody may be any of a polyclonal antibody and a monoclonal antibody as long as the antibody can recognize the peptide.

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 cells

The peptide is administered to a warm-blooded animal at a site where the antibody can be produced by administration, itself or together with a carrier or diluent. 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 performed once every 2 to 6 weeks, for a total of about 2 to 10 times. The warm-blooded animals used include, for example, monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens.

When producing monclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, a mouse with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody producing cells contained with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled peptide described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion procedure should be performed according to known methods, for example, the method of Köhler and Milstein [Nature, 256, 495 (1975)]. 639

32

Can be. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by adding and incubating at 20 to 40, preferably at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a peptide antigen is adsorbed directly or together with a carrier. An anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A is added to the monoclonal antibody bound to the solid phase. Detection method, Add the hybridoma culture supernatant to the solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add peptides labeled with radioactive substances, enzymes, etc., and detect the monoclonal antibody bound to the solid phase And the like.

Monoclonal antibodies can be selected according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a medium for selection and breeding, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or A serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. 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 antibodies Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.

(Preparation of polyclonal antibody)

The above-mentioned peptide antibody can be produced according to a method known per se or a method analogous thereto. For example, a immunizing 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. It can be produced by collecting the substance and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier-protein used for immunizing warm-blooded animals, the type of carrier peptide and the mixing ratio of carrier and hapten depend on the efficiency of the antibody against hapten immunized by cross-linking with the carrier. It is possible to crosslink any material at any ratio, if possible.For example, serum serum albumin, thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 2 with respect to 1 hapten. A method of pulling force at a rate of 0, preferably about 1 to 5 is used.

Further, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, 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 / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The dose is usually given once every 2 to 6 weeks, for a total of about 3 to 10 times.

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.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of polyclonal antibody Immunoglobulin separation and purification methods similar to those for antibody isolation and purification.

By using the cross-reactivity of an antibody that recognizes the common structure of the compound having agonist activity thus obtained, a candidate ligand is detected, and various immunoassays are used as indicators to combine various extraction methods and chromatography. By doing so, candidate ligand substances can be obtained.

(H) Methods for screening for compounds that promote or inhibit the function of orphan receptor proteins:

Using the candidate ligands obtained in (G) above, it is possible to screen for a compound that promotes the function of the orphan receptor protein (highly active agonist) or a compound that inhibits the function (antagonist). .

A compound that promotes the function of an orphan receptor protein is referred to as a “highly active agonist”. “Highly active” refers to a “(test) compound having an agonist activity” described in the above (E). This means that the cell stimulating activity (specifically, the cell stimulating activity described in the above (D), etc.) is stronger than that of.

Hereinafter, the screening method will be described in detail.

The above-mentioned (G) is obtained by using an orphan receptor protein or by constructing an expression system for a recombinant orphan receptor protein and using a receptor-binding atsey system using the expression system. Efficiently screens compounds that alter the binding between the selected ligand candidate and the orphan receptor protein (eg, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, etc.) or salts thereof be able to.

That is,

First, a case where a ligand candidate substance is brought into contact with an orphan receptor protein-expressing cell or a cell membrane fraction thereof and a test compound ((i.e., the orphan receptor protein is added to the orphan receptor protein-expressing cell or its cell membrane fraction) (D) (a) fluctuations in extracellular pH, (b) arachidonic acid release, (c) acetylcholine release,

(d) intracellular C a release, (e) fluctuation of intracellular cAMP, (f) fluctuation of intracellular cGMP, (g) inositol phosphate production, (h) fluctuations in cell membrane potential, (i) phosphorylation of intracellular proteins, (j) activation of c_fos, (k) binding of GTP r S, (1) repo overnight Compare cell stimulating activities using the expression of one gene as an index.

The test compound (b) (i.e., the orphan receptor protein-expressing cell or a cell thereof) is compared with the cell stimulating activity when the ligand candidate substance is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction. When the cell membrane fraction is contacted with a candidate compound of a compound that promotes or inhibits the function of the orphan receptor protein), the test compound (b) (or the candidate substance) ) Is likely to be a compound that promotes the function of the orphan receptor protein (so-called agonist). .

Whether or not the test compound (b) (or candidate substance) is a compound (so-called agonist) that promotes the function of the orphan receptor protein (selectively, specifically) depends on whether the orphan receptor protein and the The amount of specific binding to test compound (b) (or candidate substance) may be measured.

As a method for measuring the specific binding amount, for example, when a labeled test compound (b) (or a candidate substance) is brought into contact with an orphan receptor protein, a labeled test compound (b) (or candidate And the like, for example, a method of measuring the binding amount of the substance) to the orphan receptor protein. 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 observed, the test compound (b) (or the candidate) Substance) is recognized as a compound that promotes the function of the orphan receptor protein (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 the 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 DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells. D that encodes the desired protein part 639

36

Complementary DNA is used for the NA fragment, but is not necessarily limited to this. For example, a gene fragment or a 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 should be a baculovirus belonging to a baculovirus using an insect as a host. (Nuclear poly edrosis virus; NPV) In the evening, it is preferable to incorporate it downstream such as the SR promoter. Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, the method is carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological Chemistry, 267, 19555-19559, 1992]. be able to.

Therefore, in the above-mentioned measurement method, the orphan receptor protein may be an orphan receptor protein purified according to a method known per se, or a cell containing orphan receptor protein. 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. Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Pelling Blender ゃ polytron (manufactured by Kinematica), crushing with ultrasonic waves, or narrowing the cells while pressing with a French press. Crushing by erupting from the ground. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 min. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the membrane fraction. In the membrane fraction, the expressed orphan receptor protein and It contains a lot of 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 to 10 molecules per cell,

Preferably, it is 10 molecules.

As the labeled test compound (b) (or candidate substance), for example, [ ³ H], [ ¹²⁵ ]

Test compounds (b) (or candidate substances) labeled with [I], [ ¹⁴ C], [ ³⁵ S], etc. are used.

In order to carry out the measurement method, first, a receptor protein protein preparation is prepared by suspending a cell containing the o-phan receptor protein or a membrane fraction of the cell in a buffer suitable for screening. The buffer may be any buffer as long as it does not inhibit the binding of the candidate substance such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or Tris-HCl buffer to the receptor protein. In order to reduce non-specific binding, a surfactant such as CHAPS, Tween-80 ™ (Kaoichi Atlas), digitonin, or dexcholate can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptidyltin can be added for the purpose of suppressing the degradation of receptors and ligands by proteases. To 0.0 lm l to 10 ml of the receptor solution, an aliquot (5000 cpm to 500,000 cpm) of the labeled test compound (b) (or candidate substance) is added. 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 reaction solution is filtered through a glass fiber filter, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured using a liquid scintillation counter or a counter.

On the other hand, the test compound (b) was added to the orphan receptor protein-expressing cell or its cell membrane fraction in comparison with the cell stimulating activity when the ligand candidate substance was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction. (I.e., when the cell stimulating activity upon contact with the orphan receptor protein is weak or not observed), the test compound (b) (or Candidate substances) are compounds that inhibit the function of the orphan receptor protein (so-called (Agonist).

In addition, the test compound (b) (or the orphan receptor protein-expressing cell or the cell membrane fraction) was compared with the cell stimulating activity when the ligand candidate substance was brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction. If the cell membrane fraction is brought into contact with a candidate compound of a compound that promotes or inhibits the function of the orphan receptor protein) and a candidate ligand, if the cell stimulating activity is weak or not observed, the test is performed. Compound (b) (or candidate substance) may be a compound (a so-called antagonist) that inhibits the function of the orphan receptor protein.

Whether or not the test compound (or candidate substance) is a compound (so-called agonist) that inhibits the function of the orphan receptor protein (selectively to Specific) depends on whether the test compound (or the candidate substance) inhibits the function of the orphan receptor protein. What is necessary is just to measure the amount of specific binding.

As a method for measuring the specific binding amount, for example, a labeled test compound (b) (or a candidate substance) when a labeled test compound (b) (or a candidate substance) is brought into contact with an orphan receptor protein And the method of measuring the amount of binding to the orphan receptor protein. As a result of the measurement, if a sufficient binding amount (an increase in the binding amount of 1% or more with respect to the non-specific binding, preferably an increase in the binding amount of 10% or more) is recognized, 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 measurement method, the same method as the above-mentioned method for screening a compound that promotes the function of the orphan receptor protein (a so-called highly active agonist) and the like are used.

In addition, when a labeled ligand candidate is brought into contact with an orphan receptor protein, a labeled ligand candidate and a test compound (b) (ie, a candidate for a compound that inhibits the function of orphan receptor protein) By contacting the orphan receptor protein with the orphan receptor protein, the amount of binding of the labeled ligand candidate substance to the orphan receptor protein is measured and compared to determine the compound that inhibits the function of the orphan receptor protein. It is also possible to screen. The specific description of the screening method is as follows.

First, the orphan receptor protein used in the screening method of the present invention is Any one may be used as long as it contains the above-mentioned protein receptor protein. In order to obtain a large amount of the orphan receptor protein used for screening, an orphan receptor protein or the like which is expressed in large amounts using a recombinant is suitable.

The above-mentioned method is used to produce the orphan receptor protein, but it is preferably carried out by expressing DNA encoding the orphan receptor protein in the above-mentioned animal cells or insect cells. The complementary DNA fragment is used for the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding an orphan receptor protein into a host animal cell and to express them efficiently, the DNA fragment must be converted to a nucleopolyhedron belonging to a baculovirus using an insect as a host. Polyhedrin promoter of the disease virus (nul ear polyhedros is virus; NPV), promoter derived from SV40, retrovirus promoter, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter Preferably, it is incorporated downstream such as the SRa promoter. Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, a method described in the literature [Nambi, P. et al., The 'Journal of Biological' Chemistry (J. Biol. Cem.), 267, 19555-19559, 1992] It can be done according to.

Therefore, in the above screening method, 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 orphan receptor protein may be used.

The cell containing the orphan receptor protein refers to a host cell that expresses 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. Cell disruption methods include Potter— The method of crushing cells with an Elvehjem type homogenizer, crushing with a one-ring blender Polytron (manufactured by Kinematica), crushing by ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure with a French press, etc. can give. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for 30 minutes. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the 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 to 10 molecules per cell, and more preferably 10 to 10 molecules per cell. The higher the expression level, the higher the 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.

In order to carry out a method for screening a compound that alters the binding 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 used. is necessary. As the labeled ligand candidate substance, for example, a ligand candidate substance labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used.

Specifically, to screen for compounds that inhibit the function of orphan receptor protein, the membrane fraction of cells or cells containing orphan receptor protein is first suspended in a buffer suitable for screening. Prepare a receptor protein standard by turbidity. The buffer may be any buffer that does not inhibit the binding of the ligand to the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer of tris-hydrochloride. Surfactants such as CHAPS, Tween-80 ™ (Kao-Atras), digitonin, and dexcholate can also be added to the buffer to reduce non-specific binding. In addition, PMSF, Leptin, E-64 (manufactured by Peptide Research Institute), A protease inhibitor such as a protein may also be added. 0. 01m l~: L to the receptions evening first solution of OML, a certain amount (5000 c pm~500000 c pm) labeled added ligand candidates, simultaneously 10- ⁴ M~10_ ^1D test compound M ( b) (ie, a candidate compound that inhibits the function of orphan receptor protein). Prepare a reaction tube containing a large excess of unlabeled ligand candidate to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 ° C to 50T, preferably at about 4 to 3, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the solution is filtered through a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a glass counter. When the count (B.-NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B.) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is, for example, And 50% or less of the test compound (b) (ie, a candidate compound that inhibits the function of the orphan receptor protein) is selected as the compound that inhibits the function of the orphan receptor protein (so-called angiogonist). can do.

(I) The test compound (b) described in (Η) above (that is, a candidate compound for promoting or inhibiting the function of orphan receptor protein) and the compound that promotes or inhibits the function of orphan receptor protein :

The test compound (b) (ie, a candidate compound that promotes or inhibits the function of an orphan receptor protein) includes a natural 'non-natural peptide, a natural and non-natural protein, a natural and non-natural Non-peptidic compounds, synthetic compounds, natural and non-natural fermentation products, etc.

The compound that promotes or inhibits the function of the orphan receptor protein includes 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. Means a recognized compound, which may form a salt. Examples of the salt of the compound include salts with a physiologically acceptable base (eg, an alkali metal or the like) and an acid (organic acid, inorganic acid), and particularly preferred are physiologically acceptable acid addition salts. . Such salts include, for example, inorganic acids (eg, hydrochloric acid T / JP00 / 05639

42

, Phosphoric acid, hydrobromic acid, sulfuric acid) or 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.

In addition, compounds that promote (highly active agonist) or inhibit (antagonist) the function of orphan receptor protein obtainable by the above method (H) include the following (endogenous) ligands or biological activities possessed by the subtypes thereof. Since it has the same action as that described above, it is useful as a safe and low-toxic drug according to the ligand activity.

Antagonists against orphan receptor proteins can suppress the physiological activity of ligands or subtypes of orphan receptor proteins, and thus are useful as safe and low-toxic drugs that suppress the ligand activities. is there.

The highly active agonist against the orphan receptor protein is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the orphan receptor protein.

When an agonist and a highly active agonist that can be obtained by the method of the present invention are used as a pharmaceutical composition, they can be administered in a conventional manner. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.

The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (for example, rats, mice, egrets, sheep, pigs, pigs, cats, dogs, dogs, etc.). Can be administered.

The dosage of the ligand or its subgroup, antagonist, and agonist varies depending on the administration subject, target organ, symptom, administration method, and the like.In the case of oral administration, for example, about 0. It is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.For example, in the case of an injection, it is usually, for example, about 0.0 per day. About 1 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg It is convenient to administer the degree 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 not only provides a method for screening a compound that promotes or inhibits the function of the orphan receptor protein described in the above (H), but also uses, as an index, the common structure of (test) compounds having agonist activity. Methods for more efficiently and reliably determining ligands for orphan receptor proteins or subtypes thereof are also provided.

That is,

(i) bringing a test compound into contact with orphan receptor protein-expressing cells or a cell membrane fraction thereof, measuring the cell stimulating activity mediated by the orphan receptor protein, and (ii) comparing the cell stimulating activities of each test compound Thus, the common structure of the test compound having agonist activity is determined,

(iii) 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. Things.

More specifically,

(i) Contacting test compound (a) with orphan receptor protein-expressing cells or its cell membrane fraction, and contacting test compound (a) with cells not expressing orphan receptor protein or its cell membrane fraction In this case, each cell stimulating activity was measured,

(iii) measuring the specific binding amount of the candidate ligand substance to the orphan receptor protein deduced from the common structure possessed by the compound having agonist activity, whereby the orphan receptor protein is measured. Methods for determining quality ligands or sub-types thereof are provided.

In the determination method, (ί) an orphan receptor protein-expressing cell or a cell membrane thereof A test compound (specifically, test compound (a)) is brought into contact with the fraction, and the cell stimulating activity mediated by the orphan receptor protein is measured.

(ii) By comparing the cell stimulating activity of each test compound (specifically, test compound (a)), a (test) compound having agonist activity was obtained, and the common structure of the compound having agonist activity was estimated. With respect to the step of obtaining (or determining) the ligand candidate substance, the same method as described above is used.

Next, a method for determining whether the ligand candidate is a (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 orphan receptor protein can be determined by measuring the amount of specific binding of the candidate ligand to the orphan receptor protein. it can.

That is, there is a method of measuring the amount of binding of the labeled candidate ligand to the orphan receptor protein when the labeled candidate ligand is brought into contact with an orphan receptor protein. If a sufficient binding amount (an increase in the binding amount of 1% or more with respect to the non-specific binding, and preferably an increase in the binding amount of 10% or more) is recognized, the candidate substance is identified as ( Endogenous) is recognized as a ligand.

Conversely, when a sufficient binding amount is not observed, the ligand candidate substance has a cell-stimulating activity but has a low specific binding ability to the orphan receptor protein, ie, a nonspecific agonist. Is likely to be a similar 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-described orphan receptor protein. In order to obtain a large amount of orphan receptor protein used for screening, an orphan receptor protein or the like which is expressed in large amounts using a recombinant is suitable. The above-mentioned method is used to produce 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. D that encodes the desired protein part 639

45

The complementary DNA is used for the NA fragment, but is not necessarily limited to this. For example, a gene fragment or a 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 transferred to a nucleophilic polyhedrosis virus belonging to a baculovirus using an insect as a host. nucl ear polyhedros is virus (NPV) polyhedrin promoter — Yuichi, SV40-derived promoter, retroviral promoter, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter, SR α Preferably, it is incorporated downstream, such as a promoter. Inspection of the quantity and quality of the receptor that has occurred can be carried out in a manner known per se. For example, according to the method described in the literature [Najiibi, P. et al., The Journal of Biological Chemistry, 267, 19555-19559, 1992]. Can do it.

Therefore, in the above measurement method, the substance 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 a cell 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. Cells can be crushed by crushing the cells with a Potter-Elvehj em-type homogenizer, crushing with a Perling Blender ゃ Polytron (Kinematica), crushing with ultrasonic waves, or applying pressure with a French press, etc. And crushing by ejecting the gas from a thin nozzle. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (1500 to 300 rpm). The mixture is centrifuged at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as the membrane fraction. In the membrane fraction, the expressed orphan receptor protein and It contains a lot of membrane components such as cell-derived phospholipids and membrane proteins.

Preferably, it is 10 molecules.

Examples of labeled ligand candidate substances include [ ³ H], C ¹²⁵ I], [ ¹⁴ C],

A ligand candidate substance labeled with [ ³⁵ s] or the like is used.

To carry out the measurement method, first, a cell containing the orphan receptor protein or a membrane fraction of the cell is suspended in a buffer suitable for screening to prepare a receptor protein sample. Any buffer may be used as long as it does not inhibit the binding of the candidate protein to the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-HCl buffer. In addition, surfactants such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, and dexcholate can be added to the buffer for the purpose of reducing non-specific binding. In addition, protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing receptor degradation and ligand degradation by proteases. To 0.00 l to 'l 0 ml of the receptor solution, add a fixed amount (5000 cpn! To 500,000 cm) of the labeled candidate substance. The reaction is carried out at about 0 ° C. to 50 °, preferably about 4 ° to 37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the solution is filtered through 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 by a liquid scintillation counter or a τ-counter.

In the present specification and drawings, when bases and amino acids are indicated by abbreviations, the IUP AC-I UB Commission on Biochemical Nomenclature is based on a common abbreviation in the field, and examples thereof are as follows. See below. If there is an optical isomer of the amino acid, the L-form is indicated unless otherwise specified.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid A adenine

T thymine

G Guanin

C

RNA ribonucleic acid

mRNA Messenger ribonucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate d CTP Deoxycytidine triphosphate. ATP Adenosine triphosphate

EDTA Ethylenediaminetetraacetic acid SDS Sodium dodecyl sulfate G 1 y Glycine

A 1 a Alanin

Va 1 Valine

Leu

I 1 e

S e r serine

Th r threonine

Cy s

Me t Methionin

G 1 u Dalminic acid

As p Aspartic acid

Lys lysine

A r g Arginine

H is histidine

P h e feniralanin

T yr tyrosine T rp: Tribute fan

Pro: Proline

Λ o -n. •, // ノヽノ：ヤ ^ ノ、ノ

G 1 n: Glutamine

pG 1 u: pyroglutamic acid

HEPE S: N- [2-hydroxyethyl] piperazine-Ν '-[2-ethanesulfonic acid]

HBS S Hank's Balanced Salt Solution The sequence numbers in the sequence listing in this specification indicate the following sequences.

[SEQ ID NO: 1]

The amino acid sequence of the sample used in Example 1 described below is shown (see Table 1).

) o

[SEQ ID NO: 2]-shows the amino acid sequence of the sample used in Example 1 described later (see Table 1).

)

[SEQ ID NO: 3]

)

[SEQ ID NO: 4]

)

[SEQ ID NO: 5]

[SEQ ID NO: 6]

)

[SEQ ID NO: 7] The amino acid sequence of the sample used in Example 1 described below is shown (see Table 1).

[SEQ ID NO: 8]

[SEQ ID NO: 9]

[SEQ ID NO: 10]

[SEQ ID NO: 11]

2 shows the amino acid sequence of the sample used in Example 1 described later (see Table 1 [SEQ ID NO: 12]

[SEQ ID NO: 13]

Shows the amino acid sequence of the sample used in Example 1 described below (see Table 1)

[SEQ ID NO: 14]

[SEQ ID NO: 15]

[SEQ ID NO: 16]

The amino acid sequence of the sample used in Example 1 described below is shown (see Table 1). [SEQ ID NO: 17]

[SEQ ID NO: 18]

[SEQ ID NO: 19]

2 shows the amino acid sequence of the sample used in Example 1 described later (see Table 1 [SEQ ID NO: 20]

[SEQ ID NO: 21]

1 shows the nucleotide sequence of DNA encoding human FM-3 (Reference Example 1 described later). [SEQ ID NO: 22]

7 shows the nucleotide sequence of FM3F2 used in Reference Example 1 described later.

[SEQ ID NO: 23]

7 shows the base sequence of FM 3 R2 used in Reference Example 1 described later. Example

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but these do not limit the scope of the present invention.

Reference Example 1 Preparation of human-type FM-3 expressing CHO cells (See Example 1 of Japanese Patent Application No. 2000-52251)

The human type FM-3 was obtained as follows. The following two types of synthetic DNAs were synthesized based on the arrangement U of human FM-3 reported in Genomics 52, 223-229 (1998).

F 3F2: 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. The PCR reaction solution was rat hypothalamus cDNA solution 1 ιι \ (from 0.2 ng poly (A) ⁺ RNA), 1 丽 2 (10 MM), 11 FM3R2 (10 M), 51 1x attached reaction solution, 51 dNTP (10 mM), 11 Ex Taq (evening color) and 36 1 distilled water were added to make a total of 501. The reaction solution was subjected to a PCR reaction using a ThermalCycler 9600. The PCR conditions were: denaturation at 95 ° C for 2 minutes, and a cycle of 98-10 seconds, 65 at 10 seconds, and 90 seconds was repeated 28 times. After confirming the amplification of a PCR product of about 1 or 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). Plasmids were extracted from the E. coli obtained by subcloning using a plasmid extractor (Kurapo Co., Ltd.), the base sequence of the inserted fragment was determined, and the sequence was the same as that reported in the above-mentioned literature. It was confirmed to be 3 cDNA (SEQ ID NO: 21). Next, a human FM-3 cDNA fragment of about 1.2 kb was obtained from the plasmid after digestion with the restriction enzymes Sa1I and NheI. Furthermore, the expression vector for animal cells, ρΑΚΚΟ-111H, was digested with the restriction enzyme sites Sa1I and NheI at the multiple cloning site, followed by electrophoresis, and the vector was recovered. . The human FM_3 cDNA fragment and the expression vector prepared by the above procedure were ligated by ligation, and E. coli JM109 was transformed to obtain E. coli JM109ZpAKKOFM3.

The transformant E. coli JM109 / pAKKΔFM3 was cultured to prepare a large amount of plasmid pAK KOFM3 DNA.

After dissolving 20 ig of the plasmid DNA in 1 ml of physiological saline (PBS), inject it into a vial of Gene Transfer (Wako Pure Chemical), and vigorously shake using a Portex mixer to remove DNA. The containing ribosome was allowed to form. 1 to 2 × 10 CHO dhfr cells were seeded on a 35 mm diameter cell culture dish, and cultured for 20 hours, and the culture solution was replaced with fresh one. For each petri dish

The amount of (251) ribosome solution corresponding to 0.5 DNA was added dropwise and incubated for 16 hours to introduce the plasmid DNA. Furthermore, after replacing the medium with fresh medium and culturing for 1 day, the medium is replaced with the selection medium and culturing is continued for 3 days. Finally, the cells dispersed by trypsin digestion are dispersed at a low density in the selection medium ( 0/05639

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Deoxyr ibonucleosides (a minimum essential medium, alpha medium containing 10% dialyzed serum) containing no D ^ ribonucleosides) were selected for transformants. Only the transformants can grow in the selection medium, and the selection is repeated by repeating the passage, and CH〇-FM3 cells are established. Example 1 Detection of stimulatory activity on FM-3 expressing CH0 cells by cytosensor-Attach using various peptide samples

The FM- 3 expressing CHO cells were seeded in 2.7xl0 ⁵ cells / capsule density sites for sensor power capsule of, was charged wearing site sensors one workstation after overnight culture. A cell culture medium (low buffered RPMI1640 medium containing 0.1% serum albumin) set in the flow path of the site sensor is supplied to the cells in a cycle of pump ON (80 seconds) and pump OFF (40 seconds). In each cycle, the rate of change in extracellular ρΗ from 8 seconds after the pump was stopped for 30 seconds was calculated as the acidification rate. The time course of the acidification rate was monitored, and when the value became stable, the cells were exposed to the peptides shown in the table for 7 minutes and 2 seconds by switching the flow path. The values of the AcHcCationRate for each well were normalized to the values of the three cycles immediately before exposure to the peptide as 100%, and the cell responses were compared. The concentration of the exposed samples was 1-10.

Table 1 shows the results of detection of the stimulating activity of peptide samples on FM-3 expressing CH0 cells by cytosensory assay.

If the value of the acidification rate at the peak of the reaction exceeds 120% (で), if the value is 120% or less but the reaction is clearly recognized (△), if the reaction is not recognized (（), ), And those that can not be said are indicated by (?). As a result, CHO cells expressing FM-3 were found to be H-2980, F-8-F-H2 and Amefurashi (

Aplysia) -derived peptide Smal 1 Cardioactive Peptide B (SCPB). Of these, the response to SCPB was the strongest. table 1

The reaction of the detected peptide (H- 2980, F- 8-F- NH2, SCPB) comparing the structure of, found RX-NH ₂ structure C- terminal part a common structure Te to base.

FIG. 1 shows a comparison of the similarity of the amino acid sequences.

Endogenous ligand of FM-3 since the reaction is strongest against SCPB is C - in addition to the terminal R- X-NH ₂ structure was expected to take a structure similar to SCPB in other parts. In addition, it is considered that H-2980 and F-8-F-NH2 have partially similar structures. As a result of searching for a known peptide based on such prediction, neuromedin U (NMU-8) was found. Industrial applicability

The method of the present invention comprises contacting a test compound with orphan receptor protein-expressing cells or a cell membrane fraction thereof or orphan receptor protein expressed in orphan receptor protein-expressing cells or cell membrane fraction thereof. By measuring the cell stimulating activity via the receptor protein and comparing the cell stimulating activities of each test compound, an agonist is selected, and the structure of each agonist is compared to determine the orphan receptor protein. It is possible to obtain ligands or their subtypes, angiogonists and highly active agonists efficiently and reliably.

Claims

The scope of the claims

(iii) (i) The case where the candidate ligand substance deduced from the common structure possessed by the compound having agonist activity is brought into contact with the orphan receptor protein-expressing cell or its cell membrane fraction, and the case where the test compound (b) is Compare the cell stimulating activities when contacted with orphan receptor protein-expressing cells or their cell membrane fractions, and ② measure the specific binding of the orphan receptor protein to test compound (b) A method for screening a compound or a salt thereof that promotes or inhibits the function of the orphan receptor protein.

2. A compound obtainable by the screening method according to claim 1 or a compound thereof

3.

(ii) comparing the cell stimulating activity of each test compound (a) to obtain a compound having agonist activity,

(iii) measuring the specific binding amount of the candidate ligand substance to the orphan receptor protein deduced from the common structure possessed by the compound having agonist activity, whereby the orphan receptor protein is measured. Quality ligand or its subtype Way.