WO2006003877A1 - Ligand de récepteur - Google Patents

Ligand de récepteur Download PDF

Info

Publication number
WO2006003877A1
WO2006003877A1 PCT/JP2005/011801 JP2005011801W WO2006003877A1 WO 2006003877 A1 WO2006003877 A1 WO 2006003877A1 JP 2005011801 W JP2005011801 W JP 2005011801W WO 2006003877 A1 WO2006003877 A1 WO 2006003877A1
Authority
WO
WIPO (PCT)
Prior art keywords
gpr92
group
test substance
hydrogen atom
protein
Prior art date
Application number
PCT/JP2005/011801
Other languages
English (en)
Japanese (ja)
Inventor
Junji Ichihara
Naoko Ohmi
Shinichi Kojima
Toshiyuki Mikami
Tadahiko Yoshima
Original Assignee
Dainippon Sumitomo Pharma Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dainippon Sumitomo Pharma Co., Ltd. filed Critical Dainippon Sumitomo Pharma Co., Ltd.
Priority to JP2006528689A priority Critical patent/JPWO2006003877A1/ja
Publication of WO2006003877A1 publication Critical patent/WO2006003877A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to a novel use of G protein-coupled receptor (GPCR): GPR92 / 93, its ligand, and the like. More specifically, based on the identification of an agonist ligand of GPR92 / 93 and the discovery of a pharmaceutical use of the agonist ligand as an insulin secretagogue.
  • GPCR G protein-coupled receptor
  • Type 1 is a pathological condition based on insulin secretion dysfunction in the spleen
  • type 2 is a pathological condition mainly due to insulin resistance in insulin-sensitive tissues and abnormal insulin secretion in the spleen.
  • type 1 is a pathological condition based on insulin secretion dysfunction in the spleen
  • type 2 is a pathological condition mainly due to insulin resistance in insulin-sensitive tissues and abnormal insulin secretion in the spleen.
  • obesity and associated lifestyle diseases especially type 2 diabetes patients, have increased.
  • the spleen is thought to play a central role in blood glucose regulation.
  • Insulin a major glycemic hormone, is secreted from beta cells in the splenic islets of Langerin. ⁇ cells secrete the necessary amount of insulin quickly in response to blood glucose that rises transiently after eating.
  • Peripheral tissues such as muscle and fat regulate the increased blood glucose level by taking up sugar in response to insulin secreted from the spleen.
  • gluconeogenesis is suppressed in response to insulin, and blood glucose is regulated. Diabetes is thought to develop as a result of this cycle failure.
  • many of type 1 diabetes are those in which spleen is autoimmunely destroyed and insulin secretion becomes insufficiency, making blood glucose control impossible. It is known that it will cause blood sugar.
  • GPR92 / 93 is known as an orphan G protein coupled receptor (GPCR) whose ligand in vivo is unknown (see Non-Patent Document 1). It has been reported that GPR92 / 93 is expressed in tissues including the brain (see Non-Patent Document 2), but its function is not known.
  • GPCR G protein coupled receptor
  • lysophosphatidic acid is a kind of phospholipid and has been known as a biosynthetic intermediate of phospholipid, but since then it has been proved to be an extracellular signaling molecule for various biological reactions.
  • the biological reaction include cell division promotion, apoptosis suppression, remodeling of the cytoplasmic skeleton, induction of cell shape change, tumor cell invasion, and the like (see Non-Patent Document 3).
  • the insulin secretion promoting action of LPA was not known.
  • Non-Patent Document 1 Lee, D. K. et al., Gene, 2001 Sep 5; 275 (1): 83-91.
  • Non-Patent Document 2 Demetrios, K. Vassilatis et al., Proc. Nat. Acad. Science, USA, 2003 Ap ril 15; 100 (8): 4903-4908.
  • Non-Patent Document 3 Yoh Takuwa et al., J. Biochem., 767-771 (2002)
  • G protein-coupled receptor an agonist ligand that can be a GPR92 / 93 agonist, a medicine containing a GPR92 / 93 agonist as an active ingredient, Specifically, it is intended to provide a method for screening for insulin secretagogues, GPR92 / 93 agonists or antagonists, and a method for screening for insulin secretagogues.
  • GPCR G protein-coupled receptor
  • the present inventors have conducted extensive studies in view of the above problems, and have identified a factor that is expressed and localized in the normal spleen Langer's island. That is, the present inventors compare the genes expressed in the normal human spleen Langerhans Island with those expressed in other tissues in the normal state, thereby expressing the expression station in the normal human spleen Langerno and Nun Island. Relics I have identified the gene. Next, when the protein encoded by the gene was examined, it was found to be GPR92 / 93, an orphan GP CR.
  • the present inventors searched for a substance serving as a ligand that causes GPR92 / 93-mediated signal transduction.
  • a substance serving as a ligand that causes GPR92 / 93-mediated signal transduction As a result, it was proved to be a ligand of lysophosphatidic acid (hereinafter sometimes abbreviated as LPA), which is a kind of in vivo lipid, and its derivative power GPR92 / 93.
  • LPA lysophosphatidic acid
  • lysophosphatidic acid has an insulin secretion promoting action in spleen cells. That is, it was found that GPR92 / 93 is a GPCR having an insulin secretion-regulating action, and its agonist ligand SLPA.
  • the present invention has been completed based on the above findings.
  • the present invention relates to pharmaceutical uses and the like of GPR92 / 93 agonists:
  • Insulin secretion promoter containing GPR92 / 93 agonist as active ingredient
  • R 1 represents a hydrogen atom, an acyl group having 8 to 22 carbon atoms, or a 1-alkene binole group having 8 to 22 carbon atoms,
  • R 2 represents a hydrogen atom, myo inositol 1-yl group, 2-ammoethyl group or phosphono group,
  • R 3 represents a hydrogen atom or an acyl group having 2 to 22 carbon atoms, and at least one of R 1 and R 3 represents an acyl group having 8 to 22 carbon atoms or a 1-alkenyl group having 8 to 22 carbon atoms.
  • Insulin secretion-promoting agent which is a compound strength represented by formula (1) lysophosphatidic acid, 1-Acy ⁇ PAF, or a pharmaceutically acceptable salt thereof; [5] In formula (1), characterized in that R ⁇ R 2 and R 3 represents a combination of the following, insulin secretagogues described in [3]:
  • R 1 represents an oleoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 1 represents a palmitoyl group
  • R 2 represents a acetyl group
  • R 3 represents a 2-ammoethyl group
  • R 1 represents a docosahexaenoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 3 represents a docosahexaenoyl group, R 2 and R 1 represent a hydrogen atom;
  • R 1 represents a stearoyl group
  • R 3 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 3 represents a stearoyl group
  • R 1 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 2 represents a phosphono group
  • R 1 and R 3 represent an otatanyl group
  • R 1 represents a hydrogen atom, an acyl group having 8 to 22 carbon atoms, or a 1-alkene binole group having 8 to 22 carbon atoms,
  • R 2 represents a hydrogen atom, myo inositol 1-yl group, 2-ammoethyl group or phosphono group,
  • R 3 represents a hydrogen atom or an acyl group having 2 to 22 carbon atoms, and at least one of R 1 and R 3 represents an acyl group having 8 to 22 carbon atoms or a 1-alkenyl group having 8 to 22 carbon atoms.
  • GPR92 / 93 agonist according to [10] which is a compound represented by
  • R 1 represents an oleoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 1 represents a palmitoyl group
  • R 2 represents a acetyl group
  • R 3 represents a 2-ammoethyl group
  • R 1 represents a docosahexaenoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 3 represents a docosahexaenoyl group, R 2 and R 1 represent a hydrogen atom;
  • R 1 represents a stearoyl group
  • R 3 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 3 represents a stearoyl group
  • R 1 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 2 represents a phosphono group
  • R 1 and R 3 represent an otatanyl group
  • GPR92 / 93 or a fragment to which a ligand can bind is contacted with a test substance and a reference substance selected from Z or lysophosphatidic acid, a derivative thereof, and a pharmaceutically acceptable salt thereof, and the GPR92 / 93 Or a method for screening a GPR92 / 93 ligand, comprising selecting a compound having a higher binding activity to a fragment to which a ligand can bind than the reference substance;
  • reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;
  • R 1 represents a hydrogen atom, an acyl group having 8 to 22 carbon atoms, or a 1-alkene binole group having 8 to 22 carbon atoms,
  • R 2 represents a hydrogen atom, myo inositol 1-yl group, 2-ammoethyl group or phosphono group,
  • R 3 represents a hydrogen atom or an acyl group having 2 to 22 carbon atoms, and at least one of R 1 and R 3 represents an acyl group having 8 to 22 carbon atoms or a 1-alkenyl group having 8 to 22 carbon atoms.
  • R 2 and R 3 represent the following combinations:
  • R 1 represents an oleoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 1 represents a palmitoyl group
  • R 2 represents a acetyl group
  • R 3 represents a 2-ammoethyl group
  • R 1 represents a docosahexaenoyl group
  • R 2 and R 3 represent a hydrogen atom
  • R 3 represents a docosahexaenoyl group, R 2 and R 1 represent a hydrogen atom;
  • R 1 represents a stearoyl group
  • R 3 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 3 represents a stearoyl group
  • R 1 represents a hydrogen atom
  • R 2 represents a myo-inositol-1-yl group
  • R 2 represents a phosphono group
  • R 1 and R 3 represent an otatanyl group
  • a screening method for an insulin secretagogue comprising selecting a compound with an indicator that the test substance is a GPR92 / 93 agonist;
  • reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;
  • Insulin secretion promoter obtained by the method according to any one of [24] to [29]; [34] Insulin secretion promoter according to [33], which is a blood glucose regulator [35] The insulin secretagogue according to [33], which is an agent for improving glucose tolerance abnormality;
  • a method for screening a fat accumulation-inhibiting drug comprising selecting a compound using as an index that the test substance is a GPR92 / 93 antagonist;
  • a reference substance selected from lysophosphatidic acid, a derivative thereof, and a pharmaceutically acceptable salt thereof is added to GPR92 / 93 or a fragment to which a ligand can bind in the presence or absence of a test substance.
  • the reaction system is The
  • the screening method according to [42] which is an animal cell endogenously expressing a G protein that can be coupled to / 93 and GPR92 / 93, a homogenate of these cells, or a membrane fraction derived from those cells;
  • a method for producing a biologically-derived fatty acid derivative comprising the following steps:
  • the present invention provides an orphan G protein-coupled receptor (GPCR): GPR92 / 93 ligand, a method for screening a GPR92 / 93 agonist or antagonist using the ligand, and the like. It became possible.
  • GPR92 / 93 agonist of the present invention exhibits insulin secretion promoting activity and can be a glucose tolerance ameliorating agent or a diabetes therapeutic agent.
  • GPR92 / 93 is a protein known as a kind of GPCR, specifically, the protein represented by SEQ ID NO: 2 (human GPR92 / 93) [See Genbank Acc. No. NM — 020400; Gene, 275 (1), p83-91 (2001)]. Further, in this specification, GPR92 / 93 includes homologues and mutants. For example, homologues include proteins from other species such as mice that correspond to human proteins, and these are identified by HomoloGene (http: //www.ncbi.nlm.nih. Gov / HomoloGene /) The base sequence ability of the generated gene can be identified a priori. Specific examples include mouse GPR92 / 93 (Genbank Acc. No. XM_355812) represented by SEQ ID NO: 4 or 10.
  • Variants include naturally occurring allelic variants and non-naturally occurring variants. Specifically, (a) the amino acid sequence set forth in SEQ ID NO: 2 lacks one or more amino acids. A protein that retains GPCR function, and (b) an amino acid sequence that has 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2, A protein to be retained, (c) a DNA having a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, and an amino acid sequence encoded by the DNA that is hybridized under stringent conditions, Examples include proteins that retain the functions of GPC R.
  • GPCR function specifically refers to the same ligand-receptor interaction as natural human GPR92 / 93 represented by SEQ ID NO: 2 and activates conjugated Ga. And a protein having an activity of promoting the GDP / GTP exchange reaction of Ga.
  • the “deletion, addition or substitution” of amino acids in (a) and “80% or more sequence identity” in (d) include, for example, a protein having the amino acid sequence represented by SEQ ID NO: 2 Include naturally occurring mutations due to processing in cells, species differences of individuals from which the protein is derived, individual differences, differences between tissues, and the like, and artificial amino acid mutations.
  • amino acid modification for example, the amino acid represented by SEQ ID NO: 2
  • a conventional site-directed mutagenesis is performed on the DNA encoding the sequence, and then this DNA is expressed by a conventional method.
  • site-directed mutagenesis methods for example, a method using amber mutation (gapped 'duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), PCR using mutagenesis primers By Etc.
  • the number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications is acceptable as long as the GPCR function can be maintained.
  • Sequence identity in (b) refers to sequence identity and homology between two DNAs or two proteins.
  • the “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared.
  • the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences.
  • sequence identity is calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994) using Vector NTI.
  • sequence identity is measured using sequence analysis software, specifically analysis tools provided by Vector NTI, GENETYX-MAC, or public databases, where the public database is f
  • the public database is f
  • the sequence identity in the present invention may be 80% or more. Preferably it is 95% or more, and still more preferably 97%.
  • stringent conditions refers to, for example, 6xSSC (solution containing 0.5 M NaCl, 0.15 M trisodium citrate as lOxSSC), 45 ° C in a solution containing 50% formamide. The following conditions may be mentioned: a hybrid is formed and then washed with 2 ⁇ SSC at 50 ° C.
  • the salt concentration in the washing step should be selected from, for example, conditions of 50 ° C at 2xSSC (low stringency conditions) and conditions up to 50 ° C at 2xSSC (high stringency conditions). Can do.
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
  • the “complementary base sequence” in the above (c) is a base complementary to the base sequence of DNA encoding GPR92 / 93 based on the base pair relationship such as A: T and G: C. It is intended to mean a related polynucleotide.
  • GPR92 / 93 is used in a sense that encompasses all of the recombinant GPR92 / 93 produced by recombinant cell power containing the DNA encoding GPR92 / 93 and functional fragments thereof. .
  • DNA comprising a base sequence encoding GPR92 / 93 represents DNA encoding the amino acid sequence of GPR92 / 93, and GPR92 / 93 derived from humans and other mammals, or the GPR92 / 93 In the amino acid sequence of 93, one or more amino acids are substituted, deleted, inserted, attached or modified, and have the same ligand-receptor interaction as natural GPR92 / 93, and are conjugated. There is no particular limitation as long as it is a DNA encoding a polypeptide having an activity that promotes the GDP ′ GTP exchange reaction of the subunit.
  • GPR92 / 93 DNA encoding GPR92 / 93 derived from mammals other than humans such as ushi, pig, monkey, mouse, rat, etc. is exemplified, and these include the spleen of mammals
  • a cDNA clone of human GPR92 / 93 can be isolated as a probe from a cDNA library derived from kidney or lung cells or a genomic library.
  • GPR92 / 93 may be one in which a mutation has been partially introduced by artificial treatment such as site-directed mutagenesis based on the cDNA clone of human GPR92 / 93.
  • DNA encoding human-derived GPR92 / 93 represented by SEQ ID NO: 1 and DNA encoding mouse-derived GPR92 / 93 represented by SEQ ID NO: 3 or 9.
  • GPR92 / 93 is affinity chromatography using an anti-GPR92 / 93 antibody from a membrane-containing fraction of cells transfected with an expression vector containing DNA encoding GPR92 / 93 or a fragment thereof. Can be isolated. Alternatively, c derived from the cell
  • GPR92 / 93 cDNA clone isolated from a DNA library or genomic library is cloned into an appropriate expression vector, introduced into host cells for expression, and contained in a cell culture membrane
  • the fraction can also be purified by affinity chromatography using anti-GPR92 / 93 antibody, His-tag, GST-tag or the like.
  • GPR92 / 93 with a fluorescent substance such as GFP, G It is also possible to select only FP positive cells, that is, cells into which GPR92 / 93 has been transfected and use it for screening (Xu et al., Nat. Cell Biol, 2, 261-267 (2000)) .
  • a mutation may be partially introduced by artificial treatment such as site-directed mutagenesis based on the GPR92 / 93 cDNA clone.
  • site-directed mutagenesis based on the GPR92 / 93 cDNA clone.
  • the ligand binding domain needs to be highly conserved, it is desirable not to introduce mutations in such regions.
  • Conservative amino acid substitutions are well known, and those skilled in the art can introduce mutations into GPR92 / 93 as appropriate without changing the properties of GPR92 / 93!
  • GPR92 / 93 ligand refers to an antigen that is formed only by lysophosphatidic acid and its derivatives described later (that is, it binds to the physiological ligand binding site of the receptor and is ligand-like). ) And antagonists (substances that bind to the physiological ligand binding site of the receptor, but do not exhibit ligand-like activity) and inverse agonists (substances that bind to any site of the receptor Substances that change conformation and inactivate receptors) are also included.
  • GPR92 / 93 agonist is used as a general term for substances that bind to GPR92 / 93 and promote the activity of the receptor, and is represented by lysophosphatidic acid typified by lysophosphatidic acid described later. In addition to acid derivatives, etc., all compounds having agonist activity against GPR92 / 93 are included.
  • GPR92 / 93 antagonist is used as a general term for substances that bind to GPR92 / 93 and inhibit the signal transduction activity of the receptor, and include the aforementioned GPR92 / 93 antagonist and GPR92 / 93. Equivalent to 93 inverse ghosts.
  • the first aspect of the present invention is a GPR92 / 93 agonist comprising lysophosphatidic acid, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and an insulin secretagogue comprising a GPR92 / 93 agonist, etc. About.
  • lysophosphatidic acid (hereinafter sometimes abbreviated as “LPA”) represents 1 asinole sn glyce mouth 1-nore-3-phosphate (1-Acy ⁇ sn-glycero ⁇ 3-phosphate).
  • acyl represents a linear acyl group having 14 to 22 carbon atoms and having 0 to 6 double bonds.
  • equation (2) [0024] [Chemical 4]
  • lysophosphatidic acid derivative means that LPA has been modified to one or more of the following (i) to (vii): Examples include:
  • the 01-position acyloxy group is a hydroxyl group, a mercapto group, an amino group, an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, an alkylamino group having 1 to 4 carbon atoms, or a dialkylamino group having 1 to 4 carbon atoms. Modification to convert to group,
  • the hydroxyl group at position 2 is an acyloxy group, a mercapto group, an amino group, an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, an alkylamino group having 1 to 4 carbon atoms, or an alkyl group having 1 to 4 carbon atoms. Modification to convert to a dialkylamino group,
  • R 1 represents a hydrogen atom, an acyl group having 8 to 22 carbon atoms, or a 1-alkene binole group having 8 to 22 carbon atoms,
  • R 2 represents a hydrogen atom, myo inositol 1-yl group, 2-ammoethyl group or phosphono group,
  • R 3 represents a hydrogen atom or an acyl group having 2 to 22 carbon atoms, and at least one of R 1 and R 3 represents an acyl group having 8 to 22 carbon atoms or a 1-alkenyl group having 8 to 22 carbon atoms. ] The compound represented by this is mentioned.
  • examples of the acyl group having 8 to 22 carbon atoms include a 0 to 6 double bond! /, May! /, And a straight chain acyl group.
  • examples of the acyl group having 2 to 22 carbon atoms include straight chain acyl groups having 0 to 6 double bonds.
  • examples of the 1-alkenyl group having 8 to 22 carbon atoms include groups in which the ester moiety of the acyl group is modified to vinyl ether. That is, Plasmalogen is mentioned as a compound of the formula (1) in which R 1 represents a 1-alkenyl group.
  • Diacylglycerol pyrophosphate may be referred to as DG PP (8: 0)
  • PA phosphatidic acid
  • LPC lysophosphatidylcholine
  • PA 1,2-di (cis 9-octadecenoyl) sn-glycerol 3 phosphate sodium 3 ⁇ 4_
  • LPC includes Lushi [Lushi brain L- ⁇ lysophosphatidylcholine] derived from commercial sales.
  • examples of the compound represented by the formula (1) include lysophosphatidylcholine plasmalogen (eg, Lys phosphatidylcholine plasmalogen; for example, a commercial rush heart L-a-lysophosphatidinorecolin plasmalogen).
  • lysophosphatidylcholine plasmalogen eg, Lys phosphatidylcholine plasmalogen; for example, a commercial rush heart L-a-lysophosphatidinorecolin plasmalogen.
  • the 2-position acyloxy compound in which the 1-position acyl group is transferred to the 2-position hydroxyl group in LPA or LPI is also within the category of LPA or LPI in the present invention.
  • L-NASPA ⁇ -Palmitoyl L-serine- ⁇ hosphoric acid
  • the LPA, LPA derivative and L-NASPA of the present invention may form pharmaceutically acceptable salts.
  • alkali metal salts such as sodium salt, potassium salt or cesium salt, calcium salt or magnesium
  • Alkaline earth metal salts such as salts
  • inorganic metal salts such as zinc salts
  • organic salts such as triethylamine salts, triethanolamine salts, trihydroxymethylaminoamino salts or pyridinium salts, ammonia salts Etc.
  • GPR92 / 93 agonists in the present invention include the above-mentioned LPA, LPA derivatives or L-NASPA. That is, the above-mentioned LPA, LPA derivative or L-NASPA is a compound having an agonist activity for GPR92 / 93.
  • the agonist activity against GPR92 / 93 represents an activity that causes GPCR function by binding to GPR92 / 93, which will be described later.
  • GPR92 / 93 agonists show insulin secretion promoting activity in splenic Langerhans islet cells and are effective as drugs for improving impaired glucose tolerance, drugs for treating diabetes, and the like.
  • GPR92 / 93 agonist of the present invention preferably LPA, LPA derivative, L-NASPA or a pharmaceutically acceptable salt thereof, or GP R92 / 93 agonist obtained by the screening method of the present invention described later or
  • the antagonist is formulated into a dosage form suitable for oral or parenteral administration with a pharmaceutically acceptable carrier.
  • Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, Binders such as polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate, etc.
  • Disintegrants magnesium stearate, air mouth gill, talc, sodium lauryl sulfate, lubricants, citrate, menthol, glycyllysine Fragrances such as sodium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, stabilizers such as citrate, sodium citrate, acetic acid, methylcellulose, polybutylpyrrolidone, Ability to include suspension agents such as aluminum stearate, dispersants such as surfactants, diluents such as water, saline, orange juice, base waxes such as cacao butter, polyethylene glycolol, white kerosene, etc. It is not limited.
  • a formulation suitable for oral administration is a solution in which an effective amount of GPR92 / 93 agonist is dissolved in a diluent such as water, physiological saline or orange juice, and an effective amount of GPR92 / 93 agonist is solid.
  • a diluent such as water, physiological saline or orange juice
  • GPR92 / 93 agonist is solid.
  • An emulsion in which a solution is dispersed in an appropriate dispersion medium and emulsified. “Effective amount” as used herein means a sufficient amount of GPR92 / 93 agonist to ameliorate each disease when used to treat patients with impaired glucose tolerance, diabetes or other lifestyle-related diseases Say.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile There are injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
  • injection solutions which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
  • Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • a sustained-release preparation can be obtained using a biocompatible material such as collagen.
  • GPR92 / 93 agonist can be enclosed in a container in unit doses or multiple doses like ampoules or vials.
  • GPR92 / 93 agonists and pharmaceutically acceptable carriers can be lyophilized and dissolved or suspended in a suitable sterile vehicle just prior to use!
  • the dosage of the preparation of the present invention containing a GPR92 / 93 agonist as an active ingredient is the type of the active ingredient, the administration route, the severity of the disease, the animal species to be administered, the drug acceptability of the administration subject, Forces that vary depending on body weight, age, etc.
  • GPR92 / 93 agonists can be administered, for example, from about 0.01 to about 10 mg / kg, preferably from about 0.1 to about 500 mg Zkg per day for an adult.
  • GPR92 / 93 agonists of the present invention are also useful as screening tools for screening for GPR92 / 93 ligands, ie, GPR92 / 93 agonists or GPR92 / 93 antagonists, as described below.
  • the second aspect of the present invention relates to a screening method for GPR92 / 93 ligand, that is, a GPR92 / 93 agonist or antagonist.
  • the present invention also provides a GPR92 / 93 agonist screening system and a method for screening the ligand using the same.
  • the test substance in the screening method of the present invention may be any known compound and novel compound, for example, a compound library prepared using combinatorial chemistry techniques, solid-phase synthesis and phage. Examples thereof include a random peptide library prepared by the display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the test substance is preferably a compound having a molecular weight of 200 to 2000, and more preferably a compound having a molecular weight of 300 to 800.
  • a GPR92 / 93 agonist can be efficiently evaluated by using a fatty acid derivative such as the above-mentioned LPA derivative as a test substance.
  • the screening method of the present invention uses a reference substance selected from LPA, LPA derivatives, L-NASPA, and pharmaceutically acceptable salts thereof as an indicator of the binding activity to GPR92 / 93, and has a higher binding activity than the reference substance.
  • This method comprises selecting a test substance as a GPR92 / 93 ligand.
  • the screening method of the present invention comprises GPR92 / 93 or a fragment to which a ligand can bind (hereinafter sometimes collectively referred to as “GPR92 / 93 etc.”), test substance and Z or lysophosphatidic acid, A reference substance selected from the derivatives and pharmaceutically acceptable salts thereof is contacted, and a compound having a higher binding activity to the GPR92 / 93 or a fragment to which the ligand can bind than the reference substance. Including selecting.
  • a screening method including
  • the binding activity between the test substance and GPR92 / 93 is obtained by immobilizing a cell membrane fraction such as GPR92 / 93 on the chip, loading the test substance solution on the chip, and performing surface plasmon resonance. This is derived by measuring the binding and dissociation of the test substance to the membrane by the method, and calculating the affinity between the test substance and GPR92 / 93 from the rate of binding or dissociation or the amount of binding.
  • GPR92 / 93 or a fragment thereof is expressed in their form. It can be provided in the form bound to a cell, a cell membrane fraction of the cell, or an affinity column.
  • Examples of cells expressing GPR92 / 93 include cells transfected with an expression vector containing DNA encoding GPR92 / 93 or a fragment thereof.
  • an affinity column an anti-GPR92 / 93 antibody column, a column using a ligand, and, when GPR92 / 93 is provided as a recombinant protein, a metal chelate having a specific affinity with a His tag or a GST tag.
  • a dartathione column can be used.
  • Examples of a method for detecting the binding activity between GPR92 / 93 or a fragment thereof and a ligand include, for example, a method for detecting the amount of ligand, or a method for detecting the amount of label bound to GPR92 / 93 or a fragment thereof by labeling the ligand. The method of measuring is mentioned.
  • Examples of the labeling method of the ligand include a method of labeling with a radioisotope such as 3H, 14C, 32P and 33P.
  • a radioisotope such as 3H, 14C, 32P and 33P.
  • an LPA derivative in which the hydrogen atom of the fatty acid part is labeled with 3H or an LPA derivative in which the phosphorus atom is labeled with 32P can be used.
  • 32P-labelled LPA and 1-oleoyl [oleoyl- 9,10- 3 H] LPA (1- 01eoyl [oleoyl-9, 10- J H] LPA; NEN Life Science products commercially;. Songzhu An, et al J Biol C hem, Vol 273, Issue 14, 7906- 7910, April 3, 1998).
  • 1-Acy ⁇ PAF (Registry No .: 163005-42-3 compound) in which the hydrogen atom of the acetyl group is labeled with tritium
  • 1-Acyl-PAF compound in which the hydrogen atom of the palmitoyl group is labeled with tritium
  • 1-Acy ⁇ PAF compound of Registry No.:112015-19-7) in which the carbon atom of the palmitoyl group is labeled with 14C [See Methods in Enzymology (1987), 141 (Cell. ReguL, Pt. B), 301-13].
  • the present invention also provides a GPR92 / 93 ligand screening method using a lipid bilayer containing GPR92 / 93 and a G protein (particularly a Ga subunit) conjugated to GPR92 / 93. That is, the present invention provides a test substance and Z or lysophosphatidic acid, a derivative thereof, in a reaction system comprising a lipid bilayer containing GPR92 / 93 and an a subunit of G protein that can be conjugated to GPR92 / 93.
  • reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;
  • reaction system comprising a lipid bilayer containing GPR92 / 93 and an ⁇ subunit of G protein that can be conjugated to GPR92 / 93
  • a host eukaryotic cell transfected with an expression vector containing (ii) a DNA encoding a polypeptide in which the ⁇ subunit of G protein that can be conjugated to GPR92 / 93 is fused to the C-terminal side of GPR92 / 93
  • GPR92 / 93 And animal cells that endogenously express G protein that can be coupled to GPR92 / 93, homogenates of these cells
  • screening for GPCR agonists is performed using GTP ⁇ GDP exchange reaction in Ga or cell stimulating activity of coupled G protein as an index.
  • specific procedures such as effector selection and activity measurement method are determined depending on the mode of Ga to be conjugated.
  • adenylate cyclase is used.
  • Ga containing a region that interacts with the phospholipase C specifically, including an effector-interacting region of Ga belonging to the Gi family or Gs family
  • Go including a region that interacts with 8 specifically, including an effector interaction region of Ga belonging to the Gq family
  • Agonists can be screened.
  • G o of GPR92 / 93 is considered to belong to the Gs family.
  • animal cells eg, HEK293 cells, L1.2 cells, etc.
  • G ⁇ ⁇ y trimeric G protein containing the Ga
  • G ⁇ 92 / 93 activated G ⁇ j8 ⁇ can dissociate into G ⁇ and G j8 ⁇ Free G ⁇ y can interact with phospholipase C
  • GPR92 / 93 to be used in the screening of the present invention is an anti-GPR92 / 93 antibody derived from a membrane-containing fraction of a cell transfected with an expression vector containing DNA encoding GPR92 / 93 or a fragment thereof. It can be isolated by the affinity chromatography used. Alternatively, a DNA clone isolated using the GPR92 / 93 cDNA clone from the cell-derived cDNA library or genomic library as a probe can be cloned into an appropriate expression vector and introduced into a host cell for expression. Cell culture membrane The contained fraction can also be purified by affinity chromatography using anti-GPR92 / 93 antibody, His-tag, GST-tag or the like.
  • GFP fluorescent substance
  • a fusion protein of GPR92 / 93 by expressing a fluorescent substance such as GFP and a fusion protein of GPR92 / 93, it is possible to select only GFP positive cells, that is, cells transfected with GPR92 / 93 and use them for screening ( Xu et al., Nat. Cell Biol, 2, 261-267 (2000)).
  • a mutation may be partially introduced by artificial treatment such as site-specific mutagenesis based on the GPR92 / 93 cDNA clone.
  • the ligand binding domain needs to be highly conserved, it is desirable not to introduce mutations in such regions.
  • Conservative amino acid substitutions are well known, and those skilled in the art can introduce mutations into GPR92 / 93 as appropriate without changing the properties of GPR92 / 93.
  • the origin of the lipid bilayer membrane retaining GPR92 / 93 is not particularly limited as long as the receptor can assume the original three-dimensional structure, but preferably human, ushi, pig, monkey, mouse, rat, etc. Fractions containing cell membranes of mammalian cells such as intact cells, cell homogenates, or cell membrane fractions fractionated from the homogenates by centrifugation or the like.
  • an artificial lipid bilayer membrane prepared by an ordinary method such as a solution force prepared by mixing various lipids such as phosphatidylcholine, phosphatidylserine, and cholesterol at an appropriate ratio, preferably a ratio close to that in the cell membrane of a mammalian cell is also used. Can be preferably used in one embodiment of the present invention.
  • the Ga conjugated with GPR92 / 93 needs to have at least a region involved in binding of the Ga to GPCR and a region involved in binding of any Ga guanine nucleotide.
  • G a conjugated with GPR92 / 93 is considered to belong to the Gs family (Gs a)
  • the G a used has at least the GPCR binding region of Gs a
  • the Gs a guanonucleotide It has a binding region or a guanine nucleotide binding region derived from Ga belonging to another family. From the results of X-ray crystal structure analysis of Ga, etc., a sequence of about 5 amino acids at the C-terminus is important for binding to GPCR.
  • the guanine nucleotide binding region is the nucleotide binding site of ras protein. Homologous regions (from the N-terminal side, including amino acids motifs called P bots, G, boxes, G boxes, and G "boxes, and the beginning of a E helix and a F helix in highly helix domains) Arco It is clear.
  • Ga conjugated to GPCR belongs to the Gi family (Gi a) or Gq family (Gq ⁇ )
  • the Ga to be used has at least the Gi ⁇ or Gq a GPCR binding region, respectively. And having a guanine nucleotide-binding region of Ga belonging to the corresponding family or a Ga-derived guanine nucleotide-binding region belonging to another family.
  • Ga activation domain of the receptor interacts with the GPCR binding region of Ga, resulting in a conformational change of Ga.
  • Nin nucleotide binding domain force also dissociates GDP and binds GTP quickly.
  • binding of inverse ghosts decreases the activated Ga — GTP level because the Ga active domain is inactivated by changes in receptor conformation.
  • GTPase activity of Ga such as 35S-labeled GTP y S! / ⁇ If a GTP analog is added to the system, the presence or absence of the test substance is present.
  • GPR92 / 93 antagonists were measured by comparing the radioactivity bound to the membrane in the presence and absence of the test substance in a system coexisting with a bioactive ligand such as LPA and its derivatives. Can be screened. That is, if the radioactivity decreases in the presence of the test substance as compared to the absence of the test substance, the test substance is an antagonist.
  • screening can be performed by monitoring the binding of GTP analog to Ga using the surface plasmon resonance method or the like.
  • the aforementioned cell stimulating activity can also be measured using the effect of conjugated Ga on the effector as an index.
  • the screening system of the present invention further includes GPR92 / 93. It is necessary to include a lipid bilayer membrane containing an effector as a constituent element.
  • the conjugated Go must further include a region for interacting with the effector. This region may be the original effector interaction region of G ⁇ or may be an effector interaction region of Ga belonging to a different family. For example, for Gsa, Gq belonging to a different family includes Gq ⁇ , Gi ⁇ , G12 ⁇ and the like.
  • G o that contains effector interaction regions of G o belonging to different families (eg Gq); (eg Gs a)
  • Gq effector interaction regions of G o belonging to different families
  • Gs a As the simplest example of a chimera, about 5 amino acids at the C-terminus of Gq a Examples include those substituted with the C terminal sequence of a (Gqs a).
  • G a conjugated to GPR92 / 93 contains an effector interaction region of Gs a
  • a lipid bilayer membrane containing adenylate cyclase is used as an effector.
  • the conjugated Ga contains the effector interaction region of Gqa
  • conjugate Ga contains the effector interaction region of Gs a
  • a lipid bilayer membrane containing adenylate cyclase is used as the effector.
  • Ligand activity is evaluated using the promoting action of acid cyclase activity as an index.
  • AC adenylate cyclase
  • an effector that is, a chimera containing Ga or Ga or an effector interaction region of Gsa or Gia
  • proteins in the case of Chimera Gs a or Chia Gi a
  • ATP is added to a membrane fraction containing AC, and the amount of cAMP produced is determined using RI (1251), anti-cAMP antibody, Competing with cAMP labeled with enzymes (alkaline phosphatase, peroxidase, etc.), fluorescent substances (FITC, rhodamine, etc.), etc., or by adding [ ⁇ -32P] ATP to membrane fractions containing AC [32P]
  • RI 1251
  • anti-cAMP antibody Competing with cAMP labeled with enzymes (alkaline phosphatase, peroxidase, etc.), fluorescent substances (FITC, rhodamine, etc.), etc.
  • FITC alkaline phosphatase, peroxidase, etc.
  • FITC fluorescent substances
  • rhodamine etc.
  • the method of measuring the radioactivity after separating cAMP with an alumina column or the like is mentioned, but is not limited thereto.
  • G a Gs a
  • a system that coexists with a physiologically active ligand such as LPA Measure the AC activity in the presence and absence of the test substance. If the AC activity decreases in the presence of the test substance than in the absence of the test substance, the test sample is an antagonist of GPR92 / 93. .
  • G a G i
  • the AC activity is measured and compared in the presence and absence of the test substance, and if the AC activity increases in the presence of the test substance, the test substance is inverted by GPR92 / 93. -A strike, and an agonist if activity decreases.
  • AC activity was measured and compared in the presence and absence of the test substance in a system coexisting with a physiologically active ligand such as LPA, and the AC activity increased in the presence of the test substance than in the absence of the test substance.
  • the test sample is then an antagonist of GPR 92/93.
  • the action of Ga on AC was generated by the ability to measure intracellular cAMP levels or by labeling cells with [3H] adenine [3H It can also be evaluated by measuring the radioactivity of cAMP.
  • the amount of intracellular cAMP is measured by incubating the cells for an appropriate period of time in the presence and absence of the test substance, and then performing the above competitive immunoassay on the extract obtained by disrupting the cells. Any other known method can be used
  • a method for evaluating the amount of cAMP by measuring the expression level of a ribota gene under the control of a cAMP response element (CRE).
  • CRE cAMP response element
  • animal cells introduced with a vector containing an expression cassette linked to a DNA encoding a reporter protein downstream of a promoter containing CRE are introduced into the presence of a test substance.
  • the amount of cAMP in the cells is evaluated by measuring and comparing the expression of the reporter gene in the extract obtained by culturing the cells for an appropriate period of time in the absence and absence and disrupting the cells using a known method. Is.
  • G a is Gs a
  • the test substance can be inverted to GPR92 / 93.
  • -A strike and if it increases, it is an agony.
  • the amount of cAMP in the presence and absence of the test substance is measured and compared to screen for GPR92 / 93 antagonists. But it can. That is, if the amount of cAMP decreases in the presence of the test substance than in the absence of the test substance, the test substance is antagonist.
  • Ga is Gi a
  • intracellular cAMP or the expression level of reporter gene under CRE control
  • the test substance is an inverse agonist of GPR92 / 93. Yes, if it decreases, it will be an asset.
  • GPR92 / 93 antagonists can be screened by measuring and comparing the amount of cAMP in the presence and absence of the test substance in a system coexisting with a biologically active ligand such as LPA. . That is, if the amount of cAMP increases in the presence of the test substance than in the absence of the test substance, the test substance is an antagonist.
  • a screening system containing phospholipase C ⁇ (hereinafter also referred to as PLC ⁇ ! /)
  • an effector ie, a chimeric protein containing G a as an effector interaction region of G q a
  • the effect of the Gq a or the chimeric Gq a on the effector can be evaluated by directly measuring the PLC
  • PLC j8 activity can be determined, for example, by adding 3H-labeled phosphatidylinositol 1,4,5-diphosphate to the PLC j8-containing membrane fraction and measuring the amount of inositol phosphate produced using a known method.
  • GPR92 / 93 antagonists can be screened by measuring and comparing PLC ⁇ activity in the presence and absence of the test substance in a system coexisting with a bioactive ligand such as LPA. . That is, if the PLC activity decreases in the presence of the test substance than in the absence of the test substance, the test substance is antagonist.
  • Gq a or chimeric Gq a on PLC j8 is produced by adding [3H] inositol to the cells and producing [3H] inositol phosphate. It can also be evaluated by measuring the radioactivity of cells or measuring the intracellular Ca2 + level. Intracellular Ca2 + levels can be determined using a fluorescent probe (fora-2, indo-1, fluor-3, Calcium-Green I, etc.) after incubating the cells for an appropriate time in the presence or absence of the test substance. Optically measured or calcium sensitive photoprotein Force that can be measured using equolin, etc. Any other known method may be used. An apparatus suitable for spectroscopic measurement using a fluorescent probe is the FLIPR (Molecular Devices) system.
  • the test substance is an agonist of GPR92 / 93, If it decreases, it is an inverse ghost.
  • the GPR92 / 93 ligand screening method using GPR92 / 93 and conjugated Ga described above is performed in the presence of a ligand for GPR92 / 93, for example, LPA, the GPR92 / 93 is further used against GPR92 / 93. You can easily select a tral antagonist.
  • GPR92 / 93 ligands can be screened using various physiological actions dependent on GPR92 / 93 as indices.
  • the physiological action include activation of ERK, which is a kind of MAP kinase.
  • ERK which is a kind of MAP kinase.
  • a method of measuring whether or not a test substance promotes phosphorylation of ERK by measuring the amount of phosphorylated ERK by Western blotting or the like (Kabarowski et al. al., Proc. Natl. Acad. Sci. USA, 97, 12109-12114 (2000)).
  • Another example is a method of screening for a GPR92 / 93 ligand by measuring GPR92 / 93-dependent DNA synthesis promoting activity (cell growth promoting activity).
  • a method for screening a GPR92 / 93 ligand by measuring GPR92 / 93 dependent DNA synthesis inhibitory activity (cell growth inhibitory activity) can be mentioned.
  • the test substance is a GPR92 / 93 antigen, and if it is decreased, it is an inverse agonist.
  • the amount of Ca 2+ (or the expression level of reporter gene under SRE control) in the presence and absence of the test substance in a system coexisting with a physiologically active ligand such as LPA GPR92 / 93 antagonists can be screened. That is, if the amount of Ca 2+ (or the expression level of the reporter gene under SRE control) decreases in the presence of the test substance than in the absence of the test substance, the test substance is an antagonist.
  • a preferred embodiment of a screening system provided for the screening method of the present invention, comprising a lipid bilayer membrane comprising GPR92 / 93, and a Ga conjugated with GPR92 / 93 as a constituent element, A polypeptide comprising at least a region involved in the binding of GPR92 / 93-encoding DNA to a conjugated Ga G PCR and a region involved in the binding of any Ga guanine nucleotide.
  • an expression vector containing the encoding DNA A transfected host eukaryotic cell, a homogenate of the cell, or a membrane fraction derived from the cell.
  • the Ga is not particularly limited as long as it is conjugated with GPR92 / 93! Each gene of / Ga is known and can be easily obtained.
  • DNA encoding a polypeptide containing G ⁇ that is conjugated to GPR92 / 93 includes at least a sequence encoding a region involved in binding to a GPCR of conjugated Ga, and an arbitrary guanine nucleotide of Ga. It is necessary to have a sequence encoding a region involved in the binding of. As described above, from the results of X-ray crystal structure analysis of Ga, the GPCR binding region and the guanine nucleotide binding region are well known, and those skilled in the art can delete part of the Ga coding sequence if desired. It is easy to construct the fragment.
  • the DNA encoding Ga conjugated with GPR92 / 93 further comprises a nucleotide sequence encoding a region for interacting with a desired effector. Need to include.
  • the DNA contains a nucleotide sequence encoding an effector interaction region of Gi or Gsa.
  • the DNA contains a nucleotide sequence encoding the effector interaction region of Gqa.
  • Go gene is known and its effector interaction region is also well known.
  • DNA encoding a chimeric Ga protein by appropriately combining known genetic engineering techniques.
  • DNA encoding the chimeric protein eg, Gqs
  • a sequence encoding about 5 amino acids at the C-terminus of Gqa cDNA can be obtained by using a known technique such as PCR. Examples include those substituted with a DNA sequence encoding a C-terminal sequence.
  • DNA encoding GPR92 / 93 and DNA encoding Ga conjugated to GPR92 / 93 must be functionally linked to a promoter capable of exhibiting promoter activity in a host eukaryotic cell. Don't be.
  • the promoter used is not particularly limited as long as it can function in the host eukaryotic cell.
  • SV40-derived early promoter cytomegalovirus LTR, rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived Initial promoter, baculovirus-derived polyhedrin promoter, etc.
  • a promoter of a constituent protein gene of a eukaryote-derived cell such as a virus promoter, a j8-actin gene promoter, a PGK gene promoter, and a transferrin gene promoter.
  • the expression vector used should be capable of inserting a coding DNA between the promoter region and the terminator region, which preferably contains a transcription termination signal, that is, a terminator region downstream of the promoter.
  • the expression vector is a selectable marker gene (tetracycline, ampicillin, kanamycin, no, idaguchimycin, phosphinothricin and other drug resistance genes, auxotrophic mutation complementary genes, etc.
  • vectors used in the screening system of the present invention in addition to plasmid vectors, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses suitable for use in mammals such as humans. Also, box virus, poliovirus, Sindbis virus, Sendai virus, etc., or a vacuum virus vector suitable for use in insect cells.
  • the DNA encoding GPR92 / 93 and the DNA encoding Ga conjugated to GPR92 / 93 may be carried on two separate expression vectors and co-transfected into a host cell. Alternatively, it may be inserted dicistronic or monocistronic on one vector and introduced into a host cell.
  • the host cell is not particularly limited as long as it is a mammalian cell such as a human, monkey, mouse, rat, hamster, or a certain insect cell.
  • a mammalian cell such as a human, monkey, mouse, rat, hamster, or a certain insect cell.
  • Examples include monkey-derived cells such as Vero, human-derived cells such as HeLa, HEK293, and MCFIOA, and insect-derived cells such as S19, Sf21, and High Five.
  • Gene transfer into a host cell may be performed using any known method that can be used for gene transfer in eukaryotic cells.
  • any known method that can be used for gene transfer in eukaryotic cells.
  • calcium phosphate coprecipitation method, electroversion method, ribosome method, Examples include a microinjection method.
  • Host cells into which the gene has been introduced contain, for example, a minimal amount of about 5-20% urine fetal serum. It can be cultured using an essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Ham's F-12 medium, RPMI 1640 medium, 199 medium, Grace's insect cell culture medium, or the like.
  • the culture temperature which is preferably about 6 to about 8 for the medium, is usually about 27 to about 40 ° C.
  • the eukaryotic cells into which the DNA encoding GPR92 / 93 and the DNA encoding Ga conjugated to GPR92 / 93 obtained as described above are intact are allowed to remain intact depending on the screening method used. It may be used as a cell! /, Or a cell homogenate obtained by disrupting the cell in an appropriate buffer, or by centrifuging the homogenate under an appropriate condition (for example, about 1 After centrifugation at about 000 X g, the supernatant is recovered, and then the precipitate is recovered by centrifugation at about 100,000 X g).
  • the screening system to be used is preferably a cell force as described above. It is a membrane fraction to be prepared.
  • the screen system used is intact eukaryotic cells.
  • the host eukaryotic cell contains a vector containing an expression cassette operably linked to a DNA encoding a reporter protein downstream of a promoter region containing a cAMP response element (CRE) or a TPA response element (TRE). It must be introduced.
  • CRE is a cis element that activates gene transcription in the presence of cAMP. The ability to include a sequence containing TGACGTCA as a consensus sequence.
  • TRE is a cis element that activates gene transcription in the presence of Ca2 +, and a sequence that includes TGACTCA as a consensus sequence. As long as it retains Ca2 + responsiveness, a part of the sequence is deleted or replaced. It can be an array containing insertions or attachments.
  • a promoter sequence containing CRE or TRE The above-mentioned viral promoters and mammalian constituent protein gene promoters can be used in the same manner, and a CRE or TRE sequence is placed downstream of the promoter sequence using a restriction enzyme and DNA ligase, or using PCR or the like.
  • any known gene capable of detecting and quantifying gene expression quickly and easily can be used.
  • luciferase, ⁇ -galactosidase, 13-glucuronidase, alkaline phosphatase The ability to include DNA encoding a reporter protein such as peroxidase is not limited to these. It is more preferable that a terminator sequence is arranged downstream of the reporter gene.
  • a vector carrying such a CRE (or TRE) -reporter expression cassette a known plasmid vector or viral vector can be used.
  • An expression vector containing a reporter gene under the control of a serum response element (SRE) can also be prepared in the same manner as described above.
  • FIG. 1 Another preferred embodiment of a screening system provided for the screening method of the present invention, comprising a lipid bilayer membrane comprising GPR92 / 93, and Ga conjugated to GPR92 / 93 as a constituent element
  • a lipid bilayer membrane comprising GPR92 / 93, and Ga conjugated to GPR92 / 93 as a constituent element
  • Is an expression vector comprising DNA encoding a fusion protein in which a polypeptide containing at least a GPCR binding region of a conjugated G chain and an arbitrary guanine nucleotide binding region of Ga is linked to the C-terminal side of GPR92 / 93.
  • DNA encoding GPR92 / 93 and DNA encoding a polypeptide containing a GPCR binding region of Ga and any Ga guanine nucleotide binding region conjugated to GPR92 / 93 are as described above. Can be obtained. Those skilled in the art can construct a DNA encoding a fusion protein of GPR92 / 93 and Ga by appropriately combining known genetic engineering techniques based on these DNA sequences. In brief, DNA or other DNA DNA encoding GPR92 / 93 has been removed using PCR, etc. so that DNA encoding DNA encodes the DNA reading frame, ie, in-frame. Use to ligate. At this time, a part of the C terminus of GPR92 / 93 may be deleted, or a linker sequence such as a His tag may be inserted between GPR92 / 93 and G ⁇ .
  • the DNA encoding the obtained fusion protein is inserted into an expression vector as described above. And introduced into a host eukaryotic cell using the above-described gene transfer technique.
  • the fusion protein is expressed on the membrane of the resulting eukaryotic cell, the Ga activation domain on the third intracellular loop of the receptor and the coupled Ga receptor binding region are against GPR92 / 93. It can interact in the absence of physiological ligands to promote the GDP 'GTP exchange reaction in Ga. Therefore, Ga is constantly activated.
  • One receptor G o When using fusion protein-expressing cells for screening using the effect of Ga on the effector as an indicator, it is understood that Ga is linked to the receptor. If it interferes with the action, an amino acid sequence (for example, a thrombin sensitive sequence) that is cleaved by a specific protease is introduced at the junction between the receptor and Ga, and the fusion protein is expressed on the membrane. Later, the protease can act to separate the receptor from Ga.
  • an amino acid sequence for example, a thrombin sensitive sequence
  • any form of intact cells, cell homogenates, and membrane fractions can be appropriately selected and used depending on the screening method used.
  • Still another embodiment of the screening system provided for the screening method of the present invention comprising a lipid bilayer membrane containing GPR92 / 93, and Ga conjugated to GPR92 / 93 as a component, A cell prepared by transfecting a host animal cell that endogenously expresses the conjugated G protein with an expression vector containing DNA encoding GPR92 / 93, a homogenate of the cell, or a membrane derived from the cell Minutes.
  • an expression vector into which the DNA is inserted, and the expression vector into a host cell those described above can be used.
  • the screening system of the present invention comprises GPR92 / 93 and animal cells that endogenously express GPR92 / 93 and a G protein conjugated with GPR92 / 93, It is a cell-derived membrane fraction.
  • Preferred examples of such cells include mammalian spleen, kidney and lung-derived cells.
  • GPR92/93 as a screening system containing a lipid bilayer membrane containing GPR92 / 93 and Ga conjugated to GPR92 / 93 as a constituent element.
  • a fusion protein of conjugated Ga or purified receptor and conjugated Ga What was reconstituted in the engineered lipid bilayer membrane can be used.
  • GPR92 / 93 can be purified from membrane fractions obtained from spleen, kidney or lung-derived cells of humans or other mammals by affinity chromatography using anti-GPR92 / 93 antibody.
  • the receptor can be obtained by using an anti-GPR92 / 93 antibody, His-tag, GST-tag, or the like from a recombinant cell into which an expression vector containing DNA encoding GPR92 / 93 has been introduced. It can also be purified from sugar etc.
  • a fusion protein of the receptor and conjugated Ga can also be obtained from an anti-GPR92 / 93 antibody, His-tag, GST-tag from a recombinant cell introduced with an expression vector containing DNA encoding the fusion protein. It can be purified by affinity chromatography using the above.
  • lipids constituting the artificial lipid bilayer membrane include phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Ch), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). A mixture of one or more of these in an appropriate ratio is preferably used.
  • Add the appropriate amount of purified receptor and G ⁇ or the receptor G ⁇ fusion protein incubate for about 20-30 minutes with occasional stirring in ice, and then dialyze against an appropriate buffer.
  • Proteolibosomes can be prepared by centrifuging at about 100,000 8 for 30-60 minutes and collecting the sediment.
  • the GPR92 / 93 agonist selected by the above-described screening method of the present invention that is, the GPR92 / 93 agonist, exhibits insulin secretagogue activity in the splenic Langerhans Islet as well as the physiological ligand of GPR92 / 93. As a result, it shows an effect of improving glucose tolerance abnormality. Therefore, by combining these with appropriate additives, it can be made into a drug for improving glucose tolerance. Therefore, the present invention also provides a drug for improving impaired glucose tolerance by combining a GPR92 / 93 agonist selected by the screening method of the present invention and a pharmaceutically acceptable carrier, Or provide lifestyle-related disease treatment, especially diabetes treatment.
  • GPR92 / 93 antagonists such as GPR92 / 93 antagonists and inverse agonists selected by the above-described screening method of the present invention inhibit insulin secretion and suppress sugar uptake in adipocytes. It is thought that. Therefore, it is clear that GPR92 / 93 antagonists exhibit anti-obesity action, and therefore, they can be made anti-obesity drugs by combining them with appropriate additives.
  • Bioly-derived fatty acid derivatives are known to be difficult to produce by chemical synthesis because they contain many geometric isomers and optical isomers.
  • a method for purifying low molecular weight organic compounds by removing abundant substances such as proteins from samples derived from living organisms such as animal organs reverse phase HPLC, gel filtration, extraction with organic solvents
  • proteins by heat denaturation and acid denaturation, etc., but it is difficult to process a large amount of biological sample!
  • problems such as that only a part of the bioactive component having very low efficiency can be recovered, and that the bioactive component cannot be recovered because it is denatured and deactivated.
  • the present inventors have found a method for efficiently purifying a biologically derived fatty acid derivative from a biologically derived sample such as pig brain or spleen.
  • the present invention also provides a method for producing a biologically-derived fatty acid derivative and a low-molecular component, characterized by comprising a purification step that combines extraction using solid-phase extraction coagulant and reverse-phase HPLC. That is, the following steps:
  • a method for producing a biologically-derived fatty acid derivative and a low-molecular component containing thiophene is also within the scope of the present invention.
  • the solid-phase extraction resin is a resin that adsorbs a fatty acid derivative-containing fraction and a low-molecular component and does not adsorb a protein.
  • Oasis HLB resin manufactured by Waters
  • the f column can be displayed.
  • Examples of biological samples include mammal-derived organ samples that are not particularly limited. Mammals include, but are not limited to, rodents such as mice or rats, rabbits, dogs, pigs, cows, humans, and the like. Examples of the organ include, but are not limited to, brain, spleen, liver, heart, kidney, lung, stomach, adrenal gland, and the like.
  • Examples of the biologically derived fatty acid derivatives include linear or branched, saturated or unsaturated fatty acid derivatives having 5 to 30 carbon atoms. Specifically, a linear or branched saturated fatty acid having 5 to 30 carbon atoms, an unsaturated fatty acid having a straight chain or branched 1 to 10 double bond or triple bond having 5 to 30 carbon atoms. And esters of saturated or unsaturated fatty acids. And the ester.
  • the dalyceride may have an ester bond with 3 to 3 saturated fatty acids or unsaturated fatty acids.
  • an arbitrary hydroxyl group of the glycerin structure may form a phosphate ester.
  • the phosphate ester may be a phosphoric acid such as an alkyl ester having 1 to 6 carbon atoms or glyceride.
  • Examples include zylcholine, phosphatidylserine, or phosphatidylinositol.
  • hydrophilic organic solvent examples include methanol, acetonitrile, and mixed solvents thereof. Preferable examples include 10% to 95% methanol, 10% to 95% acetonitrile.
  • aqueous solution containing these hydrophilic organic solvents used as an eluent examples include water and a buffer solution, and the pH is preferably adjusted to 2 to 10 as appropriate. Specifically, 0.05M to 0.5 acetic acid, 0.03% to 0.3% TFA, 0.03% to 0.3% formic acid, 0.1% to 1% ammonia and the like may be contained.
  • the eluate containing hydrophilic organic solvent is usually eluted with 0.5 to 5 L.
  • the eluate can be fractionated as appropriate, and the fraction containing the desired biologically derived fatty acid derivative can be obtained using biological activity as an index.
  • the obtained fraction can be further purified by a purification method known to those skilled in the art as appropriate to obtain a single compound.
  • the purification method include reverse phase HPLC, normal phase HPLC, silica gel column chromatography, ion exchange chromatography, and recrystallization.
  • reverse Phase HPLC is mentioned.
  • LPA (22: 6) represented by formula (3) or its isomer (2-acyl form
  • the Oasis HLB resin column is washed with water and then with 2% to 10% methanol water. After that, LPA (22: 6) -containing fraction can be obtained using 10% -90% acetonitrile- 10% -90% methanol as eluent.
  • the ligand of the present invention such as LPI (18: 0) represented by the formula (6) or its isomer (2-acyl form) can also be obtained as a single compound by the same method.
  • mice were immunized according to the following schedule, and antiserum-resistant polyclonal antibodies were prepared.
  • FCA Complete Usagi (KBL JW 11 weeks)
  • FCA Compl ete Freuncfs Adjuvant
  • Tissue sections were prepared at 4-5 ⁇ m thickness from paraffin blocks of biopsy tissue samples (57-year-old male, 95-year-old male, etc.). Slide samples were deparaffinized with xylene, Hydrated again through the treatment. Subsequently, antigen activation by autoclaving was performed in Target Retrieval Solution (DAKO). Subsequent operations used Vectastain ABC-AP kit (Vector). After blocking with the attached reagent, it was incubated for 45 minutes with the primary antibody prepared in (1) above. After washing, the cells were incubated with an anti-rabbit secondary antibody (8-1000) diluted to 5 11 ⁇ 21 for 30 minutes. After washing, Vector ABC-AP (AK-5000) reagent was added, and color was developed with Vector Red (SK-5100).
  • DAKO Target Retrieval Solution
  • TLARPDATQSQRRRKTVRL (SEQ ID NO: 6) was used as the GPR92 / 93 antigen peptide, and as a result of examining the localization of expression by the same method, it became clear that it was significantly expressed in Langerno and Seungsu Island. .
  • a mixture of 11 ⁇ 1 containing 10 ⁇ g of total RNA and 7- (dT) 24 primer (manufactured by Amersham) lOOpmol was heated at 70 ° C. for 10 minutes and then cooled on ice. After cooling, the mixture is mixed with Super bcnpt Choice system for cDNA Synthesis (Gibco—BRL) [included 5xFirst Strand cDNA Buffer 4 / z 1, 0.1M DTT 2 ⁇ 1 included in the kit and the kit 10 mM dNTP Mix 1 ⁇ 1 contained in the solution was added, and the mixture was heated at 42 ° C. for 2 minutes.
  • Super Scriptll RT 21 400 U included in the kit was added to the mixed solution, and the mixed solution was heated at 42 ° C. for 1 hour and then cooled on ice. After cooling, DEPC-treated sterilized distilled water 9 1 ⁇ 1, 5xSecond Strand Reaction Buffer 30 ⁇ 1, included in the kit, lOmM dNTP Mix 3 1, included in the kit, E. coli DNA Ligase 1 ⁇ 1 (10U), E. included in the kit. E. coli DNA Polymerase I 4 1 (40 U) and E. coli RNase H 1 ⁇ 1 (2 U) contained in the kit were added, and the mixture was reacted at 16 ° C. for 2 hours.
  • the recovered aqueous layer was mixed with 7.5M ammonium acetate solution (72.5 ⁇ 1) and ethanol (362.5 ⁇ 1), and the mixture was centrifuged at 4 ° C., 14,000 rpm for 20 minutes. After centrifugation, the supernatant was discarded to obtain a DNA pellet. Thereafter, 0.5 mL of 80% ethanol was added to the DNA pellet. The mixture was centrifuged at 14,000 rpm for 5 minutes at 4 ° C, and the supernatant was discarded to obtain a DNA pellet again. To the obtained DNA pellet, 0.5 mL of 80% ethanol was added again.
  • the mixture was centrifuged at 4 ° C, 14,000 rpm for 5 minutes, and the supernatant was discarded to obtain a DNA pellet.
  • the obtained DNA pellet was dried and then dissolved in DEPC-treated sterile distilled water 121 to obtain a cDNA solution.
  • PCR is performed under the conditions of first repeating a heat insulation cycle consisting of 94 ° C for 30 seconds, then 65 ° C for 30 seconds, and then 72 ° C for 1.5 minutes, and finally warming at 72 ° C for 5 minutes. It was broken. After PCR, a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was subcloned into pT7-Blue vector (Novagen), and E.coli JM109 strain competent cell (Toyobo) was transformed with the plasmid. It is obtained by culturing transformed cells in lOOmL of LB medium containing 50 ⁇ g / mL ampicillin. The plasmid containing the base sequence of the mouse-derived gene (mGPR92) was obtained by separating and purifying from the cultured cells using QIAGEN Plasmid Maxi kit (QIAGEN).
  • the primer prepared from the cDNA prepared in Example 3 in the form of a cage the primer consisting of the nucleotide sequence represented by SEQ ID NO: 11 3 20 pmol, the primer capable of the nucleotide sequence represented by SEQ ID NO: 12 4 20 pmol, Pfe Platinum Polymerase (Invitrogen) 2.5 U, 50 ⁇ L of a reaction solution containing 5 ⁇ L of buffer attached to Pfe Platinum polymerase and 7.5 ⁇ L of dNTP mixture (2 mM) was prepared. PCR is performed at 94 ° C for 1 minute, followed by 30 heat insulation cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 1 minute 20 seconds. It was conducted.
  • PCR product showing about 1.2 kbp was recovered by agarose electrophoresis.
  • the recovered PCR product was inserted into pDONR221 (Invitrogen) using the BP reaction (Invitrogen) of GATEWAY system, and E.coli DH5 strain competent cell (Toyobo) was transformed with the plasmid. Culturing transformed cells in a kanamycin-containing medium cultivated bacterial strength by purifying the plasmid, the entry vector containing the nucleotide sequence of the ORF part of this mouse-derived gene (m GPR92) pENTR / mGPR92 Got / 93.
  • pENTR / mGPR92 / 93 was inserted into the destination vector pCAGGS-DES T (described later) using the LR reaction (Invitrogen) of the GATEWAY system, and then E.coli DH5 A strain, a competent cell (Toyobo Co., Ltd.) was transformed. Obtained by culturing colonies obtained by transformation in a medium containing ampicillin. The plasmid for introduction into animal cells was prepared by separating and purifying from the cultured cells using QIAGEN Plasmid Maxi kit (QIAGEN).
  • the destination vector pCAGGS-DEST is a mammalian cell expression vector p
  • GATEWAY reading frame B is inserted in the same direction as the promoter.
  • the gene and the introduction reagent were mixed and prepared as follows. That is, mix the GPR92 / 93 incorporation vector prepared in Example 6 0.05 ⁇ g / Opti-MEM medium 5 ⁇ 1 and Lipofectamine 0.3 ⁇ 1 / Opti-MEM medium 5 ⁇ 1 at the well of a 96-well plate at room temperature. And then allowed to stand for 30 minutes, F-12 medium 40 1 was added to obtain a gene introduction solution.
  • the medium of CHO-K1 cells which were cultured in 96-well plate the day before at 2 x 10 4 cells / 100 ⁇ 1 / well (medium: 10% FBSZF-12) and cultured at 5% CO, 37 ° C for 22-24 hours Aspirate and remove F
  • the cells were cultured at 37 ° C for 3.5 hours. Aspirate the medium, wash once with 10% FBS ZF-12 medium, add 100 1 / well of 10% FBSZF-12 medium, and add 5% CO.
  • the cells were further cultured at 37 ° C for 24 hours.
  • the culture supernatant of the cells transiently transfected with the method (1) above was removed and washed with PBS (—).
  • the seal was peeled off, and the intensity of the long-lived fluorescence of 620 nm generated by XL665 that received energy transfer from the excited cryptate and the long-lived fluorescence of 665 nm generated by the energy transfer from the excited cryptate were measured with a measuring instrument ARVO.
  • Table 1 shows the amount of cAMP for which the measured force was also calculated. As shown in Table 1, cAMP production was increased in cells in which human and mouse GPR92 / 93 were transiently introduced, and GPR92 / 93 was estimated to be a GPCR co-operating with Gs.
  • the GPCR92 / 93 gene was transiently introduced into the cells by the method shown in Example 7 (1).
  • Example 7 (2) When quantifying the amount of cAMP by the method in Example 7 (2), after washing with PBS (—) at the beginning, various concentrations of LPA SniM IBMX / lmg / ml BSA / PBS (—) 50 ⁇ I / well The mixture was added and allowed to stand at room temperature for 30 minutes. Thereafter, the amount of cAMP was quantified by the method (2).
  • GPR92 / 93 ligands (agonist and inverse agonist) can be evaluated by using a test substance instead of LPA.
  • ICR mice (CLEA Japan, SPF, o 71) from the spleen islets were isolated by collagenase digestion method. The procedure is shown below. ICR mice bred under free-feeding conditions are opened under Nembutal anesthesia, and the lower abdominal vena cava is cut to cause blood loss. Place the duodenum so that the common bile duct is exposed, and ligate the common bile duct just before the duodenum. Insert a force fluid from the liver side of the common bile duct, inject 3 ml of 0.02% collagenase solution, and then remove the spleen. After digestion at 37 ° C for 20 minutes in a 5 ml collagenase solution, vigorously penetrate to disrupt the spleen.
  • La Islet was transferred into KRB (containing 0.5% BSA), sized, and incubated in a thermostat set at 37 ° C for 30 minutes. Pre-heat KRB with LPA added to each condition in a 37 ° C constant temperature bath. After completion of the incubation, 5 islands of La Islet were added in a Z tube, incubated for exactly 30 minutes and then transferred to ice to stop the secretion reaction. The supernatant of each tube was collected, and the insulin concentration was measured using a Levis insulin kit mouse (Shibagoat). The results are shown in Table 4.
  • DMSO is a solvent-only negative control
  • GLP-1 is a peptide compound known to have an insulin secretion-promoting action, that is, a positive control.
  • LPA showed insulin secretion promoting activity in a dose-dependent manner.
  • LPA Insulin secretion promoting activity at 10 ⁇ was almost the same as GLP-1 (100 ⁇ ).
  • Example 2 GPR92 / 93 expression strain prepared in (1) is seeded in a flask, and 10% FBS (manufactured by Gibco) is cultured in the medium until 60 to 70% confluent. Cells are collected and buffer A (50 mM
  • HEPES HEPES
  • 10 mM 2-ME, ImM PMSF 0.25 M sucrose
  • Homogenize 400 rpm, 20 strokes
  • centrifuge 100000 g for 60 minutes
  • resuspend the resulting precipitate in buffer A again.
  • This suspension is layered on 35% (mass / vol) sucrose in buffer A and centrifuged at 45000 g for 45 minutes. Collect the interface fraction, suspend in buffer A, and centrifuge at 100000g for 60 minutes.
  • the resulting precipitate is suspended in buffer A containing 20 ⁇ g / ml aprotinin and used in the following assembly.
  • GPR92 / 93-expressing cell membrane fraction (eg, 1-10 ⁇ g) prepared in the above procedure is dissolved in 10 mM acetate buffer (pH 4) and fixed to the matrix on the surface of Biacore sensor chip CM5 via carboxyl groups. Turn into.
  • GPR92 / 93 ( ⁇ _020400; SEQ ID NO: 13) was amplified by PCR using the following primers.
  • AtGPR92M GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCatgttagccaacagc tcctcaac (SEQ ID NO: 15)
  • AtGPR92R GGGGACCACTTTGTACAAGAAAGCTGGGTCAtcagagggcggaatcctggg gacac (Eye number 16)
  • the fragment obtained was subjected to the BP reaction between PDONR201 and the GATEWAY system.
  • PENTR / GPR92 / 93 was obtained.
  • the nucleotide sequence of the obtained clone was confirmed.
  • a GPR92 / 93 expression vector was constructed from pENTR / GPR92 / 93 by a method commonly used by those skilled in the art.
  • the clones thus obtained were expanded and cultured, and plasmid DNA for introduction was purified using the Qiagen MAXI kit.
  • CHO-K1 cells were seeded in 96-well plates at 2xl0 4 cells / well and cultured in a C02 incubator for 24 hours using a medium not containing antibiotics.
  • 50 ng of GPR92 / 93 expression plasmid DNA (GP R92 / 93 / pCAGGS) was diluted in 51 OPTI-MEM, and 0.31 LipofectAmine was diluted in 51 OPT-MEM. Both were mixed and allowed to stand at room temperature for 30 minutes, and then 40 1 serum-free F12 medium was added and added to a well washed once with serum-free F12 medium.
  • the medium was replaced with F12 medium supplemented with 10% FBS (washed once), and cultured in a C02 incubator for 24 hours.
  • the cells were washed with PBS ( ⁇ ) and replaced with Hanks Hepes solution containing 50 ⁇ l of 0.5 mM IBMX containing the porcine organ activity fraction. After 15 minutes, Hanks Hepes solution was aspirated and 40 ⁇ l of Lysis buffer (1% Triton X100, 50 mM phosphate buffer (pH 7.0), 0.2% BSA) was added to lyse the cells. Of the cell lysates, 101 was used for cAMP quantitative assay.
  • a column (900 ml capacity) packed with 300 g of Oasis HLB resin from Waters was preliminarily washed with 1 L of methanol, and then equilibrated with 2 L or more of water. The centrifugation supernatant was passed through an equilibrated Oasis HL B resin column and adsorbed. Wash the column with 5 L of water (C1 and After washing with 5 L of 5% aqueous methanol (labeled C2), the active ingredient was eluted by passing 4 L of 90% acetonitrile- 10% methanol (labeled C3).
  • the eluate C3 was purified by reverse phase liquid chromatography. 4 L of eluate C3 was concentrated under reduced pressure using an evaporator, and the concentrate was dissolved in a small amount of methanol. The concentrated solution was passed through a ZORBAX Eclipse XDB-C18 column manufactured by Agilent, and the active ingredient was eluted by increasing the acetonitrile concentration in a 0.1% trichloroacetic acid / acetononitrile solvent system. The gradient conditions were implemented according to the following conditions.
  • Each fraction 50 1 was concentrated under reduced pressure using a speed-back concentrator, and then dissolved in 11 DMSO, and the activity was measured by the assay method using GPR92 / 93-expressing CHO-K1 cells. Activity was eluted in fraction 64 and fraction 65. The amount of cA MP produced was 1.9 nM and 3.1 nM, respectively.
  • Each fraction 101 was concentrated under reduced pressure using a speed-back concentrator, and then the activity was measured by the Atsy method (see Example 13) using GPR92 / 93-expressing CHO-K1 cells dissolved in 1 ⁇ 1 DMSO. . Activity was eluted in fraction 16. The amount of cAMP produced was 32 nM.
  • Fraction 16 (700 1) was concentrated under reduced pressure using a speed-back concentrator, dissolved in 200 1 methanol, passed through an Atlantis column manufactured by Waters, and the acetonitrile concentration was adjusted with a 0.1% trichloroacetic acid / acetonitrile solvent system. The active ingredient was eluted by boosting [].
  • the gradient conditions were as follows.
  • Each fraction 201 was concentrated under reduced pressure using a speed-back concentrator, and then dissolved in 11 DMSO, and the activity was measured by the assay method using GPR92 / 93-expressing CHO-K1 cells. Activity was eluted in fractions 41 and 47. The respective cA MP production was 4.3 nM and 5.1 nM.
  • a Micomass QTOF2 mass spectrometer equipped with a Nanospray ion source and a Thermo Electoron ion trap mass spectrometer LCQ were used for the analysis.
  • the sample was dissolved in 0.1% AcOH-H20 / MeOH / AcCN (l: 1: 1), and Nanospray measurement was performed at a flow rate of 200 nL / min.
  • the capillary temperature was set to 150 ° C and the ionization voltage was set to 1.8 kV, and measurements were performed in both positive and negative modes. In QTOF2, only the positive ion mode was measured.
  • the activity of the structurally similar compounds of the active compounds was measured by the Atsy method (see Example 13) using GPR92 / 93-expressing CHO-K1 cells. The activity measurement results are shown in Table 1.
  • the GPR92 / 93 agonist of the present invention exhibits insulin secretion promoting activity, is useful as a glucose tolerance ameliorating agent and a diabetes therapeutic agent, and can also be used as a research reagent.
  • the screening method of the present invention makes it possible to search for GPR92 / 93 agonists and antagonists that can be drug candidates.
  • SEQ ID NO: 5 Antigenic peptide
  • SEQ ID NO: 6 antigenic peptide
  • SEQ ID NO: 7 PCR primer
  • SEQ ID NO: 8 PCR primer SEQ ID NO: 11: PCR primer SEQ ID NO: 12: PCR primer SEQ ID NO: 15: PCR primer SEQ ID NO: 16: PCR primer

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Diabetes (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Endocrinology (AREA)
  • Molecular Biology (AREA)
  • Obesity (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Emergency Medicine (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un médicament en vue d’améliorer une tolérance anormale au glucose ou un médicament antidiabétique comprenant un agoniste de GPR92/93 en tant qu’ingrédient actif ; un procédé de sélection d’un médicament en vue d’améliorer une tolérance anormale au glucose ou d’un médicament antidiabétique suivant le fait qu’une substance test soit un ligand de GPR92/93 en tant qu’indication ; et ainsi de suite.
PCT/JP2005/011801 2004-06-30 2005-06-28 Ligand de récepteur WO2006003877A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006528689A JPWO2006003877A1 (ja) 2004-06-30 2005-06-28 受容体リガンド

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2004192733 2004-06-30
JP2004-192733 2004-06-30
JP2004-271320 2004-09-17
JP2004271320 2004-09-17

Publications (1)

Publication Number Publication Date
WO2006003877A1 true WO2006003877A1 (fr) 2006-01-12

Family

ID=35782685

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/011801 WO2006003877A1 (fr) 2004-06-30 2005-06-28 Ligand de récepteur

Country Status (2)

Country Link
JP (1) JPWO2006003877A1 (fr)
WO (1) WO2006003877A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006090289A3 (fr) * 2005-02-28 2006-12-07 Alphabeta Ab Composes destines a reduire l'agregation de beta-peptide amyloide
WO2012086406A1 (fr) * 2010-12-22 2012-06-28 花王株式会社 Sécrétagogue de glp-1
JP2019520789A (ja) * 2016-04-14 2019-07-25 マース インコーポレーテッドＭａｒｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Ｇｐｒ９２の調節因子の同定方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092104A1 (fr) * 2001-04-25 2002-11-21 Biosynergen, Inc. Composition pharmaceutique a base d'acide lysophosphatidique
WO2003021262A1 (fr) * 2001-08-28 2003-03-13 Takeda Chemical Industries, Ltd. Procede de criblage
WO2003082335A1 (fr) * 2002-04-03 2003-10-09 Sumitomo Pharmaceuticals Co., Ltd. Nouveau dispositif de controle du sucre dans le sang et procede de criblage associe
JP2004517604A (ja) * 2000-05-18 2004-06-17 インサイト・ゲノミックス・インコーポレイテッド Ｇタンパク質結合受容体
WO2005009469A1 (fr) * 2003-07-28 2005-02-03 Sumitomo Pharmaceuticals Co., Ltd. Nouveau medicament de regulation de la glycemie et procede de criblage associe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004517604A (ja) * 2000-05-18 2004-06-17 インサイト・ゲノミックス・インコーポレイテッド Ｇタンパク質結合受容体
WO2002092104A1 (fr) * 2001-04-25 2002-11-21 Biosynergen, Inc. Composition pharmaceutique a base d'acide lysophosphatidique
WO2003021262A1 (fr) * 2001-08-28 2003-03-13 Takeda Chemical Industries, Ltd. Procede de criblage
WO2003082335A1 (fr) * 2002-04-03 2003-10-09 Sumitomo Pharmaceuticals Co., Ltd. Nouveau dispositif de controle du sucre dans le sang et procede de criblage associe
WO2005009469A1 (fr) * 2003-07-28 2005-02-03 Sumitomo Pharmaceuticals Co., Ltd. Nouveau medicament de regulation de la glycemie et procede de criblage associe

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LEE DK ET AL: "Discovery and mapping of ten novel G protein-coupled receptor genes.", GENE., vol. 275, 2001, pages 83 - 91, XP004307114 *
TAKUWA Y ET AL: "The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities.", J BIOCHEM., vol. 131, no. 6, 2002, pages 767 - 771, XP009028822 *
VASSILATIS DK ET AL: "The G protein-coupled receptor repertoires of human and mouse.", PROC NATL ACAD SCI USA., vol. 100, no. 8, 2003, pages 4903 - 4908, XP002992972 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006090289A3 (fr) * 2005-02-28 2006-12-07 Alphabeta Ab Composes destines a reduire l'agregation de beta-peptide amyloide
AU2006217544B2 (en) * 2005-02-28 2012-03-22 Alphabeta Ab Compounds for reducing aggregation of amyloid beta-peptide
AU2006217544B8 (en) * 2005-02-28 2012-04-05 Alphabeta Ab Compounds for reducing aggregation of amyloid beta-peptide
WO2012086406A1 (fr) * 2010-12-22 2012-06-28 花王株式会社 Sécrétagogue de glp-1
JP2012144518A (ja) * 2010-12-22 2012-08-02 Kao Corp Ｇｌｐ−１分泌促進剤
JP2019520789A (ja) * 2016-04-14 2019-07-25 マース インコーポレーテッドＭａｒｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Ｇｐｒ９２の調節因子の同定方法
US11237177B2 (en) 2016-04-14 2022-02-01 Mars, Incorporated Methods for identifying modulators of GPR92
JP7039483B2 (ja) 2016-04-14 2022-03-22 マース インコーポレーテッド Ｇｐｒ９２の調節因子の同定方法

Also Published As

Publication number Publication date
JPWO2006003877A1 (ja) 2008-04-17

Similar Documents

Publication Publication Date Title
Fernández-Salas et al. p53 and tumor necrosis factor α regulate the expression of a mitochondrial chloride channel protein
Gruber et al. Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland
Dell'Angelica et al. GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex
Denti et al. RanBPM is a phosphoprotein that associates with the plasma membrane and interacts with the integrin LFA-1
Martinez-Garay et al. The novel centrosomal associated protein CEP55 is present in the spindle midzone and the midbody
US20040077535A1 (en) Novel physiologically active peptide and use thereof
JP5554925B2 (ja) ヒト非小細胞肺癌における転座及び変異体ｒｏｓキナーゼ
US20070202520A1 (en) Novel lipase
JP2004113151A (ja) 癌遺伝子及びその用途
KR100553300B1 (ko) 혈관신생 및 심혈관형성의 촉진 또는 억제 방법
JP3497133B2 (ja) Ａ−３３関連抗原およびそれらの薬理学的使用
Johnson et al. Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs
US7396920B2 (en) Tumour suppressor and uses thereof
Ruder et al. EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin
McKenzie et al. Casein kinase Iε associates with and phosphorylates the tight junction protein occludin
WO2011156252A2 (fr) Sulfatation de protéines de voie wnt
Perez-Villar et al. Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin α (Rch1α)
WO2006003877A1 (fr) Ligand de récepteur
Wang et al. Characterization of Ceap-11 and Ceap-16, Two Novel Splicing–Variant–Proteins, Associated with Centrosome, Microtubule Aggregation and Cell Proliferation
KR20100076992A (ko) Ｔｒｐｃ 도메인과 ｓｅｓｔｄ1 도메인의 복합체 및 이를 포함하는 방법 및 용도
WO2003066860A1 (fr) Inhibiteurs d'angiogenese
AU2003231196C1 (en) Androgen-regulated PMEPA1 and cancer
EP1641922B1 (fr) Choline-phosphotransferases et ethanolamine-phosphotransferases nouvellement identifiees
Yuchi et al. Structural insights into disease mutations of the ryanodine receptor
US20080241129A1 (en) Adipocyte secreted protein ccdc80 is a potent stimulator of bone formation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006528689

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase