WO2006003877A1 - Receptor ligand - Google Patents

Abstract

It is intended to provide a drug for improving abnormal glucose tolerance or an antidiabetic drug comprising a GPR92/93 agonist as the active ingredient; a method of screening a drug for improving abnormal glucose tolerance or an antidiabetic drug depending on the fact that a test substance is a GPR92/93 ligand as an indication; and so on.

Description

 Specification

 Receptor ligand

 Technical field

 [0001] 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.

<o

 Background art

 [0002] Diabetes is classified into type 1 and type 2 based on its pathology. That is, type 1 is a pathological condition based on insulin secretion dysfunction in the spleen, and type 2 is a pathological condition mainly due to insulin resistance in insulin-sensitive tissues and abnormal insulin secretion in the spleen. In recent years, with the westernization of eating habits and increased social stress, obesity and associated lifestyle diseases, especially type 2 diabetes patients, have increased.

 [0003] 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. β 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. Furthermore, in the liver, gluconeogenesis is suppressed in response to insulin, and blood glucose is regulated. Diabetes is thought to develop as a result of this cycle failure. In fact, 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.

[0004] From the above viewpoint, it is considered that promotion of insulin secretion in the spleen greatly contributes to the effect of improving the diabetic condition. As drugs with such a mechanism of action, sulfo-lurea drugs are currently being promoted, but since there is no insulin secretion regulating action according to blood sugar level, even if blood sugar falls Insulin secretion promoting effect continues and low blood There is a risk of falling into sugar, coma and even death. Currently, there are no safe drugs that have the characteristics that insulin secretion is promoted according to the blood glucose level, and the development of such drugs is desired.

 [0005] By the way, 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.

 On the other hand, lysophosphatidic acid (LPA) 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. Examples of 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). However, 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)

 Disclosure of the invention

 Problems to be solved by the invention

[0006] The problem to be solved by the present invention is as follows: G protein-coupled receptor (GPCR): 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. Means for solving the problem

[0007] 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.

 [0008] Next, the present inventors searched for 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. Furthermore, it was found that 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.

 [0009] That is, the present invention relates to pharmaceutical uses and the like of GPR92 / 93 agonists:

 [1] Insulin secretion promoter containing GPR92 / 93 agonist as active ingredient;

[2] The insulin secretagogue according to [1], wherein the GPR92 / 93 agonist is lysophosphatidic acid, a derivative thereof, L-NASPA or a pharmaceutically acceptable salt thereof;

 [3] Lysophosphatidic acid or its derivative power Formula (1):

 [0010] [Chemical 1]

[In the formula, 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 insulin secretagogue according to [2], which is a compound represented by

[4] Insulin secretion-promoting agent according to [3], which is a compound strength represented by formula (1) lysophosphatidic acid, 1-Acy 卜 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]:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a hydrogen atom;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group;

6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, R ¹ and R ³ represent an otatanyl group;

 [6] The insulin secretagogue according to any one of [1] to [5], which is a blood glucose regulator;

 [7] The insulin secretion promoter according to any one of [1] to [5], which is a drug for improving glucose tolerance abnormality;

 [8] The insulin secretion promoter according to any one of [1] to [5], which is a drug for lifestyle-related diseases;

 [9] The insulin secretion promoter according to any one of [1] to [5], which is a therapeutic agent for diabetes;

 [10] GPR92 / 93 agonist containing lysophosphatidic acid, a derivative thereof, L-NASPA or a pharmaceutically acceptable salt thereof as an active ingredient;

[11] Lysophosphatidic acid or its derivative power Formula (1):

[Chemical 2]

[In the formula, 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

 [12] The compound strength represented by the formula (1): an insulin secretagogue according to [11], which is lysophosphatidic acid, 1-Acy 卜 PAF, or a pharmaceutically acceptable salt thereof;

[13] In formula (1),

The GPR92 / 93 agonist according to [11], wherein R ² and R ³ represent the following combinations:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a hydrogen atom;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group;

6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, R ¹ and R ³ represent an otatanyl group;

 [14] 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;

[15] The following steps (a) to (d):

(a) Contact the test substance with GPR92 / 93 or a fragment to which the ligand can bind Process,

 (b) measuring the binding activity of the test substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind;

 (c) comparing the measured value of (b) with the measured value when a reference substance is used as a test substance; and

 (d) a step of selecting, as a GPR92 / 93 ligand, a test substance whose measured value of (b) is equal to or greater than the measured value of (c) based on the result of (c),

Including the screening method according to [14];

[16] The following steps (a) to (c):

 (a) contacting a reference substance and a fragment to which GPR92 / 93 or a ligand can bind in the presence and absence of a test substance;

 (b) Measure the binding activity of the reference substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind, and compare the measured value in the presence of the test substance with the measured value in the absence of the test substance. Process, and

 (c) selecting as a GPR92 / 93 ligand a test substance in which the measured value of (b) in the presence of the test substance is larger than the measured value of (b) in the absence of the test substance, [14] the screening method according to [14];

 [17] In a reaction system comprising a lipid bilayer containing GPR92 / 93 and an α subunit of a G protein that can be conjugated to GPR92 / 93, the test substance and sputum or lysophosphatidic acid, its derivatives and their derivatives Contacting a reference substance selected from pharmaceutically acceptable salts, and selecting a compound that is higher in GDP 'GTP exchange reaction of the subunit or cell stimulating activity of the G protein than the reference substance. GPR92 / 93 ligand screening method;

 [18] The reaction system is

(0) Host eukaryotic cells transfected with an expression vector containing DNA encoding GPR92 / 93, GO GPR92 / 93 C-terminal polymorphic G protein α-subunit fused to GPR92 / 93 Host eukaryotic cells transfected with an expression vector containing DNA encoding the peptide, Gii) Traited with an expression vector containing DNA encoding GPR92 / 93 A host animal cell that endogenously expresses a G protein that can be coupled to GPR92 / 93, or Gv) an animal cell that endogenously expresses a G protein that can be coupled to GPR92 / 93 and GPR92 / 93, The screening method according to [17], which is a homogenate of these cells or a membrane fraction derived from those cells;

[19] The following steps (a) to (d):

 (a) contacting a test substance with a reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;

 (b) measuring the GDP · GTP exchange reaction of the subunits in (a) or the cell stimulating activity of the G protein;

 (c) A step of comparing the measured value of (b) with the measured value when a reference substance selected from lysophosphatidic acid, its derivatives and pharmaceutically acceptable salts thereof is used as a test substance. ,as well as

 (d) a step of selecting, as a GPR92 / 93 agonist, a test substance whose measured value of (b) is equal to or greater than the measured value of (c) based on the result of (c),

Including the screening method according to [17] or [18];

[20] The following steps (a) to (c):

 (a) a reference substance selected from lysophosphatidic acid, its derivatives and pharmaceutically acceptable salts thereof, and a lipid bilayer containing GPR92 / 93 and GPR92 / in the presence and absence of the test substance Contacting a reaction system comprising an α subunit of a G protein that can be conjugated to 93,

 (b) measuring the GDP-GTP exchange reaction of the subunit in (a) or the cell stimulating activity of the G protein, and comparing the measured value in the presence of the test substance with the measured value in the absence of the test substance; as well as

 (c) the measurement value of (b) in the presence of the test substance is smaller than the measurement value of (b) in the absence of the test substance! /, the step of selecting the test substance as a GPR92 / 93 antagonist The screening method according to [17] or [18], comprising:

[21] Lysophosphatidic acid or its derivative power Formula (1):

[Chemical 3]

[In the formula, 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 screening method according to any one of [17] to [20], which is a compound represented by:

 [22] The compound power represented by the formula (1): the screening method according to [21], which is lysophosphatidic acid, 1-Acy 卜 PAF, or a pharmaceutically acceptable salt thereof;

[23] In formula (1),

R ² and R ³ represent the following combinations:

[21] the insulin secretagogue according to:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a hydrogen atom;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group;

6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, R ¹ and R ³ represent an otatanyl group;

 [24] A screening method for an insulin secretagogue, comprising selecting a compound with an indicator that the test substance is a GPR92 / 93 agonist;

[25] Contact the test substance with GPR92 / 93 or a fragment to which the ligand can bind. The method according to [24], wherein a compound that binds to the GPR92 / 93 or the fragment is selected;

 [26] In a reaction system comprising a lipid bilayer containing GPR92 / 93 and an α subunit of G protein that can be conjugated to GPR92 / 93, the GDP / GTP exchange reaction of the subunit or the G protein The method according to [24], comprising a step of comparing cell stimulating activity in the presence and absence of a test substance;

[27] The reaction system is

 (0) Host eukaryotic cells transfected with an expression vector containing DNA encoding GPR92 / 93, GO GPR92 / 93 C-terminal polymorphic G protein α-subunit fused to GPR92 / 93 Host eukaryotic cells transfected with an expression vector containing DNA encoding the peptide, Gii) G protein endogenously coupled with GPR92 / 93, transfected with an expression vector containing DNA encoding GPR92 / 93 Gv) GPR92 / 93 and animal cells that endogenously express G protein that can be conjugated to GPR92 / 93, homogenates of those cells, or membrane fractions derived from those cells A screening method according to [26],

[28] The following steps (a) to (d):

 (a) contacting a test substance with a reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;

 (b) a step of measuring the GDP · GTP exchange reaction of the subunit in (a) or the cell stimulating activity of the G protein; and

 (c) selecting a test substance having cell stimulating activity as a GPR92 / 93 agonist based on the result of (b),

Including the screening method according to [26] or [27];

 [29] The method according to [24], comprising evaluating whether the test substance is a GPR92 / 93 agonist by the method according to any one of [14] to [19];

 [30] The screening method according to any one of [24] to [29], wherein the insulin secretagogue is a blood glucose regulator;

[31] Any of [24] to [29], wherein the insulin secretagogue is a drug for improving glucose tolerance The screening method according to 1;

 [32] The screening method according to any one of [24] to [29], wherein the insulin secretagogue is a therapeutic agent for diabetes;

 [33] 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;

 [36] The insulin secretagogue according to [33], which is a drug for lifestyle-related diseases;

 [37] The insulin secretagogue according to [33], which is a therapeutic agent for diabetes;

 [38] Fat accumulation inhibitor containing a GPR92 / 93 antagonist as an active ingredient;

 [39] The fat accumulation-suppressing drug according to [38], which is an anti-obesity drug;

[40] 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;

 [41] 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 method according to [40], wherein the compound that inhibits the binding of the reference substance and the GPR92 / 93 or the fragment is selected; and [42] in the presence or absence of the test substance, Criteria selected from lysophosphatidic acid, its derivatives and their pharmaceutically acceptable salts in a reaction system comprising a lipid bilayer containing GPR92 / 93 and an α subunit of G protein that can be conjugated to GPR 92/93 43. A method according to [40], wherein the compound is contacted and a compound that inhibits the GDP′GTP exchange reaction of the subunit of the reference substance or the cell stimulating activity of the G protein is selected. ] The reaction system is The

(0) Host eukaryotic cells transfected with an expression vector containing DNA encoding GPR92 / 93, GO GPR92 / 93 C-terminal polymorphic G protein α-subunit fused to GPR92 / 93 Transform with an expression vector containing DNA encoding the peptide Host eukaryotic cells, Gii) host animal cells endogenously expressing a G protein that can be coupled to GPR92 / 93, transformed with an expression vector containing DNA encoding GPR92 / 93, or Gv) GPR92 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;

 [44] The screening method according to any one of [40] to [43], wherein the fat accumulation inhibitor is an anti-obesity agent;

 [45] Fat accumulation inhibitor obtained by the method according to any one of [40] to [43]; [46] Fat accumulation inhibitor according to [45], which is an anti-obesity agent [47] A method for producing a biologically-derived fatty acid derivative comprising the following steps:

(a) contacting the solid-phase extraction resin with the biological sample to adsorb the biological fatty acid derivative and the low molecular component; and

 (b) A step of eluting the fatty acid derivative-containing fraction and low-molecular components from an aqueous solution containing 10% to 95% of a hydrophilic organic solvent.

 Further features of the present invention and advantages of the present invention are described in more detail below. The invention's effect

 [0013] 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. In addition, the 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.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0014] In the present specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC—IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

[0015] In this specification, "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.

 Here, “retaining 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.

In addition, 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. As a technique for artificially performing the “deletion, addition or substitution” of amino acids in the above (b) (hereinafter sometimes referred to as amino acid modification), for example, the amino acid represented by SEQ ID NO: 2 For example, a conventional site-directed mutagenesis is performed on the DNA encoding the sequence, and then this DNA is expressed by a conventional method. Here, as 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.

 [0017] "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. Here, 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. Such 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. Note that 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 For example, it can be generally used at the homepage address http://www.ddbj.nig.ac.jp The sequence identity in the present invention may be 80% or more. Preferably it is 95% or more, and still more preferably 97%.

[0018] Regarding the “stringent conditions” in (c) above, the high-prisition dies used here are, for example, by Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd Edition (Molecular Cloning 2nd Edition). edition), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory press), etc. “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. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6). 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.

 In the present specification, 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. .

[0019] "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. In addition to the coding region of human GPR92 / 93 cDNA, 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 Alternatively, 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. In addition, 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.

 Specific examples include 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.

[0020] 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. In addition, by expressing a fusion protein of 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)) . Alternatively, a mutation may be partially introduced by artificial treatment such as site-directed mutagenesis based on the GPR92 / 93 cDNA clone. However, since 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!

 [0021] Unless otherwise specified, "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.

 In the present specification, the “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.

 In the present specification, the term “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.

 [0023] (D GPR92 / 93 agonist

 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.

In the present specification, lysophosphatidic acid (hereinafter sometimes abbreviated as “LPA”) represents 1 asinole sn glyce mouth 1-nore-3-phosphate (1-Acy 卜 sn-glycero 卜 3-phosphate). Here, “acyl” represents a linear acyl group having 14 to 22 carbon atoms and having 0 to 6 double bonds. Specifically, equation (2): [0024] [Chemical 4]

 LPA (18: 1) represented by the formula (3):

[0025] [Chemical 5] H

 LPA (22: 6) represented by

[0026] In the present specification, lysophosphatidic acid derivative (LPA 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,

 (ii) a modification that converts the carbon atom at position 1 into a carbo group or a thio group;

(iii) modification to convert ester at the 1-position acyloxy group to amide or thioester,

 (iv) 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,

 (v) a modification in which an ester is converted to an amide or thiester in a compound in which the hydroxyl group at the 2-position is acylated;

 (vi) a modification that condenses the 3-position phosphate group with choline, phosphate or inositol, or

 (vii) Partial bond strength of ester bond at the l-position acyloxy group Modification to plasmalogen converted to a bull ether bond.

 [0027] Specifically as LPA or LPA derivative, the following formula (1):

[0028] [Chemical 6]

[In the formula, 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.

 Here, 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.

In addition, 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.

 As a compound represented by Formula (1), in addition to the above-mentioned LPA, Formula (5):

[Chemical 7]

1-O-palmitoyl 2—0-acetylyl sn glyceryl 3 phosphorylcholine (1-O-palmitoyl-2-O-acetyl-sn-glyceryl-j-phosphorylcnoline; 1-Acyl-PAF) ^ Formula (6 ):

[Chemical 8]

 1-lysophosphatidyl-sn-glyceguchi-3-phosphono- (1-D-myo-inositol) (hereinafter sometimes abbreviated as LPI (18: 0)), formula (7):

 [0031] [Chemical 9]

 Diacylglycerol pyrophosphate (may be referred to as DG PP (8: 0)), phosphatidic acid (PA), lysophosphatidylcholine (LPC), etc. Is mentioned. Here, as PA, 1,2-di (cis 9-octadecenoyl) sn-glycerol 3 phosphate sodium ¾_ (1,2-di (cis—9-octadecenoyl) -sn-glycerol 3-pnospnate sodium saltj In addition, LPC includes Lushi [Lushi brain L-α lysophosphatidylcholine] derived from commercial sales.

 In addition, 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).

 Further, in the compounds represented by the above formulas (5) to (7), compounds in which the 1-position, 2-position, 2-hydroxyl group, or 3-position phosphate group of the glycerol skeleton is modified are also LPA. Examples of modifications included in the derivatives include the above (i) to (vii).

 In addition, 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.

[0032] or the following equation (4):

[0033] [Chemical 10] ( ⁴ )

 Ν-Palmitoyl L-serine-ρhosphoric acid (L-NASPA) is also included in the GPR92 / 93 agonist of the present invention.

 The LPA, LPA derivative and L-NASPA of the present invention may form pharmaceutically acceptable salts. Specifically, 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.

[0034] Specific examples of 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. Here, the agonist activity against GPR92 / 93 represents an activity that causes GPCR function by binding to GPR92 / 93, which will be described later.

 [0035] As shown in the Examples of the present specification, 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.

[0036] 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.

 [0037] 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. Capsules, sachets or tablets contained as condyles, suspensions in which an effective amount of GPR92 / 93 agonist is suspended in an appropriate dispersion medium, and effective amounts of GPR92 / 93 agonist are dissolved. 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.

 [0038] Suitable formulations for parenteral administration (eg, intravenous injection, intraarterial injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile There are 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. Alternatively, a sustained-release preparation can be obtained using a biocompatible material such as collagen.

 [0039] The preparation of the GPR92 / 93 agonist can be enclosed in a container in unit doses or multiple doses like ampoules or vials. Alternatively, GPR92 / 93 agonists and pharmaceutically acceptable carriers can be lyophilized and dissolved or suspended in a suitable sterile vehicle just prior to use!

 [0040] 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.

[0041] (Π) Screening method The 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.

 That is, 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. For example, 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.

(1) Screening by measurement of binding activity

 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. That is, 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.

 Specifically, the following steps (a) to (d):

(a) contacting the test substance with GPR92 / 93 or a fragment to which a ligand can bind; (b) measuring the binding activity of the test substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind;

 (c) comparing the measured value of (b) with the measured value when a reference substance is used as a test substance; and

 (d) a step of selecting, as a GPR92 / 93 ligand, a test substance whose measured value of (b) is equal to or greater than the measured value of (c) based on the result of (c),

 A screening method including

 In this case, the binding activity between the test substance and GPR92 / 93, for example, 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.

 [0043] Further, the following steps (a) to (c):

 (a) contacting a reference substance and a fragment to which GPR92 / 93 or a ligand can bind in the presence and absence of a test substance;

 (b) Measure the binding activity of the reference substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind, and compare the measured value in the presence of the test substance with the measured value in the absence of the test substance. Process, and

 (c) selecting as a GPR92 / 93 ligand a test substance in which the measured value of (b) in the presence of the test substance is larger than the measured value of (b) in the absence of the test substance, Examples include screening methods.

 Reference materials such as LPA, L-NASPA, 1-Acyl-PAF, LPI, DGPP (8: 0), LPC, and LPC plasmalogen used here should be appropriately labeled with radioactive isotopes as described below. Labeled compounds are also included in the concept of LPA or LPA derivatives.

 The ability to be a GPR92 / 93 ligand and a GPR92 / 93 agonist selected by the above method and whether it is a GPR92 / 93 antagonist can be confirmed by a screening method using the G protein described below.

[0044] In the above-mentioned! /, Deviation mode, 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. In addition, as 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. Alternatively, a dartathione column can be used.

[0045] 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. Specifically, 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. For example, 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).

 In addition, 1-Acy 卜 PAF (Registry No .: 163005-42-3 compound) in which the hydrogen atom of the acetyl group is labeled with tritium, or 1-Acyl-PAF (compound in which the hydrogen atom of the palmitoyl group is labeled with tritium ( Registry No.:112602-69-4), 1-Acy 卜 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].

 [0046] (2) Screening by measuring signal transduction

GPR92 / 93 couples with certain trimeric G proteins and transmits signals into cells. Therefore, 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. From L-NASPA and their pharmaceutically acceptable salts Screening for a GP R92 / 93 ligand, comprising contacting a selected reference substance, and selecting a compound whose GDP 'GTP exchange reaction of the subunit or cell stimulating activity of the G protein is higher than that of the reference substance Includes methods.

 Specifically, the following steps (a) to (d):

 (a) contacting a test substance with a reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;

 (b) measuring the GDP · GTP exchange reaction of the subunits in (a) or the cell stimulating activity of the G protein;

 (c) comparing the measured value of (b) with the measured value when a reference substance is used as a test substance; and

 (d) a step of selecting, as a GPR92 / 93 agonist, a test substance whose measured value of (b) is equal to or greater than the measured value of (c) based on the result of (c),

And a method for screening for GPR92 / 93 agonists.

 In addition, the following steps (a) to (c):

 (a) contacting a reference system, a lipid bilayer containing GPR92 / 93, and a reaction system comprising a G protein α-subunit capable of coupling to GPR92 / 93 in the presence and absence of a test substance ,

 (b) measuring the GDP-GTP exchange reaction of the subunit in (a) or the cell stimulating activity of the G protein, and comparing the measured value in the presence of the test substance with the measured value in the absence of the test substance; as well as

 (c) the measurement value of (b) in the presence of the test substance is smaller than the measurement value of (b) in the absence of the test substance! /, the step of selecting the test substance as a GPR92 / 93 antagonist And a screening method for GPR92 / 93 antagonists.

In the above, specifically, “reaction system comprising a lipid bilayer containing GPR92 / 93 and an α subunit of G protein that can be conjugated to GPR92 / 93” is specifically described as (DNA encoding 0 GPR92 / 93) A host eukaryotic cell transfected with an expression vector containing (ii) a DNA encoding a polypeptide in which the α subunit of G protein that can be conjugated to GPR92 / 93 is fused to the C-terminal side of GPR92 / 93 Host eukaryotic cells transfected with the expression vector Or (iii) a host animal cell that endogenously expresses a G protein that can be coupled to GPR 92/93, transfected with an expression vector containing DNA encoding GPR92 / 93, or (iv) GPR92 / 93 And animal cells that endogenously express G protein that can be coupled to GPR92 / 93, homogenates of these cells, or membrane fractions derived from these cells.

[0048] In general, screening for GPCR agonists is performed using GTP · GDP exchange reaction in Ga or cell stimulating activity of coupled G protein as an index. When the cell stimulation activity of G protein is used as an index, specific procedures such as effector selection and activity measurement method are determined depending on the mode of Ga to be conjugated. Usually, adenylate cyclase is used. In the case of using a 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), for example, by measuring the amount of cAMP, the phospholipase C | In the case of using Go including a region that interacts with 8 (specifically, including an effector interaction region of Ga belonging to the Gq family), for example, by measuring the amount of intracellular calcium ions, Agonists can be screened.

 As shown in the examples of the present invention, G o of GPR92 / 93 is considered to belong to the Gs family.

[0049] In addition, animal cells (eg, HEK293 cells, L1.2 cells, etc.) that endogenously express the trimeric G protein (G α β y) containing the Ga are used as a source of Ga. G α92 / 93 activated G α j8 γ can dissociate into G α and G j8 γ Free G β y can interact with phospholipase C | 8 to increase intracellular calcium ion concentration Therefore, ligand staring can be performed using intracellular calcium ions as an index regardless of the conjugated Ga family.

[0050] 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. In addition, 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)). Alternatively, a mutation may be partially introduced by artificial treatment such as site-specific mutagenesis based on the GPR92 / 93 cDNA clone. However, since 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.

 [0051] 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. In addition, 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.

[0052] 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. Specifically, since 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, and 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. On the other hand, 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.

 On the other hand, when Ga conjugated to GPCR belongs to the Gi family (Gi a) or Gq family (Gq α), the Ga to be used has at least the Gi α 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.

[0053] When a physiological ligand or agonist for GPR92 / 93 binds to the receptor, the 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. On the other hand, binding of inverse ghosts decreases the activated Ga — GTP level because the Ga active domain is inactivated by changes in receptor conformation. Here, instead of GTP, it is not hydrolyzed by GTPase activity of Ga such as 35S-labeled GTP y S! / ヽ If a GTP analog is added to the system, the presence or absence of the test substance is present. By measuring and comparing the radioactivity bound to the membrane below, it is possible to screen the GPR92 / 93 or the inverse antigen. That is, if the radioactivity increases in the presence of the test substance, the test substance is an agonist, and if the radioactivity decreases, it is an inverse ghost. For methods using 35S-labeled GTP y S (G ΤΡ [γ ³ ]), see Heise et al., Molecular Pharmacology, 60, 11 73-1180 (2001), or Im et al., Molecular Pharmacology, 57, 753. -759 (2000).

 In addition, 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.

[0054] Alternatively, screening can be performed by monitoring the binding of GTP analog to Ga using the surface plasmon resonance method or the like.

[0055] The aforementioned cell stimulating activity can also be measured using the effect of conjugated Ga on the effector as an index. In this case, 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 Gα 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 α, Gi α, G12 α and the like. G o that contains effector interaction regions of G o belonging to different families (eg Gq); (eg 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).

[0056] When 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. On the other hand, when the conjugated Ga contains the effector interaction region of Gqa, it is necessary to use a lipid bilayer membrane containing phospholipase Cβ as an effector.

 In addition, when the 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.

[0057] A screening system containing adenylate cyclase (hereinafter also referred to as AC) as an effector (that is, a chimera containing Ga or Ga or an effector interaction region of Gsa or Gia) For proteins (in the case of Chimera Gs a or Chia Gi a)! In the meantime, the effect of Ga on the effector can be evaluated by directly measuring AC activity. Any known technique can be used to measure AC activity.For example, 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 [α-32P] ATP to membrane fractions containing AC [32P] The method of measuring the radioactivity after separating cAMP with an alumina column or the like is mentioned, but is not limited thereto. If G a is Gs a, measure the AC activity in the presence and absence of the test substance and compare them.If the AC activity decreases in the presence of the test substance, the test substance is an inverse PR of GPR92 / 93, If activity increases, it is an agonist. 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. .

 On the other hand, when G a is 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. In addition, 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.

[0058] When intact eukaryotic cells are used as a screening system, 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

[0059] As another embodiment, there is also 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). The expression vectors used here will be described in detail later. In summary, 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.

[0060] Therefore, when G a is Gs a, if the intracellular cAMP level (or the expression level of the reporter gene under the control of CRE) decreases in the presence of the test substance, the test substance can be inverted to GPR92 / 93. -A strike, and if it increases, it is an agony. In addition, in a system where physiologically active ligands such as LPA and its derivatives coexist, 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.

 On the other hand, when Ga is Gi a, if intracellular cAMP (or the expression level of reporter gene under CRE control) increases in the presence of the test substance, the test substance is an inverse agonist of GPR92 / 93. Yes, if it decreases, it will be an asset. In addition, 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.

 [0061] On the other hand, a screening system containing phospholipase C β (hereinafter also referred to as PLC β! /) As an effector (ie, a chimeric protein containing G a as an effector interaction region of G q a ( In the case of the chimeric Gq a), the effect of the Gq a or the chimeric Gq a on the effector can be evaluated by directly measuring the PLC | 8 activity. 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. Can be evaluated. Measure PLC β activity in the presence and absence of the test substance '' and compare it.If the PLC β activity increases in the presence of the test substance, the test substance is an agonist of GPR 92/93. If it decreases, it is an inverse ghost. In addition, GPR92 / 93 antagonists can be screened by measuring and comparing PLC β 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.

[0062] When intact eukaryotic cells are used as a screening system, the action of 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.

[0063] In another embodiment, by measuring the expression level of a reporter gene under the control of a TPA (12 O-tetradecanol phorbol 13 acetate) response element (TRE) up-regulated by Ca ²⁺ There is also a method for evaluating the amount of Ca2 +. The expression vectors used here will be described in detail later. In summary, eukaryotic cells into which a vector containing an expression cassette linked to a DNA encoding a reporter protein is introduced downstream of a promoter containing TRE are treated as test substances. Intracellular Ca2 + levels are 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 presence and absence of cells and disrupting the cells using known methods. That's it.

[0064] Therefore, if the intracellular Ca ²⁺ level (or the expression level of the reporter gene under TRE control) increases in the presence of the test substance, the test substance is an agonist of GPR92 / 93, If it decreases, it is an inverse ghost. In addition, by measuring and comparing the amount of Ca ²⁺ (or the expression level of the reporter gene under TRE control) in the presence and absence of the test substance in a system coexisting with a bioactive ligand such as LPA Can screen GPR92 / 93 Antagost. That is, if the amount of Ca ²⁺ (or the expression level of a reporter gene under TRE control) decreases in the presence of the test substance than in the absence of the test substance, the test substance is an antagonist.

 [0065] If 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.

[0066] In another embodiment, GPR92 / 93 ligands can be screened using various physiological actions dependent on GPR92 / 93 as indices. Examples of the physiological action include activation of ERK, which is a kind of MAP kinase. Specifically, in the presence of GPR92 / 93, 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)). [0067] 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). In addition, a method for screening a GPR92 / 93 ligand by measuring GPR92 / 93 dependent DNA synthesis inhibitory activity (cell growth inhibitory activity) can be mentioned.

 Specifically, in the presence of GPR92 / 93, [3H] thymidine uptake or MTT ((3-4, 5-dimethylthazol- 2-yl) — 2, 5-diphenyltetrazolium bromide) (J. Biol. Chem., 276 (44), p41325—p41335 (2001)).

[0068] There is also a method for evaluating cell-stimulating activity of G protein by measuring the expression level of a rebolite gene under the control of a serum-response element (SRE) (An et al " J. Biol, Chem., 273,7906-7910, (1999); An et al "Mol. Pharmacol., 55, 787-794 (1999)). The expression vector used here will be described in detail later. In summary, eukaryotic cells into which a vector containing an expression cassette linked with a DNA encoding a reporter protein is introduced downstream of a promoter containing SRE are examined. Measure the expression of the reporter gene in the extract obtained by culturing for an appropriate period of time in the presence and absence of the substance and disrupting the cells using a known method. Is to evaluate. Therefore, if the amount of reporter gene under SRE control is increased, the test substance is a GPR92 / 93 antigen, and if it is decreased, it is an inverse agonist. In addition, by measuring and comparing 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.

[0069] 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. With 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.

 [0070] 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 Gα 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.

 [0071] In a screening system using the action of Ga on an effector as an index, 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. When adenylate cyclase is used as an effector, the DNA contains a nucleotide sequence encoding an effector interaction region of Gi or Gsa. On the other hand, when phospholipase Cβ is used as an effector, the DNA contains a nucleotide sequence encoding the effector interaction region of Gqa. Each Go gene is known and its effector interaction region is also well known. Therefore, those skilled in the art can easily construct a DNA encoding a chimeric Ga protein by appropriately combining known genetic engineering techniques. As the simplest example of 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.

[0072] 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. For example, SV40-derived early promoter, cytomegalovirus LTR, rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived Initial promoter, baculovirus-derived polyhedrin promoter, etc. And 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. Furthermore, it is desirable to have an appropriate restriction enzyme recognition site, preferably a unique restriction enzyme recognition site that cuts the vector at only one site. In addition, the expression vector is a selectable marker gene (tetracycline, ampicillin, kanamycin, no, idaguchimycin, phosphinothricin and other drug resistance genes, auxotrophic mutation complementary genes, etc.

) May be further contained.

 [0073] As 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.

[0074] 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.

 [0075] 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. Specifically, COP, L, C127, Sp2 / 0, NS-1, NIH3 T3, ST2 and other mouse-derived cells, rat-derived cells, BHK, CHO and other hamster-derived cells, COSl, COS3, COS7, CV1, 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.

 [0076] Gene transfer into a host cell may be performed using any known method that can be used for gene transfer in eukaryotic cells. For example, calcium phosphate coprecipitation method, electroversion method, ribosome method, Examples include a microinjection method.

[0077] 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.

 [0078] 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).

 [0079] For example, when the ligand characteristics of a test substance are evaluated by directly measuring GTP y S binding activity or effector activity, the screening system to be used is preferably a cell force as described above. It is a membrane fraction to be prepared. On the other hand, when assessing the ligand characteristics of a test substance by measuring intracellular cAMP level (or expression level of cAMP-responsive reporter) and intracellular Ca2 + level (or expression level of Ca2 + -responsive reporter). In addition, the screen system used is intact eukaryotic cells.

[0080] In addition, when the ligand activity is evaluated using the expression level of a cAMP-responsive reporter (when the effector is adenylate cyclase) or Ca2 + -responsive reporter (when the effector is phospholipase Cβ) as an index, 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. As long as it retains cAMP responsiveness, deletion, substitution, It may be an array containing insertions or attachments. On the other hand, 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. As 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. Can be inserted. As the reporter gene under the control of CRE or TRE, any known gene capable of detecting and quantifying gene expression quickly and easily can be used.For example, luciferase, β-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. As 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.

 [0081] 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 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. Host eukaryotic cells, homogenates of the cells or membrane fractions derived from the cells.

 [0082] 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 Gα.

[0083] 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. When 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.

 [0084] 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.

 [0085] For the receptor-expressing Ga fusion protein-expressing cells, any form of intact cells, cell homogenates, and membrane fractions can be appropriately selected and used depending on the screening method used.

 [0086] 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.

 As a method for introducing GPR92 / 93-encoding DNA, an expression vector into which the DNA is inserted, and the expression vector into a host cell, those described above can be used.

 [0087] In yet another embodiment of the present invention, 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.

[0088] In still another embodiment of the present invention, purified GPR 92/93 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. Alternatively, 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. Similarly, 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.

 [0089] Examples of 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.

[0090] For example, an artificial lipid bilayer membrane (proteoribosome) incorporating a receptor and Ga or a receptor-specific Ga fusion protein is a mixed lipid chromatophore of PC: PI: Ch = 12: 12: 1 Aliquot the appropriate amount of the solution into a glass tube, evaporate the black mouth form with nitrogen gas vapor, dry the lipid into a film, add an appropriate buffer to suspend, and then homogenize by sonication. Disperse and add a buffer containing a surfactant such as sodium cholate to completely suspend the lipid. Add the appropriate amount of purified receptor and G α or the receptor G α 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.

[0091] 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.

 In addition, 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.

(III) Process for producing biologically derived fatty acid derivative and low molecular weight compound

 Biologically-derived fatty acid derivatives are known to be difficult to produce by chemical synthesis because they contain many geometric isomers and optical isomers. On the other hand, as 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 There are known methods for removing proteins by heat denaturation and acid denaturation, etc., but it is difficult to process a large amount of biological sample! However, there are 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.

[0093] 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.

 That is, 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) contacting the solid-phase extraction resin with the biological sample to adsorb the biological fatty acid derivative and the low molecular component; and

 (b) a step of eluting biologically derived fatty acid derivative-containing fractions and low-molecular components with an aqueous solution containing 10% to 95% hydrophilic organic solvent;

 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.

Here, 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. Specifically, Oasis HLB resin (manufactured by Waters), etc. 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.

 [0094] 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. In addition, in the dalyceride, 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. As phosphoric acid (one OP (= 0) (OH)), diphosphoric acid (one OP (= 0) 0-P (= 0) (OH)), phosphati

 twenty two

 Examples include zylcholine, phosphatidylserine, or phosphatidylinositol.

 [0095] Examples of the hydrophilic organic solvent include methanol, acetonitrile, and mixed solvents thereof. Preferable examples include 10% to 95% methanol, 10% to 95% acetonitrile. Examples of the aqueous solution containing these hydrophilic organic solvents used as an eluent 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.

 Normally, 100 to 500 g of solid phase extraction resin is used per 1 kg of biological sample, and 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. Examples of the purification method include reverse phase HPLC, normal phase HPLC, silica gel column chromatography, ion exchange chromatography, and recrystallization. Preferably reverse Phase HPLC is mentioned.

 [0096] For example, for 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.

 [0097] The ability to describe the present invention more specifically with reference to the following examples. These are merely examples, and do not limit the scope of the present invention in any way.

 Example 1

 [0098] (Expression localization of GPR92 / 93 in normal human spleen Langernoens island)

 (1) Production of Usagi-derived polyclonal antibodies

As GPR92 / 93 antigen peptide, the amino acid sequence: AQSERSAVTTDATRPD (SEQ ID NO: 5) a partial peptide synthesized by a known method, was Make a sequence by mass spectrometry and HPLC ₀

 According to a known method, animals were immunized according to the following schedule, and antiserum-resistant polyclonal antibodies were prepared. In other words, two rabbits (KBL JW 11 weeks old) were used, and FCA (Complete Usagi (KBL JW 11 weeks)) was used on the first day of breeding, and dissolved in FCA (Compl ete Freuncfs Adjuvant) on the first day of breeding. 200 μg / animal of the antigen peptide was injected intraperitoneally, and the antigen peptide dissolved in FCA (Complete Freund's Adjuvant) on days 14, 28, 42, 57, 70, 84 The animals were injected intraperitoneally, and blood was collected for confirmation of antibody titer on the 49th and 63rd day of breeding. On day 91, blood was collected at 50 to 70 ml / animal, and purified with a column on which an antiserum was obtained and an antigen peptide was immobilized to obtain a primary antibody, which was purified with 0.01% azido sodium. It was replaced with PBS containing and stored frozen until use.

 (2) Immunohistochemical staining with Usagi antibody

Tissue sections were prepared at 4-5 μm 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 1½1 for 30 minutes. After washing, Vector ABC-AP (AK-5000) reagent was added, and color was developed with Vector Red (SK-5100).

 The results are shown in Table 1. As shown in Table 1, GPR92 / 93 was strongly expressed in the spleen compared to other tissues. Furthermore, when the expression distribution in the spleen tissue was examined in detail, it was found that the expression distribution was localized in the islets of Langeron, with little expression in the acinar.

 Furthermore, 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. .

[0099] [Table 1]

Example 2

[0100] (Preparation of cDNA)

Total RNA was extracted from mouse Islet tissue using RNeasy Mini Kit (QIAGEN). A mixture of 11 μ 1 containing 10 μg 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 μ1 included in the kit and the kit 10 mM dNTP Mix 1 μ1 contained in the solution was added, and the mixture was heated at 42 ° C. for 2 minutes. Further, 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 μ1, 5xSecond Strand Reaction Buffer 30 μ1, included in the kit, lOmM dNTP Mix 3 1, included in the kit, E. coli DNA Ligase 1 μ1 (10U), E. included in the kit. E. coli DNA Polymerase I 4 1 (40 U) and E. coli RNase H 1 μ 1 (2 U) contained in the kit were added, and the mixture was reacted at 16 ° C. for 2 hours. Next, add 2 μ1 (10 U) of T4 DNA Polymerase contained in the kit to the mixture, react this mixture at 16 ° C for 5 minutes, and add 0.5 M EDTA 10 1 to the mixture. Was added. Next, a phenol / black mouth / isoamyl alcohol solution (Nitsubon Gene Co., Ltd.) 162 μ1 was added to the mixture and mixed. The mixture was transferred to Phase Lock Gel Light (manufactured by Eppendorf) which had been previously centrifuged at 14,000 rpm for 30 seconds at room temperature, and centrifuged at 14,000 rpm for 2 minutes at room temperature. After centrifugation, 1451 aqueous layers were collected in an Eppendorf tube. The recovered aqueous layer was mixed with 7.5M ammonium acetate solution (72.5 μ1) and ethanol (362.5 μ1), 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.

Example 3

(Cloning of mouse-derived genes)

1 μL of the cDNA prepared in Example 2, a primer consisting of the base sequence shown by SEQ ID NO: 7, 1 20 pmol, a primer consisting of the base sequence shown by SEQ ID NO: 8, 20 pmol, TaKaRa Ex- Taq polymerase (Takara Shuzo) ) A 50 μL reaction solution containing 5 μL of buffer attached to 2U and TaKaRa Ex-Taq polymerase and dNTP mixture (2.5 mM) 4 _i uL attached to TaKaRa Ex-Taq polymerase was prepared. 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 μg / 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).

 Example 4

 [0102] (Determination of the nucleotide sequence of a mouse-derived gene)

 Using the plasmid containing the PCR product (about 1.2 kbp) obtained in Example 3 as a saddle, Thermo Sequence II dye terminator kit (Amersham Pharmacia Biotech) and ABI373D NA sequence reader (PE Applied Biosystems) In accordance with Sanger's method [F.Sanger'S.Nicklen'AR and oulson, Proceedings of National Academy of science UbA (1977), 74, 5463-5467] The base sequence of this gene (mGPR92) derived from was determined.

 Example 5

 [0103] (Construction of mouse-derived gene expression vector)

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 μL of a reaction solution containing 5 μL of buffer attached to Pfe Platinum polymerase and 7.5 μL 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. After PCR, a 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. After confirming that there are no errors in the nucleotide sequence, 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). It was confirmed by digestion with a restriction enzyme that the target expression vector was obtained, and this plasmid was named pCAGGS / mGPR92 / 93. The destination vector pCAGGS-DEST is a mammalian cell expression vector p

After smoothing the Xhol site of CAGGS, GATEWAY reading frame B is inserted in the same direction as the promoter.

 Example 6

[0104] (Human-derived gene expression vector)

 Commercially available EST clone [IMAGE clone: ID = 4335693 (5 ′) (ResGen)] force It was prepared by excising the protein coding region using a restriction enzyme and connecting it to a vector. It was confirmed by sequence analysis that the amino acid sequence encoded by the coding region introduced here was identical to the amino acid sequence described in SEQ ID NO: 2.

 Example 7

[0105] (Conjugated G protein)

 (1) Transient introduction of GPR92 / 93 into cells

For transient gene introduction, 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 μg / Opti-MEM medium 5 μ 1 and Lipofectamine 0.3 μ 1 / Opti-MEM medium 5 μ 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 μ 1 / well (medium: 10% FBSZF-12) and cultured at 5% CO, 37 ° C for 22-24 hours Aspirate and remove F

 2

 After washing twice with -12 medium, add 50 1 / well of the above gene transfer solution and add 5% CO

 2

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.

 2. The cells were further cultured at 37 ° C for 24 hours.

 (2) Measurement of cAMP production

The culture supernatant of the cells transiently transfected with the method (1) above was removed and washed with PBS (—). Add 0.5 mM IBMX / lmg / ml BSA / PBS (-) 50 μ 1 / well for 30 minutes at room temperature After standing, remove the solution and add 80 μl / well lysis buffer (l'M KF / l.25% Triton X100 / 5 OmM phosphate buffer (pH 7.0)) to lyse the cells. It was. Transfer 20 1 / well of this lysate to a 384-well white plate, and add 20 ml of cAMP diluent (0, 0.8, 2.4, 8, 24, 80, 320 nM) for standard curve preparation to the same 384-well plate. / well added. cAMP-XL665 conjugate and anti-c AMP cryptate conjugate solution of HTRF reagent set (CIS bio international), a cAMP quantification kit, is diluted 40-fold with lmg / ml BSAZPBS (-), and each 10 μ 1 I weir is added. Then, it was sealed and allowed to stand at room temperature. 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.

[Table 2]

(Here, the data represents the average value measured at η = 3)

 Example 8

 [0107] (Concentration-dependent increase in cAMP by addition of LPA)

 The GPCR92 / 93 gene was transiently introduced into the cells by the method shown in Example 7 (1). 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 μI / 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).

 The results are shown in Table 3. In GPR92 / 93-introduced cells, an increase in the amount of cAMP depending on the LPA concentration added was observed.

[0108] [Table 3]

Comforted paper cAMP production (p mol / wel I)

 PA concentration M)

 One (mock introduced cell) Human GPR92 / 93 introduced cell Mouse GPR92 / 93 introduced cell

 0.03 ± 0.01 2.06 ± 0.14 2.64 ± 0.42

 .01 0.04 ± 0.01 2.63 ± 0.22 3.12 ± 0.07

 .1 0.05 ± 0.01 4.59 ± 0.52 5.60 ± 0.30

 0.08 ± 0.03 7.67 ± 0.65 8.90 ± 0.71

(Here, the measured value is the average value of n = 3.)

 Here, GPR92 / 93 ligands (agonist and inverse agonist) can be evaluated by using a test substance instead of LPA.

 Example 9

[0109] (Insulin secretion in isolated La Island)

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. Make up to 50 ml with RPMI1640 medium (10% FCS, with antibiotics). After suspension, centrifuge for lOOOrpmXl minutes and remove the supernatant. After repeating the same procedure twice more, under the actual microscope observation, pick up La Island using a pipetman and incubate overnight at 37 ° C, 5% CO / 95% Air in RIMI medium. Provided.

 2

 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.

[0110] [Table 4]

 Additives DMS0 GLP-1 (lOOnM) LPA (2 μΜ) LPA (10 μΜ)

Insulin secretion 3338 ± 1534 7264 ± 4959 4171 ± 1720 6312 ± 2904 In Table 2, DMSO is a solvent-only negative control, and GLP-1 is a peptide compound known to have an insulin secretion-promoting action, that is, a positive control. As shown in Table 2, LPA showed insulin secretion promoting activity in a dose-dependent manner. LPA: Insulin secretion promoting activity at 10 μΜ was almost the same as GLP-1 (100 μΜ).

 Example 10

 [0111] (Screening by Binding Atsay,

 (1) Preparation of cell membrane fraction

 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

Suspend in HEPES (pH 7.0), 10 mM 2-ME, ImM PMSF, 0.25 M sucrose). Homogenize with a Potter type homogenizer (400 rpm, 20 strokes), centrifuge at 100000 g for 60 minutes, and 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 μg / ml aprotinin and used in the following assembly.

 (2) Receptor binding assembly

 [3H] LPA suspended in MultibufferGVPlate (Miripore) in reaction buffer (50 mM Hepes pH7.4, 12.5 mM magnesium acetate, 3.125 mM magnesium chloride, 0.125 mg / ml BSA) and diluted test substance (DMSO solution) Click Incubate at 30 ° C, and add the membrane fraction prepared as described above. After incubation at 30 ° C, add an equal volume of 20% TCA, and aspirate the supernatant to precipitate the protein. After washing several times with 10% TCA, the membrane dried at 37 ° C is punched out and measured with a γ counter. If the radioactivity in the test substance is greater than that in a control using only DMSO as the test substance, the test substance can be determined to be a GPR92 / 93 ligand.

 Example 11

[0112] (Screening using Biacore)

(1) Preparation of cell membrane fraction The GPR92 / 93-expressing strain described in Example 2 (1) is seeded in a flask, and 10% FBS (Gibco) is cultured in the medium until it becomes 60-70% confluent. The cells are collected and suspended in buffer A (50 mM HEPE S (pH 7.0), lOmM2-ME, ImM PMSF, 0.25M sucrose). Homogenize with a Potter type homogenizer (400 rpm, 20 strokes), centrifuge at 100000 g for 60 minutes, and resuspend the resulting precipitate in buffer A again. This suspension is layered on 35% (mass / vol) success 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. Suspend the resulting precipitate in buffer A containing 20 μg / ml aprotinin and use it in the following assembly.

(2) Measurement of coupling at via core

 The general method described in the literature [Anal Biochem. 1998 Dec 15; 265 (2): 340-50. Markgren PO et al.] Is used. GPR92 / 93-expressing cell membrane fraction (eg, 1-10 μg) 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.

 Run HBS buffer (Amersham Falmacia Baotech Co., Ltd.) through the sensor chip at a flow rate of 201 Z and record the knock ground value. The intermediate force is also switched to the test substance dissolved in the HBS buffer at a concentration of 10ηΜ to 10 / ζM for 1 minute, and the change in value associated with drug binding is recorded. Switch back to HBS buffer without drug and record the change in value as the bound drug dissociates. Binding and dissociation rates, or maximum binding capacity Calculate the affinity between the test substance and GPR92 / 93.

Example 12

Plasmid DNA production method

 GPR92 / 93 (ΝΜ_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. Furthermore, 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.

 Example 13

[0114] Atsay method

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. After culturing in a C02 incubator for 4 hours, 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 μl of 0.5 mM IBMX containing the porcine organ activity fraction. After 15 minutes, Hanks Hepes solution was aspirated and 40 μl 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. 10 μl of the reaction solution was placed in a 384-well white plate, and 5 μl each of cAMP-XL665 conjugate and Mab anti-cAMP-Cryptate conjugate were added. After reacting for 2 hours at room temperature, cAMP was quantified by the TR-FRET method using a multiplate reader ARVO.

 Example 14

 [0115] Isolation of active ingredients LPA and LPI from porcine brain tissue

 Nine fresh pig brains (wet weight 924.9 g) were collected. On that day, 5 L of physiological saline was added to 9 fresh brains, homogenized, cooled to 4 ° C, and centrifuged at 9,000 G for 30 minutes to obtain 4,277 g of supernatant.

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). Next, pass 3L of acetonitrile, 10% methanol, 10% 0.1M acetic acid (labeled C4), 80% acetonitrile, 10% methanol, 10% 0.1% formic acid, 3L (labeled C5), 80% Acetonitrile-10% methanol-10% 0.1% Trichloroacetic acid TFA3L was passed through (labeled C6). As a result of measuring the activity of each eluate, it was found that the active ingredient was eluted in 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.

(Zorbax-C 18-HPLC conditions)

L Cloth: Applied Biosystems Integral HPL

Column: ZORBAX Eclipse XDB- C18 4.6x250mm (Agilent)

Mobile phase: A: 0.1% TFA-H2O B: 0.08% TFA- 90% ACN

0% B (0min) -20% B (10min) -60% B (40min) -100% B (40min) -100% B (5min)

Flow rate: l.OmL / min

Fractionation: l.Omin / Fr

Detection: UV220nm, 280nm

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.

 800 μl of each of the above-mentioned fraction 64 and fraction 65 was concentrated under reduced pressure using a speed-back concentrator, dissolved in 200 1 methanol, passed through an Xterra column manufactured by Waters, and 0.1% trichloroacetic acid / acetonitrile solvent. The active ingredient was eluted by increasing the acetonitrile concentration in the system. The gradient conditions were carried out according to the following conditions.

(Xterra-HPLC conditions) LC instrument: Applied Biosystems Integral HPLC

Column: Xterra (registered trademark) RP18 4.6x150mm (Waters)

Mobile phase: A: 0.1% TFA-H2O B: 0.08% TFA- 90% ACN

 0% B (0min) -50% B (5min) -58% B (8min) -70% B (24min) -75% B (5min) -100% B (10min)

-100% (3min)

Flow rate: 0.8mL / min

Fractionation: l.Omin / Fr

Detection: UV220nm, 280nm

 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 μ1 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.

 (Atlantis -HPLC conditions)

 L Cloth: Applied Biosystems Integral HPL

Column: Atlantis® dC18 4.6x150mm (Waters)

Mobile phase: A: 0.1% TFA-H2O B: 0.08% TFA- 90% ACN

 0% B (0min) -50% B (5min) -58% B (8min) -70% B (24min) -100% B (5min)

Flow rate: 0.8mL / min

Fractionation: 0.3min / Fr

Detection: UV220nm, 280nm

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. Example 15

Structural analysis of porcine brain tissue-derived active ingredients LPA and LPI

For the mass analysis, 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. In LCQ, 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.

 (1) Structural analysis of active fraction 41

 When the mass spectrum of the active fraction 41 was obtained, the peak m / z 599.9 considered to be [MH]-was detected in the negative ion mode, and [M + H] + = 601.7, [M + Na] in the positive ion mode. +,

[2M + H1 +, [M + H-H20] + was detected, and the molecular weight of this component was determined to be 600, indicating that it was purified to a single component. From the fragment ions m / z419 and m / z241 detected in the negative ion MS / MS spectrum, it was estimated to be inositol phospholipid.From the fragment ions of m / z315 and m / z341, the candidate ligand component was (18: 0)). When MS / MS spectrum of lysophosphatidylinositol (LPI (18: 0)) sample was obtained, the fragment ions matched and the active component of MW600 was determined to be lysophosphatidylinositol (LPI (18: 0)).

 (2) Structure analysis of active fraction 47

When the mass spectrum of active fraction 47 was obtained, the [M- H]-and [2M- H]-peaks were detected in the negative ion spectrum, and [M + H] +, [2M + Since the peaks of H1 +, [3M + H] +, [M + Na] +, [M + H-H20] + were detected, the molecular weight of this component could be determined to be 482, and it was purified into a single component. It was a great help. In the MS / MS spectrum from the positive ion m / z 483.8, a -98 fragment was detected, suggesting the presence of a phosphate group. In the MS / MS spectrum from the negative ion m / z 481.8, a fragment ion of m / z 153 characteristic of glycerol phosphate was detected, and this component is lysophospholipid, a kind of lysophospholipid Docosahexaenoy 卜 LPA (LPA (22: 6))). In this MS / MS spectrum, the binding site of the acyl group (sn-1 or sn-2) cannot be determined, and both are possible. MS / MS spectrum of DocosahexaenoyenoLPA (LPA (22: 6)) preparation prepared by enzymatic synthesis (Tokumur, A. et al., Biochem J. 2002 Aug l; 365 (Pt 3): 617-28.) As a result, the fragment ions coincided and the active component of MW482 was determined to be Docosahexaenoy LPA (LPA (22: 6)).

 Example 16

[0117] Activity of various related compounds

 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.

[0118] [Table 5]

* (CAMP amount in the presence of the test substance) (cAMP amount in the absence of the test substance)

 Industrial applicability

[0119] 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. In addition, the screening method of the present invention makes it possible to search for GPR92 / 93 agonists and antagonists that can be drug candidates.

 Sequence listing free text

[0120] 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

Claims

The scope of the claims

 [1] An insulin secretagogue that contains a GPR92 / 93 agonist as an active ingredient.

 [2] The insulin secretagogue according to claim 1, wherein the GPR92 / 93 agonist is lysophosphatidic acid, a derivative thereof, L-NASPA, or a pharmaceutically acceptable salt thereof.

[3] Lysophosphatidic acid or its derivative power Formula (1):

 [Chemical 11]

[In the formula, 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 insulin secretion promoter of Claim 2 which is a compound represented by these.

 [4] The insulin secretagogue according to claim 3, wherein the compound is represented by the formula (1): lysophosphatidic acid, 1-Acy の PAF, or a pharmaceutically acceptable salt thereof.

[5] In equation (1),

The insulin secretagogue according to claim 3, characterized in that R ² and R ³ represent the following combinations:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a water element;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; 6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, and R ¹ and R ³ represent an otatanyl group.

 [6] The insulin secretion promoter according to any one of claims 1 to 5, which is a blood glucose regulator.

 [7] The insulin secretagogue according to any one of claims 1 to 5, wherein the insulin secretagogue is a drug for improving glucose tolerance.

 [8] The insulin secretagogue according to any one of claims 1 to 5, which is a drug for lifestyle-related diseases.

 [9] The insulin secretagogue according to any one of claims 1 to 5, which is a therapeutic agent for diabetes.

 [10] A GPR92 / 93 agonist comprising lysophosphatidic acid, a derivative thereof, L-NASPA or a pharmaceutically acceptable salt thereof as an active ingredient.

[11] Lysophosphatidic acid or its derivative power Formula (1):

 Equation (1):

 [Chemical 12]

[In the formula, 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 GPR92 / 93 agonist according to claim 10, which is a compound represented by the following formula:

[12] Compound power represented by the formula (1): lysophosphatidic acid, 1-Acy 又 は PAF or pharmacy thereof 12. A GPR92 / 93 agonist according to claim 11 which is a top acceptable salt.

[13] In equation (1),

Characterized in that R ² and R ³ represents a combination of the following, GPR92 / 93 agonist of Claim 11:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a hydrogen atom;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group;

6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, and R ¹ and R ³ represent an otatanyl group.

 [14] 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 the ligand can bind than the reference substance.

 [15] The following steps (a) to (d):

 (a) contacting the test substance with GPR92 / 93 or a fragment to which a ligand can bind;

 (b) measuring the binding activity of the test substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind;

 (c) comparing the measured value of (b) with the measured value when a reference substance is used as a test substance; and

 (d) a step of selecting, as a GPR92 / 93 ligand, a test substance whose measured value of (b) is equal to or greater than the measured value of (c) based on the result of (c),

The screening method according to claim 14, comprising:

[16] The following steps (a) to (c):

 (a) contacting a reference substance and a fragment to which GPR92 / 93 or a ligand can bind in the presence and absence of a test substance;

 (b) Measure the binding activity of the reference substance in (a) to GPR92 / 93 or a fragment to which the ligand can bind, and compare the measured value in the presence of the test substance with the measured value in the absence of the test substance. Process, and

 (c) selecting as a GPR92 / 93 ligand a test substance in which the measured value of (b) in the presence of the test substance is larger than the measured value of (b) in the absence of the test substance, The screening method according to claim 14.

[17] In a reaction system comprising a lipid bilayer containing GPR92 / 93 and an α protein subset of G protein that can be conjugated to GPR92 / 93, the test substance and sputum or lysophosphatidic acid, its derivatives and their pharmacology Contacting a reference substance selected from the above-mentioned acceptable salts, and selecting a compound whose GDP 'GTP exchange reaction of the subunit or the cell stimulating activity of the G protein is higher than that of the reference substance. Screening method for GPR92 / 93 ligand.

[18] The reaction system is

 (0) Host eukaryotic cells transfected with an expression vector containing DNA encoding GPR92 / 93, GO GPR92 / 93 C-terminal polymorphism of G protein α-subunit fused to GPR92 / 93 Host eukaryotic cells transformed with an expression vector containing DNA encoding the peptide, Gii) G protein endogenously transferred with an expression vector containing DNA encoding GPR92 / 93 and capable of coupling to GPR92 / 93 Gv) GPR92 / 93 and animal cells that endogenously express G protein that can be conjugated to GPR92 / 93, homogenates of those cells, or membrane fractions derived from those cells The screening method according to claim 17, wherein

[19] The following steps (a) to (d):

 (a) contacting a test substance with a reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;

(b) measuring the GDP · GTP exchange reaction of the subunits in (a) or the cell stimulating activity of the G protein; (c) A step of comparing the measured value of (b) with the measured value when a reference substance selected from lysophosphatidic acid, its derivatives and pharmaceutically acceptable salts thereof is used as a test substance. ,as well as

 (d) a step of selecting, as a GPR92 / 93 agonist, a test substance having a measured value of (b) equal to or greater than the measured value of (C) based on the result of (C),

 The screening method according to claim 17 or 18, comprising:

[20] The following steps (a) to (c):

 (a) a reference substance selected from lysophosphatidic acid, its derivatives and pharmaceutically acceptable salts thereof, and a lipid bilayer containing GPR92 / 93 and GPR92 / in the presence and absence of the test substance Contacting a reaction system comprising an α subunit of a G protein that can be conjugated to 93,

 (b) measuring the GDP-GTP exchange reaction of the subunit in (a) or the cell stimulating activity of the G protein, and comparing the measured value in the presence of the test substance with the measured value in the absence of the test substance; as well as

 (c) the measurement value of (b) in the presence of the test substance is smaller than the measurement value of (b) in the absence of the test substance! /, the step of selecting the test substance as a GPR92 / 93 antagonist A screening method according to claim 17 or 18, comprising:

[21] Lysophosphatidic acid or its derivative power Formula (1):

 Equation (1):

 [Chemical 13]

[Wherein, R ¹ represents a hydrogen atom, an acyl group having 8 to 22 carbon atoms, or a 1-alkenyl 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 screening method in any one of Claims 17-20 which is a compound represented by these.

 [22] The screening method according to claim 21, which is lysophosphatidic acid, 1-Acy 卜 PAF, or a pharmaceutically acceptable salt thereof, represented by the compound represented by formula (1).

[23] In equation (1),

The screening method according to claim 21, characterized in that R ² and R ³ represent the following combinations:

1) R ¹ represents an oleoyl group, R ² and R ³ represent a hydrogen atom;

2) R ¹ represents a palmitoyl group, R ² represents a acetyl group, and R ³ represents a 2-ammoethyl group;

3) R ¹ represents a docosahexaenoyl group, R ² and R ³ represent a hydrogen atom;

4) R ³ represents a docosahexaenoyl group, R ² and R ¹ represent a hydrogen atom;

5) R ¹ represents a stearoyl group, R ³ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group;

6) R ³ represents a stearoyl group, R ¹ represents a hydrogen atom, and R ² represents a myo-inositol-1-yl group; or

7) R ² represents a phosphono group, and R ¹ and R ³ represent an otatanyl group.

 [24] A method for screening an insulin secretagogue, comprising selecting a compound using as an index that the test substance is a GPR92 / 93 agonist.

[25] GPR92 / 93 or a fragment to which a ligand can bind is contacted with a test substance, and the G

A compound that binds to PR92 / 93 or the fragment is selected.

4. The method according to 4.

 [26] In a reaction system comprising a lipid bilayer containing GPR92 / 93 and an α subunit of G protein that can be conjugated to GPR92 / 93, the GDP / GTP exchange reaction of the subunit or cells of the G protein 25. The method of claim 24, comprising comparing the stimulating activity in the presence and absence of the test substance.

 [27] The reaction system is

(0 Host eukaryotes transfected with expression vectors containing DNA encoding GPR92 / 93 Biological cells, host Eukaryotic cells transfected with an expression vector containing DNA encoding a polypeptide in which the α subunit of G protein that can be conjugated to GPR92 / 93 is coupled to the C-terminal side of GO GPR92 / 93, Gii ) Host animal cell that endogenously expresses G protein that can be conjugated to GPR92 / 93, or Gv) conjugated to GPR92 / 93 and GPR92 / 93, transferred with an expression vector containing DNA encoding GPR92 / 93 27. The screening method according to claim 26, which is an animal cell that endogenously expresses a G protein that can be produced, a homogenate of those cells, or a membrane fraction derived from those cells.

[28] The following steps (a) to (d):

 (a) contacting a test substance with a reaction system comprising a lipid bilayer containing GPR92 / 93 and an OC subunit of G protein that can be conjugated to GPR92 / 93;

 (b) a step of measuring the GDP · GTP exchange reaction of the subunit in (a) or the cell stimulating activity of the G protein; and

 (c) selecting a test substance having cell stimulating activity as a GPR92 / 93 agonist based on the result of (b),

 The screening method according to claim 26 or 27, comprising:

[29] The method according to claim 24, comprising evaluating whether the test substance is a GPR92 / 93 agonist by the method according to any one of claims 14 to 19.

[30] The screening method according to any one of claims 24 to 29, wherein the insulin secretagogue is a blood glucose regulator.

 [31] The screening method according to any one of claims 24 to 29, wherein the insulin secretagogue is an agent for improving glucose tolerance.

[32] The screening method according to any one of [24] to [29], wherein the insulin secretagogue is a therapeutic agent for diabetes.

 [33] An insulin secretagogue obtained by the method according to any one of claims 24 to 32.

[34] The insulin secretagogue according to claim 33, which is a blood glucose regulator.

[35] The insulin secretion-promoting agent according to claim 33, which is an agent for improving glucose tolerance abnormality.

[36] The promotion of insulin secretion according to claim 33, which is a therapeutic agent for lifestyle-related diseases medicine.

 34. The insulin secretagogue according to claim 33, which is a therapeutic agent for diabetes.