JP7326832B2 - Screening method for substance that enhances oral sensation of oil - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for screening substances that enhance the oral sensation of oils and fats. Substances that enhance the sensation of fats and oils in the oral cavity are useful in the field of foods and the like.

Fats and oils are one of the nutrients contained in foods, and cause a unique and pleasant sensation in the oral cavity. However, fats and oils are high in calories, and there is a problem that excessive intake of fats and oils causes diseases such as hyperlipidemia and obesity. Therefore, there has been a demand for the development of a technology that enhances the sensation of fats and oils in the oral cavity.

Patent Document 1 and Patent Document 2 disclose a screening method for a substance exhibiting a fat-like taste using GPR113, which is a G protein-coupled receptor (GPCR).

Patent Document 3 discloses a screening method for a substance that enhances the oral sensation of oils and fats using GPR120, which is a G protein-coupled receptor (GPCR).

International publication 2016/210162 pamphlet WO2017/030862 Pamphlet Patent Publication No. 2016/214135 pamphlet

An object of the present invention is to provide a screening method for a substance that enhances the sensation of fat in the oral cavity.

As a result of intensive studies to solve the above problems, the present inventors found that GPR120/GPR113 strongly responds to substances such as GPR120 agonists that enhance the oral sensation of oils and fats, and completed the present invention. .

That is, the present invention can be exemplified as follows.
[1]
A method for screening a target substance,
The following steps (A) to (C):
(A) contacting GPR120/GPR113 with a test substance;
(B) measuring the response of said GPR120/GPR113 to said test substance; and (C) identifying said test substance as a target substance based on said response;
including
identifying the test substance as a target substance if the response is observed;
The target substance is a substance that enhances the oral sensation of fats and oils,
A method, wherein said GPR120/GPR113 is a combination of GPR120 and GPR113 proteins.
[2]
The above method, wherein the response is binding of the GPR120/GPR113 to the test substance or activation of the GPR120/GPR113 by the test substance.
[3]
The above-described method, wherein the GPR120/GPR113 is used in a form supported by cells, cell membranes, artificial lipid bilayer vesicles, or artificial lipid bilayer membranes.
[4]
The above method, wherein the GPR120/GPR113 is used in a cell-loaded form.
[5]
The above method, wherein the cells are animal cells.
[6]
The above method, wherein the cell has an α subunit of a G protein.
[7]
The above method, wherein the α subunit is Gas or Gαq.
[8]
The method, wherein steps (B) and (C) are carried out by steps (B1) and (C1), respectively:
(B1) the step of measuring the degree of activation D1 of the GPR120/GPR113 when the step (A) is carried out;
(C1) A step of identifying the test substance as a target substance based on the degree of activation D1. [9]
The method, wherein the step (C1) is performed by the following step (C2):
(C2) the degree of activation D1 and the degree of activation of GPR120/GPR113 in control conditions D
identifying the test substance as a target substance based on the difference from 2;
[10]
The method, wherein the control condition is the following condition (C2-1) or (C2-2):
(C2-1) Conditions under which the GPR120/GPR113 and the test substance are not brought into contact;
(C2-2) Conditions for contacting the GPR120/GPR113 with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in the step (A).
[11]
The above method, further comprising the step of measuring the degree of activation D2.
[12]
The above method, wherein the test substance is identified as a target substance if the degree of activation D1 is higher than the degree of activation D2.
[13]
The above method, wherein the response is measured using intracellular calcium concentration or intracellular cAMP concentration as an indicator.
[14]
The method, wherein the GPR120 protein is a protein according to (A) or (B) below:
(A) a mammalian GPR120 protein;
(B) Chimeric proteins of two or more mammalian GPR120 proteins.
[15]
The method, wherein the GPR120 protein is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(b) a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and having responsiveness to;
(c) A protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having responsiveness to a target substance in combination with GPR113 protein.
[16]
The method, wherein the GPR113 protein is a protein according to (A) or (B) below:
(A) a mammalian GPR113 protein;
(B) Chimeric proteins of two or more mammalian GPR113 proteins.
[17]
The method, wherein the GPR113 protein is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4;
(b) in the amino acid sequence shown in SEQ ID NO: 4, containing an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues, and in combination with GPR120 protein against a target substance a responsive protein;
(c) A protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and having responsiveness to a target substance in combination with GPR120 protein.
[18]
The above method, wherein the target substance is a GPR120 agonist.
[19]
Furthermore, the above method, comprising the step of evaluating the function of the identified target substance to enhance the oral sensation of fats and oils.
[20]
The above method, wherein the evaluation is performed by sensory evaluation.
[21]
Cells into which the gene encoding the GPR120 protein and the gene encoding the GPR113 protein have been introduced.
[22]
The cell, which is an animal cell.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently screen for a substance that enhances the oral sensation of fats and oils.

FIG. 4 shows the response of GPR113-only expression cells, GPR120-only expression cells, GPR120/GPR113 co-expression cells, and GPR120/mGluR4 co-expression cells to linoleic acid, measured using intracellular calcium concentration as an index. Compound 1 (4-[(4-Fluoro-4'-methyl Fig. 1 shows the response to [1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid). Compound 2 (2-[3-fluoro-(pyridin-3- yloxy)phenyl]-1,2-benzisothiazol-3(2H)-one-1,1-dioxide).

The present invention will be described in detail below.

The method of the present invention is a screening method for a substance that enhances the oral sensation of oils and fats using GPR120/GPR113. A substance that enhances the sensation of fat in the oral cavity is also called a “target substance”. In the method of the present invention, GPR120/GPR113 can be used to identify the target substance (that is, to identify whether the test substance is the target substance). Specifically, in the method of the present invention, the target substance can be identified (that is, whether the test substance is the target substance) can be identified based on the response of GPR120/GPR113 to the test substance. Specifically, the method of the present invention comprises (A) the step of contacting GPR120/GPR113 with a test substance; (B) the step of measuring the response of said GPR120/GPR113 to said test substance; and (C) identifying the test substance as the target substance based on the response.

<1> Target Substance The term “target substance” refers to a substance that enhances the sensation of fat in the oral cavity. That is, the target substance has the function of enhancing the oral sensation of fats and oils. "Oral sensation of grease" refers to the sensation of grease felt on the tongue. The sensation in the oral cavity of fats and oils includes the coating feeling of fats and oils. The “feeling of a coating of fats and oils” refers to the feeling that fats and oils cover the tongue and cling to the tongue. In addition, the period from 0 to 2 seconds after the oil is included in the oral cavity is also called "top", the period from 2 to 5 seconds is "middle", and the period after 5 seconds is "last". . The target substance may be, for example, a substance that enhances the oral sensation of fat in one or more periods selected from top, middle, and last. The target substance may be, for example, a substance that enhances the sensation of fat in the oral cavity when blended with an object containing fat (for example, food or drink). Objects containing fats and oils will be described later. The target substance may or may not exhibit the sensation in the oral cavity of fats and oils in the absence of fats and oils.

A target substance may be, for example, a GPR120 agonist. That is, the GPR120 agonist may function as a substance (target substance) that enhances the sensation of fat in the oral cavity. A "GPR120 agonist" refers to a substance that elicits a GPR120 protein response.

The target substance may consist of a single component (ie, a pure substance) or may consist of a combination of two or more components (ie, a mixture). A "mixture" is also referred to as a "composition". When the target substance is a mixture, as long as the mixture has the function of enhancing the oral sensation of oils and fats, each component constituting the mixture independently has the function of enhancing the oral sensation of oils and fats. may or may not have.

<2> Test Substance The term “test substance” refers to a substance used in the method of the present invention as a target substance candidate. A test substance is not particularly limited. A test substance may consist of a single component (ie, a pure substance) or may consist of a combination of two or more components (ie, a mixture). When the test substance is a mixture, there are no particular restrictions on the number of types or composition ratio of the components that constitute the mixture. The test substance may be a known substance or a new substance. The test substance may be natural or man-made. A test substance can be, for example, a compound library generated using combinatorial chemistry techniques. Test substances include, for example, alcohols, ketones, aldehydes, ethers, esters, hydrocarbons, sugars, organic acids, nucleic acids, amino acids, peptides, and various other organic or inorganic components. Test substances also include, inter alia, existing food additives. "Existing food additive" means a substance that is approved for use as a food additive. As the test substance, one test substance may be used, or two or more test substances may be used in combination. By contacting GPR120/GPR113 with two or more components together and carrying out the method of the present invention, it is possible to identify whether the combination of those components as a whole is the target substance. "GPR120/GPR113 combining two or more ingredients
The case of contacting with” includes the case of contacting a mixture of test substances with GPR120/GPR113, and the case of contacting two or more test substances together with GPR120/GPR113.

Test substances may be selected to include substances such as those exemplified above, eg, existing food additives. That is, as a test substance, for example, one existing food additive may be used, two or more food additives may be used in combination, one or more food additives and It may be used in combination with one or more other ingredients.

Test substances may also be selected to include substances other than free fatty acids, for example. That is, the method of the present invention may exclude cases where the test substance consists of free fatty acids. Also, the method of the present invention may exclude cases where the test substance contains free fatty acids.

Test substances may also be selected to include, for example, substances other than known substances of interest. A “known target substance” refers to a substance that is known to have a function of enhancing the sensation of fat in the oral cavity. That is, the method of the present invention may exclude cases where the test substance consists of a known target substance. Also, the method of the present invention may exclude cases where the test substance contains a known target substance. Known target substances include the known GPR120 agonists linoleic acid, 4-[(4-Fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid, 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1,2-benzisothiazol-3(2H)-one-1,1-dioxide.

<3>GPR120/GPR113
"GPR120/GPR113" refers to the combination of GPR120 and GPR113 proteins.

"GPR120 protein" refers to a protein encoded by the GPR120 gene. The GPR120 protein is a G protein-coupled receptor (GPCR). The GPR120 protein is also simply referred to as "GPR120". As the GPR120 protein, one GPR120 protein may be used, or two or more GPR120 proteins may be used in combination.

"GPR113 protein" refers to a protein encoded by the GPR113 gene. The GPR113 protein is a G protein-coupled receptor (GPCR). The GPR113 protein is also simply referred to as "GPR113". As the GPR113 protein, one GPR113 protein may be used, or two or more GPR113 proteins may be used in combination.

GPR120 protein and GPR113 protein are also collectively referred to as "GPCR". The GPR120 gene and the GPR113 gene are also collectively referred to as "GPCR gene".

GPR120/GPR113 has responsiveness to target substances. That is, the combination of both GPCRs (that is, GPR120 protein and GPR113 protein) has responsiveness to the target substance. In other words, each GPCR has responsiveness to a target substance in combination with other GPCRs. Each GPCR may or may not have responsiveness to a target substance independently. For example, the GPR120 protein alone may have responsiveness to a target substance. Also, for example, the GPR113 protein alone does not have to be responsive to the target substance. GPR120/GPR113 may have higher responsiveness to a target substance than each GPCR. For example, GPR120/GPR113 may have higher responsiveness to a target substance than GPR120 protein. Also, for example, GPR120/GPR113 may have higher responsiveness to a target substance than GPR113 protein. “Having responsiveness to a target substance” may mean being activated by a target substance.

Regarding the responsiveness to the target substance, the description of the measurement of the response of GPR120/GPR113 to the target substance in the method of the present invention can be applied mutatis mutandis.

GPCR genes and GPCRs include GPCR genes and GPCRs of various organisms. Examples of organisms include animals such as mammals. Specific examples of animals such as mammals include Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Canis lupus familiaris (dog), Felis catus (cat), Bos taurus (bovine), Sus
scrofa (pigs), Pan troglodytes (chimpanzees), Macaca fascicularis (cynomolgus monkeys), Equus caballus (horses). Animals such as mammals particularly include humans. Nucleotide sequences of GPCR genes and amino acid sequences of GPCRs of various organisms can be obtained from public databases such as NCBI and Ensembl. The nucleotide sequence of the human GPR120 gene (cDNA) and the amino acid sequence of the GPR120 protein are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the human GPR113 gene (cDNA) and the amino acid sequence of the GPR113 protein are shown in SEQ ID NOs: 3 and 4, respectively.

That is, the GPCR gene may be, for example, a gene having a nucleotide sequence encoding the amino acid sequence of the GPCR exemplified above (eg, the nucleotide sequence of the GPCR gene of the organism exemplified above, such as the nucleotide sequence of SEQ ID NO: 1 or 3). . In addition, the GPCR may be, for example, a protein having the amino acid sequence of the GPCR exemplified above (eg, the amino acid sequence of the GPCR of the organism exemplified above, such as the amino acid sequence of SEQ ID NO: 2 or 4). The expression "having a (amino acid or base) sequence" means "including the (amino acid or base) sequence" unless otherwise specified, and includes the case of "consisting of the (amino acid or base) sequence". do.

GPCRs also include chimeric GPCRs. Chimeric GPCRs include chimeric GPR120 protein and chimeric GPR113 protein. The term "GPCR" referred to in the description of the chimeric GPCR refers to the GPR120 protein in the case of the chimeric GPR120 protein, and to the GPR113 protein in the case of the chimeric GPR113 protein. A "chimeric GPCR" refers to a GPCR chimeric protein (ie, a chimeric protein of two or more GPCRs). In other words, "chimeric GPCR" refers to a protein having a GPCR chimeric sequence (ie, a protein having two or more GPCR chimeric sequences). A “GPCR chimeric sequence” refers to a chimeric sequence of GPCR amino acid sequences (ie, a chimeric sequence of two or more GPCR amino acid sequences). The term "chimeric sequence of GPCR" specifically refers to an amino acid sequence of a GPCR, the partial sequence of which is replaced with one or more partial sequences of the amino acid sequence of another GPCR. . Amino acid sequence substitutions in constructing a chimeric GPCR can be made between corresponding sites in the amino acid sequence of the GPCR. The term "corresponding site in the amino acid sequence of GPCR" refers to the site arranged at the corresponding position in the alignment of the amino acid sequences of those GPCRs. Chimeric GPCRs include, for example, chimeric proteins of the GPCRs exemplified above (specifically, chimeric proteins of two or more GPCRs selected from the GPCRs exemplified above). Specific examples of chimeric GPCRs include mammalian chimeric GPCRs (specifically, chimeric proteins of two or more GPCRs selected from mammalian GPCRs). That is, a GPCR is a protein having, for example, a chimeric sequence of the amino acid sequences of the GPCRs exemplified above (specifically, a chimeric sequence of two or more amino acid sequences selected from the amino acid sequences of the GPCRs exemplified above). There may be. Chimeric GPCRs that are responsive to the target substance in combination with other GPCRs can be selected.

The number of types of GPCRs constituting the chimeric GPCR is not particularly limited. The number of types of GPCRs constituting the chimeric GPCR may be two, three or more.

The composition ratio of each GPCR in the chimeric GPCR is not particularly limited. The composition ratio of each GPCR can be appropriately set within a range in which the total composition ratio of the GPCRs constituting the chimeric GPCR does not exceed 100%. The composition ratio of each GPCR is, for example, 1% or more, 3% or more, 5% or more, 10% or more, 20% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more % or more, 95% or more, 97% or more, or 99% or more, 99% or less, 97% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50 % or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, or 1% or less, or any consistent combination thereof. “Constituent ratio of each GPCR” refers to the ratio of the number of amino acid residues derived from each GPCR to the total number of amino acid residues constituting the chimeric GPCR. In addition, when the amino acid residue constituting the chimeric GPCR corresponds to the conserved sequence of the GPCRs constituting the chimeric GPCR, the amino acid residue may be considered to be derived from any of these GPCRs.

The distribution of sites derived from each GPCR in the chimeric GPCR is not particularly limited. In the chimeric GPCR, the sites derived from each GPCR may be concentrated at one site, or may be distributed at two or more sites. For example, when a chimeric GPCR is designed by replacing the internal amino acid sequence of a certain GPCR (GPCRA) with the amino acid sequence of another GPCR (GPCRB), the amino acid sequence of GPCRA is the N-terminal side and the C-terminal side of the chimeric GPCR. remain dispersed.

Similarly, GPCR genes include chimeric GPCR genes. The description of chimeric GPCRs can also be applied mutatis mutandis to chimeric GPCR genes.

As long as the original function of the GPCR gene is maintained, the GPCR gene exemplified above, for example, a gene having the nucleotide sequence of the GPCR gene of the organism exemplified above, such as the nucleotide sequence of SEQ ID NO: 1 or 3, or a chimeric sequence thereof may be a variant of Similarly, as long as the original function of the GPCR is maintained, the GPCR exemplified above, for example, a protein having the amino acid sequence of the GPCR of the organism exemplified above, such as the amino acid sequence of SEQ ID NO: 2 or 4, or a chimeric sequence thereof; may be a variant of Note that such a variant in which the original function is maintained is sometimes referred to as a "conservative variant". The terms "GPR120 gene" and "GPR113 gene" are intended to encompass the above-exemplified GPR120 gene and GPR113 gene, respectively, as well as conservative variants thereof. Similarly, the terms "GPR120 protein" and "GPR113 protein" are intended to encompass the GPR120 protein and GPR113 protein exemplified above, respectively, as well as conservative variants thereof. Conservative variants include, for example, homologues and artificial variants of the GPCR genes and GPCRs exemplified above.

In addition, the GPCR gene specified in the biological species from which it is derived is not limited to the GPCR gene itself found in the biological species, but includes genes having the nucleotide sequence of the GPCR gene found in the biological species and conservative variants thereof. shall be Similarly, the GPCR identified in the biological species from which it is derived is not limited to the GPCR itself found in the biological species, but includes proteins having the amino acid sequence of the GPCR found in the biological species and conservative variants thereof. do. These conservative variants may or may not be found in the species. For example, the term "mammalian GPCR" is intended to encompass proteins having the amino acid sequence of GPCRs found in mammals and conservative variants thereof. Also, for example, the term "mammalian chimeric GPCR" is intended to encompass proteins having chimeric sequences of amino acid sequences of GPCRs found in mammals and conservative variants thereof. In other words, the GPCRs that constitute the "mammalian chimeric GPCR" are not limited to the GPCRs themselves found in mammals, but may be conservative variants thereof.

“Original function is maintained” means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "Maintaining the original function" of a gene means that a variant of the gene encodes a protein that retains the original function. That is, the term "original function is maintained" for each GPCR gene means that the gene variant encodes a protein (GPCR) that is responsive to a target substance in combination with other GPCRs. In addition, the term "original function is maintained" for each GPCR means that the GPCR variant has responsiveness to a target substance in combination with other GPCRs.

Whether a combination of GPCRs has responsiveness to a target substance can be confirmed, for example, by measuring the response of the combination when the combination is brought into contact with the target substance.

Examples of conservative variants are given below.

GPCR gene homologues or GPCR homologues can be easily obtained from public databases by, for example, BLAST search or FASTA search using the above-exemplified nucleotide sequence of the GPCR gene or the above-exemplified amino acid sequence of the GPCR as a query sequence. . Homologs of GPCR genes can be obtained, for example, by PCR using chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of these known GPCR genes as primers.

As long as the original function of the GPCR gene is maintained, the above amino acid sequence (for example, the amino acid sequence of the GPCR of the above-exemplified organism such as the amino acid sequence of SEQ ID NO: 2 or 4, or a chimeric sequence thereof) has one or several It may be a gene encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added at the position. For example, the encoded protein may be lengthened or truncated at its N-terminus and/or C-terminus. The above-mentioned "one or several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. It means preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

Substitutions, deletions, insertions and/or additions of one or several amino acids described above are conservative mutations that maintain normal protein function. A typical conservative mutation is a conservative substitution. Conservative substitutions are between Phe, Trp and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile and Val when the substitution site is a hydrophobic amino acid, which is a polar amino acid. between Gln and Asn, between Lys, Arg, and His when it is a basic amino acid, and between Asp and Glu when it is an acidic amino acid, when it is an amino acid having a hydroxyl group are mutations that replace each other between Ser and Thr. Substitutions regarded as conservative substitutions specifically include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln
, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu mentioned. In addition, the substitution, deletion, insertion, or addition of amino acids as described above may be caused by naturally occurring mutations (mutants or variants) such as those based on individual differences in organisms from which genes are derived, or differences in species. included.

In addition, as long as the original function is maintained, the GPCR gene is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more of the entire amino acid sequence. ,
More preferably, it may be a gene encoding a protein having an amino acid sequence with an identity of 97% or more, particularly preferably 99% or more.

In addition, the GPCR gene is prepared from the above nucleotide sequence (for example, the nucleotide sequence of the GPCR gene of the above-exemplified organism such as the nucleotide sequence of SEQ ID NO: 1 or 3, or a chimeric sequence thereof) as long as the original function is maintained. It may also be a gene, such as DNA, that hybridizes under stringent conditions with the obtained probe, such as a complementary sequence to all or part of the above base sequence. “Stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs with high identity, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% Conditions that hybridize DNAs with more than % identity but do not hybridize DNAs with lower identities, or normal Southern hybridization washing conditions of 60°C, 1×SSC, 0.1% SDS , preferably 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, washing once, preferably 2 to 3 times. be able to.

As mentioned above, the probes used for the hybridization may be part of the complementary sequence of the gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the gene described above as a template. For example, a DNA fragment with a length of about 300 bp can be used as a probe. When using a DNA fragment with a length of about 300 bp as a probe, washing conditions for hybridization include 50° C., 2×SSC, and 0.1% SDS.

In addition, since the degeneracy of codons differs depending on the host, the GPCR gene may be one in which arbitrary codons are replaced with equivalent codons. That is, the GPCR gene may be, for example, a variant of the GPCR gene exemplified above due to the degeneracy of the genetic code. For example, the GPCR gene may be modified to have optimal codons according to the codon usage of the host used.

In the present invention, the term "gene" is not limited to DNA and may include any polynucleotide as long as it encodes the protein of interest. Namely, "GPCR gene"
By may mean any polynucleotide that encodes a GPCR. A GPCR gene may be DNA, RNA, or a combination thereof. A GPCR gene may be single-stranded or double-stranded. A GPCR gene may be single-stranded DNA or single-stranded RNA. A GPCR gene may be a double-stranded DNA, a double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. A GPCR gene may contain both DNA and RNA residues in a single polynucleotide strand. When the GPCR gene contains RNA, the descriptions of DNA such as the nucleotide sequences exemplified above may be appropriately read in accordance with RNA. GPCR genes may or may not contain introns. The mode of the GPCR gene can be appropriately selected according to various conditions such as its utilization mode.

The “identity” between amino acid sequences is calculated by blastp using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). means identity between amino acid sequences. In addition, "identity" between base sequences means the identity between base sequences calculated by blastn using the default Scoring Parameters (Match/Mismatch Scores = 1, -2; Gap Costs = Linear). do.

In addition, a GPCR may have a part or all of a conserved sequence of a GPCR (that is, a conserved sequence of amino acid sequences of two or more GPCRs). "GPCR" referred to in the description of conserved sequences refers to GPR120 protein in the case of conserved sequences possessed by GPR120 protein, and to GPR113 protein in the case of conserved sequences possessed by GPR113 protein. GPCRs have, for example, part or all of the conserved sequences of the above-exemplified GPCRs (specifically, the conserved sequences of the amino acid sequences of two or more GPCRs selected from the above-exemplified GPCRs). good too. GPCRs specifically include, for example, part or all of conserved sequences of mammalian chimeric GPCRs (specifically, conserved sequences of amino acid sequences of two or more GPCRs selected from mammalian GPCRs). may have. Conserved sequences can be determined by alignment of the subject amino acid sequences. Some or all of the conserved sequences include regions that are 10% or more, 30% or more, 50% or more, 70% or more, 90% or more, or 100% of the conserved sequences.

In addition, GPCRs may contain other amino acid sequences in addition to the amino acid sequences of GPCRs as described above. That is, the GPCR may be a fusion protein between the above-described GPCR amino acid sequence and other amino acid sequences. Other amino acid sequences are not particularly limited as long as GPR120/GPR113 has responsiveness to the target substance. Other amino acid sequences include, for example, tag sequences such as His tags and V5 epitope tags. Other amino acid sequences may be linked, for example, to the N-terminus or C-terminus of the GPCR, or both.

GPCR can be used in any form that can be used for screening target substances. Specifically, GPCRs can be used in any form as long as GPR120/GPR113 can come into contact with the test substance and GPR120/GPR113 has responsiveness to the target substance. The mode of use of GPCR can be appropriately set according to various conditions such as embodiments of the method of the present invention.

GPCR may be used in a form isolated to a desired degree, such as a purified product or a crudely purified product, or may be used in a form contained in a material. GPCRs may be specifically used, for example, in the form carried by a structure. Examples of structures include cells, cell membranes, artificial lipid bilayer vesicles, and artificial lipid bilayer membranes. Structures include, among others, cells. In other words, a GPCR is a cell having a GPCR, a cell membrane having a GPCR, an artificial lipid bilayer vesicle having a GPCR, or a structure having (carrying) a GPCR, such as an artificial lipid bilayer having a GPCR. Can be used in any form. Constructs having these GPCRs may also be used, for example, in a form isolated to a desired degree, or in a form contained in a material. Also, the GPCR may form part of an instrument. That is, GPCRs can also be used, for example, in the form of instruments comprising GPCRs. Instruments with GPCRs include, for example, instruments with immobilized GPCRs and instruments with GPCR-containing structures (lipid bilayer membranes, etc.). Examples of instruments with lipid bilayer membranes include chips with arrayed lipid bilayer membranes (WO2005/000558; Watanabe R. et al., Arrayed lipid bilayer chambers allow single-molecule analysis
of membrane transporter activity. Nat Commun. 2014 Jul 24;5:4519.；Kamiya K. et
al., Preparation of artificial cell membrane and single ion channel measurement, Electrochemistry, 83, 1096-1100 (2015)) and an ion channel measurement device equipped with a lipid bilayer membrane prepared by the droplet contact method (Kawano R. et al. al., Automated Parallel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3, No. 1995 (2013)). All of these forms of GPCRs are included within the scope of GPCRs used in the methods of the present invention.

A GPCR can be produced, for example, by expressing a GPCR gene. GPCR gene expression may be performed, for example, using cells or using a cell-free protein synthesis system. For GPCR gene expression using cells, the description of GPCR-containing cells described later can be referred to. The expressed GPCR can be appropriately obtained in the form described above and used in the method of the present invention.

A cell having a GPCR is also referred to as "the cell of the present invention". GPCRs can function, for example, by localizing to the cell membrane. Thus, a cell of the invention may have a GPCR, eg, in the cell membrane.

GPCRs are expressed from GPCR genes. Thus, the cells of the invention have GPCR genes. Specifically, the cells of the present invention have a GPCR gene that can be expressed. It is sufficient that the cells of the present invention have a GPCR gene to express the GPCR. That is, the cells of the present invention may or may not have the GPCR gene after expression of the GPCR. In other words, the cell of the present invention is a cell that expresses a GPCR gene, and also a cell that expresses a GPCR. In addition, "expression of GPCR gene" and "expression of GPCR" can be used synonymously.

The cells of the present invention may have one copy of each GPCR gene, or two or more copies of the GPCR gene. In addition, the cells of the present invention may have one GPCR gene for each GPCR gene, or may have two or more GPCR genes. In addition, the cells of the present invention may have one type of GPCR for each GPCR, or may have two or more types of GPCRs.

The cell of the present invention may originally have a GPCR gene, or may have been modified to have a GPCR gene.

Cells that inherently have a GPCR gene include cells of organisms from which the GPCR gene is derived, such as taste cells of mammals such as humans. A cell that inherently has a GPCR gene can be obtained, for example, from an organism or tissue containing the cell.

Cells modified to have a GPCR gene include cells into which a GPCR gene has been introduced. The cell of the present invention may be, for example, a cell introduced with the GPR120 gene and/or the GPR113 gene. The cells of the present invention may be, for example, cells into which the GPR120 gene and the GPR113 gene have been introduced.

The cells of the present invention and cells used to obtain them (eg, cells into which a GPCR gene has been introduced or into which a GPCR gene has been introduced) are also collectively referred to as "host cells."

The host cell is not particularly limited as long as it can express a functional GPCR and can therefore be used for screening of the target substance. Host cells include, for example, bacterial, fungal, plant, insect, and animal cells. Preferred host cells include eukaryotic cells such as fungal, plant, insect and animal cells. More preferred host cells include animal cells. Animals include, for example, mammals, birds, and amphibians. Mammals include, for example, rodents and primates. Rodents include, for example, Chinese hamsters, hamsters, mice, rats and guinea pigs. Primates include, for example, humans, monkeys, and chimpanzees. Birds include, for example, chickens. Amphibians include, for example, Xenopus laevis. Also, the tissue or cells from which the host cells are derived are not particularly limited. Tissues or cells from which host cells are derived include, for example, ovary, kidney, adrenal gland, tongue epithelium, olfactory epithelium, pineal gland, thyroid, melanocytes. Chinese hamster cells include, for example, Chinese hamster ovary-derived cell lines (CHO). Specific examples of CHO include CHO-DG44 and CHO-K1. Human cells include, for example, human embryonic kidney cell-derived cell lines (HEK). Specific examples of HEK include HEK293 and HEK293T. Monkey cells include, for example, African green monkey kidney cell-derived cell lines (COS). Specific examples of COS include COS-1. Xenopus laevis cells include, for example, Xenopus laevis oocytes. Examples of insect cells include Spodoptera frugiperda-derived cells such as Sf9, Sf21 and SF+, and Trichoplusia ni-derived cells such as High-Five. A host cell is an individual, independent cell (e.g., a free cell)
or may form an aggregate such as a tissue.

A GPCR gene can be obtained by cloning from an organism having the GPCR gene. Nucleic acids such as genomic DNA and cDNA containing the same gene can be used for cloning. GPCR genes can also be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

The obtained GPCR gene can be used as it is or after being modified as appropriate. That is, the variant can be obtained by modifying the GPCR gene. Gene modification can be performed by a known technique. For example, site-directed mutagenesis can be used to introduce a desired mutation at a desired site in DNA. Thus, for example, by site-directed mutagenesis, the coding region of a gene can be altered such that the encoded protein contains substitutions, deletions, insertions, and/or additions of amino acid residues at specific sites. . As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol.,
154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, GPCR gene variants may be obtained directly by chemical synthesis.

There are no particular restrictions on the form of introduction of the GPCR gene into the host cell. The GPCR gene only needs to be retained in the host cell so as to be expressible. Specifically, for example, when a GPCR gene is introduced in a form such as DNA that requires transcription, the GPCR gene may be retained in the host cell so as to be expressible under the control of a promoter that functions in the host cell. . In the host cell, the GPCR gene may exist extrachromosomally or may be introduced onto the chromosome. When two or more genes are introduced, each gene should be maintained in the host cell so that it can be expressed.

A promoter for expressing a GPCR gene is not particularly limited as long as it functions in host cells. A "promoter that functions in a host cell" refers to a promoter that has promoter activity in a host cell. The promoter may be a host cell-derived promoter or a heterologous promoter. The promoter may be the native promoter of the GPCR gene or the promoter of other genes. The promoter may be a promoter stronger than the native promoter of the GPCR gene. For example, promoters that function in animal cells include SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter. As the promoter, a highly active type of a conventional promoter may be obtained and used by using various reporter genes. Methods for evaluating the strength of promoters and examples of strong promoters are described in Goldstein et al.

A GPCR gene can be introduced into a host cell, for example, using a vector containing the gene. A vector containing a GPCR gene is also referred to as a “GPCR gene expression vector”. A GPCR gene expression vector can be constructed, for example, by ligating a DNA fragment containing a GPCR gene with a vector. By introducing an expression vector for the GPCR gene into the host cell, the gene can be introduced into the host cell. A vector may be provided with a marker such as a drug resistance gene. The vector may also contain an expression control sequence such as a promoter for expressing the inserted gene. A vector can be appropriately selected according to various conditions such as the type of host cell and the mode of introduction of the GPCR gene. For example, vectors that can be used for gene introduction into animal cells include plasmid vectors and virus vectors. Viral vectors include, for example, retroviral vectors and adenoviral vectors. Plasmid vectors include, for example, pcDNA series vectors (pcDNA3.1, etc.; Thermo Fisher Scientific), pBApo-CMV series vectors (Takara Bio), and pCI-neo (Promega). Depending on the type and structure of the vector, the vector can be integrated into the host cell chromosome, autonomously replicated outside the host cell chromosome, or temporarily retained outside the host cell chromosome. For example, vectors having a viral origin of replication, such as the SV40 origin of replication, can autonomously replicate extrachromosomally in animal cells. Specifically, for example, the pcDNA series vectors have an SV40 replication origin and can autonomously replicate extrachromosomally in host cells (COS-1, HEK293T, etc.) expressing the SV40 large T antigen.

A GPCR gene can also be introduced into a host cell by, for example, introducing a nucleic acid fragment containing the gene into the host cell. A nucleic acid fragment containing a GPCR gene is also referred to as a "GPCR gene fragment". Such fragments include linear DNA and linear RNA. Examples of linear RNA include mRNA and cRNA.

A method for introducing a nucleic acid such as a vector or a nucleic acid fragment into a host cell can be appropriately selected according to various conditions such as the type of host cell. For example, methods for introducing nucleic acids such as vectors and nucleic acid fragments into host cells such as animal cells include the DEAE dextran method, the calcium phosphate method, the lipofection method, the electroporation method, and the microinjection method. Moreover, when the vector is a viral vector, the vector can be introduced into the host cell by infecting the host cell with the vector (virus).

Alternatively, cells that originally have a GPCR gene may be modified to increase expression of the GPCR gene and used. “Increased gene expression” means that the expression level of the same gene per cell is increased compared to unmodified cells. As used herein, "unmodified cells" refer to control cells that have not been modified to increase expression of the target gene. Unmodified cells include wild-type cells and modified cells. Techniques for increasing GPCR gene expression include increasing the copy number of the GPCR gene and improving the transcription efficiency and translation efficiency of the GPCR gene. An increase in GPCR gene copy number can be achieved by introducing the GPCR gene into a host cell. Introduction of the GPCR gene can be performed as described above. The GPCR gene to be introduced may be derived from the host cell or heterologous. Improving the transcription efficiency and translation efficiency of GPCR genes can be achieved by modifying expression regulatory sequences of genes such as promoters. For example, an improvement in transcription efficiency of a GPCR gene can be achieved by replacing the promoter of the GPCR gene with a stronger promoter.

The cells of the present invention may have other arbitrary properties as long as they can be used for screening of target substances. Such properties include, for example, properties useful for measuring the response of GPR120/GPR113 to a target substance. The description of the properties of the cells of the present invention can be applied mutatis mutandis to the use of GPR120/GPR113 in other embodiments. Other embodiments of using GPR120/GPR113 include the use of artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes having GPR120/GPR113.

The cells of the present invention may or may not have, for example, taste receptors other than GPR120/GPR113 (also referred to as “other taste receptors”). It may be preferred that the cells of the invention have no other taste receptors. Cells that do not have other taste receptors include cells that do not have genes encoding other taste receptors, and cells that have genes encoding other taste receptors but do not express the same genes. cells without For example, the cells of the present invention may be those that do not originally have other taste receptors, or those that have been modified so as not to have other taste receptors. Modification of cells so that they do not have other taste receptors can be achieved, for example, by knocking out genes encoding other taste receptors.

Also, the cells of the present invention may have, for example, proteins involved in signal transduction. In other words, the cells of the invention may have genes encoding proteins involved in signal transduction. Proteins involved in signal transduction include, for example, G proteins, adenylate cyclase, protein kinase A, phospholipase C, inositol 1,4,5-trisphosphate (IP3) receptors, and calcium channels. As a G protein, G
It includes the α subunit of the protein (Gα protein). Gα proteins include Gαs and Gαq. Gαq includes Gα15 and Gα16. Cells with Gas may, for example, also have adenylate cyclase. Cells with Gαq may also have phospholipase C, for example. Cells with phospholipase C may, for example, also have IP3 receptors. Cells with phospholipase C, for example, may also have calcium channels. G proteins also include chimeric G proteins (ie, chimeric proteins of two or more G proteins).
Chimeric G proteins include chimeric Gα proteins (i.e., two or more G
alpha protein chimeric protein). Chimeric Gα proteins include chimeric proteins of two or more Gα proteins of different origins and/or types. Chimeric Gα proteins specifically include chimeric proteins of Gas or Gαq and other Gα proteins. Specific examples of such chimeric Gα proteins include chimeric proteins of Gαq and transducin or gustducin. More specific examples of such chimeric Gα proteins include Gα15-trans48LD (JP-A-2015-192664) and Gα16-gust44 (JP-A-2016-214135). "Gαs" includes Gas and other Gα
Chimeric proteins with proteins may also be included. "Gαq" may also include chimeric proteins of Gαq and other Gα proteins.

Proteins involved in signal transduction and genes encoding them include proteins and genes of various organisms. Examples of organisms include animals such as mammals. Specific examples of animals such as mammals include those described above, such as Homo sapiens (human), Mus musculus (mouse), and Rattus norvegicus (rat). Animals such as mammals particularly include humans. Amino acid sequences of proteins involved in signal transduction in various organisms and nucleotide sequences of genes encoding them can be obtained from public databases such as NCBI and Ensembl. The same is true for each component of the chimeric protein.

In addition, the cells of the present invention may have components depending on the parameter to be measured, for example. Such components include probes such as calcium indicators and reporter genes such as the luciferase gene. When a probe such as a calcium indicator is expressed from a gene, the cell of the present invention may have a gene encoding the probe.

The cells of the present invention may originally have the properties exemplified above, or may be modified to have the properties exemplified above. For modification of cells, the description of modification of cells related to GPCR genes, such as introduction of GPCR genes, can be applied mutatis mutandis. The gene as exemplified above may be a host cell-derived gene or a heterologous gene. In addition, the genes exemplified above may or may not have the same origin as the GPCR gene. When two or more genes are introduced, each gene should be maintained in the host cell so that it can be expressed. Those genes may all be retained on a single expression vector, or all may be retained on the chromosome, for example. In addition, those genes may be maintained separately on multiple expression vectors, or may be separately maintained on single or multiple expression vectors and chromosomes, for example. The genes exemplified above and the proteins encoded by them may have, for example, the base and amino acid sequences of known genes and proteins, respectively. In addition, the genes and proteins encoded by the above-exemplified genes may be, for example, conservative variants of known genes and proteins, respectively. For conservative variants of genes and proteins, the descriptions for GPCR genes and conservative variants of GPCRs can be applied mutatis mutandis.

The “original function” of Gas may be the function of activating adenylate cyclase by coupling with GPR120/GPR113 (eg, GPR120 protein in response to a target substance) in response to a target substance. Activation of adenylate cyclase may, for example, increase intracellular cAMP levels. Specifically, the “original function” of Gas is the function of increasing the intracellular cAMP concentration by coupling with GPR120/GPR113 (for example, the GPR120 protein in response to the target substance) in response to the target substance. may

The “original function” of Gαq may be the function of activating phospholipase C by coupling with GPR120/GPR113 (eg, GPR120 protein in response to a target substance) in response to a target substance. Phospholipase C activation may, for example, increase intracellular IP3 levels and/or intracellular diacylglycerol (DAG) levels. An increase in intracellular IP3 concentration and/or intracellular DAG concentration may, for example, increase intracellular calcium concentration. That is, the "original function" of Gαq specifically refers to intracellular IP3 concentration and/or intracellular DAG coupled with GPR120/GPR113 in response to the target substance (for example, GPR120 protein in response to the target substance). It may be a function of increasing concentration, or a function of increasing intracellular calcium concentration by coupling with GPR120/GPR113 (for example, GPR120 protein in response to a target substance).

A cell having a GPCR gene can be used as a cell having a GPCR (the cell of the present invention) as is, or after expressing the GPCR gene as appropriate. That is, when a cell having a GPCR gene already expresses the GPCR gene, the cell may be used as it is as a cell having the GPCR (the cell of the present invention). In addition, GPCR-containing cells (the cells of the present invention) can be obtained by allowing GPCR gene-containing cells to express the GPCR gene. For example, by culturing a cell having a GPCR gene, the GPCR gene can be expressed, thereby obtaining a cell having the GPCR (the cell of the present invention). Specifically, for example, after introduction (eg, transfection) of the GPCR gene, the host cell can be continued to be cultured to express the GPCR gene. The medium composition and culture conditions are not particularly limited as long as the cells having the GPCR gene can be maintained (eg, grown) and the GPCR gene can be expressed. Upon culturing, the cells with the GPCR gene may or may not proliferate. The medium composition and culture conditions can be appropriately set according to various conditions such as the type of host cells. Cultivation can be carried out using, for example, normal media and normal conditions used for culturing cells such as animal cells, as they are or with appropriate modifications. For example, media that can be used for culturing animal cells specifically include Opti-MEM medium (Thermo Fisher Scientific), DMEM medium, RPMI 1640 medium, and CD293 medium. Cultivation can be performed, for example, at 36° C. to 38° C. in an atmosphere containing CO ₂ such as 5% CO ₂ by static culture. In addition, if necessary, a selective drug or an expression inducer can be used.

Expression of a GPCR can be confirmed by measuring the response to a target substance when combined with another GPCR. Expression of GPCR can also be confirmed by measuring the amount of mRNA transcribed from the GPCR gene, or by detecting GPCR by Western blotting using an antibody.

The cells of the invention can be used in the methods of the invention, for example, as is (as contained in the culture) or harvested from the culture medium. In addition, the culture and the cells collected therefrom may be used in the method of the present invention after being subjected to appropriate treatments such as washing, concentration, dilution, and immobilization. Thus, the cells of the present invention may be used, for example, in a form isolated to a desired degree, or in a form contained in a material. The same is true for other constructs with GPCRs.

Cell membranes containing GPCRs can be prepared, for example, from the cells of the present invention. GPCR-containing cell membranes are specifically obtained, for example, as membrane fractions when the cells of the present invention are disrupted.

In addition, GPCRs can be used to produce artificial lipid bilayer vesicles and artificial lipid bilayer membranes having GPCRs. For example, an artificial lipid bilayer vesicle or an artificial lipid bilayer membrane having a GPCR can be prepared by incorporating a GPCR into a pre-prepared artificial lipid bilayer vesicle or artificial lipid bilayer membrane. In addition, for example, artificial lipid bilayer vesicles and artificial lipid bilayer membranes having GPCRs can be prepared by preparing GPCRs in artificial lipid bilayer vesicles and artificial lipid bilayer membranes using GPCRs as raw materials. can. For the preparation of artificial lipid bilayer membrane vesicles having GPCRs and artificial lipid bilayer membranes, GPCRs in appropriate forms such as membrane fractions having GPCRs can be used. Artificial lipid bilayer membrane vesicles and artificial lipid bilayer membranes can be produced, for example, by known means. For example, methods for producing artificial lipid bilayer membranes include the Montal-Mueller method and the droplet contact method (Kawano R. et al., Automated Parallel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3, No. 1995 ( 2013)
). For example, US2018-0095071 discloses that an artificial lipid bilayer membrane was prepared using a crude membrane fraction obtained from cultured cells. An artificial lipid bilayer vesicle may have a GPCR, eg, in its membrane. Lipid bilayer vesicles include liposomes.

Membranes such as cell membranes and artificial lipid bilayer membranes can be used, for example, so that spaces separated by the membrane are created. Such membranes can be used, for example, to separate two spaces, such as two wells. That is, such a membrane can be used to provide a reaction system comprising two spaces, e.g., two wells, the two spaces being separated from each other by the membrane. can. Such two spaces may have at least part of their boundary separated by such a membrane. Such a reaction system may be provided, for example, as an apparatus as described above.

In the methods of the invention, a combination of both GPCRs (ie GPR120 protein and GPR113 protein) is utilized. Both GPCRs may or may not be produced together. When both GPCRs are produced together, they can be used as they are in the method of the present invention. Moreover, when both GPCRs are not produced together, these GPCRs can be appropriately combined after production and used in the method of the present invention. Specifically, both GPCRs may be used in the method of the present invention in the coexistence of the GPCRs so that the GPCRs can function together. For example, when both GPCRs are used in the form of being carried on a structure, these GPCRs can be used in the form of being carried by a single structure. Both GPCRs can be used, for example, in a single cell-loaded form. Such cells are obtained, for example, by co-expressing both GPCRs in a host cell.

<4> Method of the present invention The method of the present invention can be performed in vitro.

First, GPR120/GPR113 and the test substance can be brought into contact.

The system in which the contact between GPR120/GPR113 and the test substance is carried out is also called "reaction system". Contacting GPR120/GPR113 with the test substance can be carried out in a suitable liquid. The liquid in which the contact between GPR120/GPR113 and the test substance is carried out is also called "reaction liquid". That is, for example, GPR120/GPR113 and the test substance can be brought into contact by coexisting GPR120/GPR113 and the test substance in an appropriate reaction solution. Specifically, for example, GPR120/GPR113 (e.g., in the form of cells having GPR120/GPR113, etc., as exemplified above) and a test substance are dissolved, suspended, dispersed, etc. in a suitable liquid medium and allowed to coexist. This allows contact between GPR120/GPR113 and the test substance. Liquid media include aqueous media such as water and aqueous buffers. When two or more components are brought into contact with GPR120/GPR113 together, the contact between those components and GPR120/GPR113 may or may not start at the same time. That is, for example, after contacting one component with GPR120/GPR113 is initiated, another component may be added to the reaction system. Reaction conditions (conditions for contacting GPR120/GPR113 with a test substance) are not particularly limited as long as the target substance can be screened. The reaction conditions can be appropriately set according to various conditions such as the type of use of GPR120/GPR113, the type of test substance, and the method of measuring the response of GPR120/GPR113. As the reaction conditions, for example, known reaction conditions for measuring interactions between substances such as interactions between proteins and ligands may be used as they are or after being modified as appropriate. The concentration of the test substance can be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, or 1 μM to 10 mM. The concentration of GPR120/GPR113 can be, for example, 1 pg/mL to 10 mg/mL. In addition, when cells having GPR120/GPR113 are used, the concentration of cells having GPR120/GPR113 may be, for example, 10 cell/mL to 10,000,000 cell/mL. Contact between GPR120/GPR113 and test substance may or may not be terminated at an appropriate time. Contact between GPR120/GPR113 and the test substance may generally be continued at least until measurement of the response of GPR120/GPR113 to the test substance. The duration of contact between GPR120/GPR113 and the test substance is, for example, 0.1 seconds or longer, 0.5 seconds or longer, 1 second or longer, 5 seconds or longer, 10 seconds or longer, 20 seconds or longer, 30 seconds or longer, and 40 seconds. 50 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more, 24 hours or less, 12 hours 6 hours or less, 2 hours or less, or 1 hour or less, or any compatible combination thereof. Specifically, the duration of contact between GPR120/GPR113 and the test substance is, for example, 1
hours to 6 hours. The reaction system contains other components in addition to GPR120/GPR113 (e.g., cells having GPR120/GPR113 in the form exemplified above) and test substances, as long as the target substance can be screened. you can Other components can be appropriately set according to various conditions such as the usage form of GPR120/GPR113, the type of test substance, and the method of measuring the response of GPR120/GPR113. Other components include salts such as calcium salts, carbon sources such as glucose, other medium components, and pH buffers. Moreover, a copper ion is mentioned as another component.

The response of GPR120/GPR113 to the test substance can then be measured. A response of GPR120/GPR113 to a substance is also referred to as "a substance elicits a response of GPR120/GPR113".

Responses of GPR120/GPR113 to test substances include binding of GPR120/GPR113 to test substances and activation of GPR120/GPR113 by test substances.

The timing of measuring the response of GPR120/GPR113 to the test substance is not particularly limited as long as the response of GPR120/GPR113 to the test substance is measurable when the test substance is the target substance. The timing of measuring the response of GPR120/GPR113 to the test substance can be appropriately set according to various conditions such as the mode of use of GPR120/GPR113, the type of test substance, and the method of measuring the response of GPR120/GPR113. Specifically, the timing for measuring the response of GPR120/GPR113 to the test substance is a suitable time from the time when contact between GPR120/GPR113 and the test substance begins until the time when the response of GPR120/GPR113 to the test substance disappears. It can be time. The timing of measuring the response of GPR120/GPR113 to the test substance is, for example, the time when the maximum response of GPR120/GPR113 to the test substance is obtained (for example, the time when the amount of binding of the test substance to GPR120/GPR113 is maximized, The time point at which the degree of activation of GPR120/GPR113 by the test substance is maximal). In addition, the timing for measuring the response of GPR120/GPR113 to the test substance is, for example, 0.1 second after the start of contact between GPR120/GPR113 and the test substance.
After 5 seconds, after 1 second, after 5 seconds, after 10 seconds, after 20 seconds, after 30 seconds, after 40 seconds, after 50 seconds, after 1 minute, after 2 minutes, After 3 minutes, after 5 minutes, after 10 minutes, after 30 minutes, after 1 hour, or after 2 hours, up to 24 hours, up to 12 hours, up to 6 hours , 2 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, or 1 minute; It may be a combination that does not. Specifically, the timing of measuring the response of GPR120/GPR113 to the test substance may be, for example, from 1 second to 10 minutes after the start of contact between GPR120/GPR113 and the test substance.

Based on the response of GPR120/GPR113 to the test substance, it can then be identified whether the test substance is the substance of interest. That is, based on the response of GPR120/GPR113 to the test substance, the test substance can be identified as the target substance.

Specifically, when a GPR120/GPR113 response to a test substance is observed, the test substance can be identified as the target substance. That is, for example, when binding of GPR120/GPR113 to a test substance or activation of GPR120/GPR113 by a test substance is observed, the test substance can be identified as a target substance.

The activation of GPR120/GPR113 by the test substance is the degree of activation of GPR120/GPR113 (degree of activation D1) when the step (A) is performed (i.e., under the conditions in which GPR120/GPR113 is brought into contact with the test substance). can be determined as an index. That is, the step (B) may be, for example, (B1) a step of measuring the degree of activation D1. Further, the step (C) may be, for example, (C1) a step of identifying whether the test substance is the target substance based on the degree of activation D1.

Specifically, the activation of GPR120/GPR113 by the test substance is the degree of activation of GPR120/GPR113 (in conditions of contacting GPR120/GPR113 with the test substance) when the step (A) is carried out (activity It can be determined by comparing the degree of activation D1) with the degree of activation of GPR120/GPR113 in control conditions (degree of activation D2). That is, the step (C1) may be, for example, (C2) a step of identifying whether the test substance is the target substance based on the difference between the degree of activation D1 and the degree of activation D2.

"Control condition" refers to the following condition (C2-1) or (C2-2):
(C2-1) Conditions under which GPR120/GPR113 and the test substance are not brought into contact;
(C2-2) Conditions for contacting GPR120/GPR113 with a test substance, where the concentration of the test substance is lower than the concentration of the test substance in step (A) above.

In other words, the activation of GPR120/GPR113 by a test substance can be determined using, for example, the difference in the degree of activation of GPR120/GPR113 due to the presence or absence of the test substance or the difference in concentration.

The condition (C2-1) includes conditions before contacting GPR120/GPR113 with the test substance. The above condition (C2-1) is a condition after contacting GPR120 / GPR113 with the test substance, in which the test substance is substantially (for example, completely) removed from the reaction system, and GPR120 / Also included are conditions in which the GPR113 response is substantially (eg, completely) abolished. The concentration of the test substance under the condition (C2-2) is not particularly limited as long as the concentration allows measurable difference between the degree of activation D1 and the degree of activation D2. The concentration of the test substance under the condition (C2-2) is, for example, 90% or less, 70% or less, 50% or less, 30% or less, 20% or less, 10% of the test substance concentration in the step (A). Below, 5% or less, or 1% or less concentration. Other than the presence or absence or concentration of the test substance, the control condition is not particularly limited as long as the response of GPR120/GPR113 to the test substance can be evaluated. Control conditions can be, for example, identical to the conditions of step (A) above, except for the presence or concentration of the test substance.

The method of the invention may comprise the step of measuring the degree of activation D2. The degree of activation D1 and the degree of activation D2 may be measured with a time lag in a single reaction system, or may be measured in separate reaction systems simultaneously or with a time lag. The degree of activation D2 may be measured before or after the degree of activation D1. For example, after measuring the degree of activation D2, a test substance may be added to the reaction system to measure the degree of activation D1.

When the degree of activation D1 is high, it can be determined that the test substance activates GPR120/GPR113. Specifically, when the degree of activation D1 is higher than the degree of activation D2, it can be determined that the test substance has activated GPR120/GPR113. For example, the ratio of the degree of activation D1 to the degree of activation D2 is 112% or more, 113% or more, 114% or more, 115% or more, 117% or more, 120% or more, 125% or more, 130% or more, 140% 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, or 300% or more, GPR120/GPR113 by the test substance
It may be determined that the activation of Specific examples of the ratio of the degree of activation D1 to the degree of activation D2 include the ΔF/F value described in Examples.

The method for measuring the response of GPR120/GPR113 to test substances is not particularly limited. A method for measuring the response of GPR120/GPR113 to a test substance can be appropriately selected according to various conditions such as the type of use of GPR120/GPR113 and the type of response to be measured. That is, the response of GPR120/GPR113 to a test substance can be measured, for example, by a suitable technique capable of measuring binding between GPR120/GPR113 and the test substance or activation of GPR120/GPR113 by the test substance.

The method for measuring the binding between GPR120/GPR113 and the test substance is not particularly limited. The binding between GPR120/GPR113 and a test substance can be measured by known techniques for measuring binding between substances such as binding between proteins and ligands. Such techniques include, for example, Isothermal Titration Calorimetry (ITC), Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance (NMR) Fluorescence Correlation Spectroscopy. Spectroscopy; FCS).

There is no particular limitation on the method for measuring the activation of GPR120/GPR113 by the test substance. Activation of GPR120/GPR113 by a test substance can be measured, for example, by known techniques for measuring activity of receptors such as GPCRs. Examples of such techniques include a method of measuring intracellular calcium concentration and a method of measuring intracellular cAMP concentration. That is, activation of GPR120/GPR113 by a test substance can be measured using, for example, intracellular calcium concentration or intracellular cAMP concentration as an indicator. Thus, activation of GPR120/GPR113 by a test substance
Specifically, for example, using cells having GPR120/GPR113, intracellular calcium concentration or intracellular cAMP concentration can be measured as an indicator. For example, when GPR120/GPR113 is activated by a test substance, it may couple with Gas to activate adenylate cyclase, thus increasing intracellular cAMP levels. Also, for example, when GPR120/GPR113 is activated by a test substance, it may couple with Gαq to activate phospholipase C, thereby increasing intracellular IP3 concentration and/or intracellular diacylglycerol concentration. An increase in intracellular IP3 concentration and/or intracellular diacylglycerol concentration may, for example, increase intracellular calcium concentration. Techniques for measuring intracellular cAMP concentration include, for example, ELISA and reporter assay. Reporter assays include, for example, luciferase assays. According to the reporter assay, the intracellular cAMP concentration can be measured using a reporter gene (luciferase gene, etc.) that is configured to be expressed depending on the cAMP concentration.
Techniques for measuring intracellular calcium concentration include, for example, calcium imaging. In calcium imaging, a calcium indicator can be used to measure intracellular calcium concentration. Calcium indicators include calcium-sensitive fluorescent dyes and calcium-sensitive fluorescent proteins. Examples of calcium-sensitive fluorescent dyes include Fura 2 and Fluo 4. Examples of calcium-sensitive fluorescent proteins include Cameleon, TN-XL, GCaMP, and G-GECO. Specific examples of Cameleon include Yellow Cameleon 2.60 (YC2.60) (PNAS vol.101:10554-10559 (2004)). Detection of a signal (for example, fluorescence) in calcium imaging can be performed using a detector suitable for the type of signal, such as a fluorescence detector. Examples of fluorescence detectors include confocal laser microscopes such as FV1200 (manufactured by Olympus) and high-throughput screening systems such as FDSS7000 (manufactured by Hamamatsu Photonics). The FDSS7000 supports multi-well plate measurements such as 96-well and 384-well plates, and can test multiple test substances at once. "Calcium concentration" may mean free calcium ion concentration.

The description of measurement of activation of GPR120/GPR113 using cells having GPR120/GPR113 can also be applied mutatis mutandis to the use of GPR120/GPR113 in other embodiments. Other embodiments of using GPR120/GPR113 include the use of artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes having GPR120/GPR113. Examples of using GPR120/GPR113 in other embodiments include, in particular, cases in which GPR120/GPR113 is used in a form having an internal space.

That is, for example, using artificial lipid bilayer membrane vesicles having GPR120/GPR113, the activation of GPR120/GPR113 can be measured by the same method as in the case of using cells having GPR120/GPR113. In such cases, "cell" in the description of measurement of GPR120/GPR113 activation using GPR120/GPR113-bearing cells can be read as "artificial lipid bilayer vesicles".

In addition, for example, a cell membrane having GPR120/GPR113 or a membrane such as an artificial lipid bilayer membrane is used so that a space separated by the membrane is generated, and GPR120/GPR120/GPR120/GPR120/ Activation of GPR113 can be measured. Specifically, for example, if such a membrane is used to separate two spaces, i.e., such a membrane is a reaction system comprising two spaces, the two spaces being the When used to provide a membrane-separated reaction system, activation of GPR120/GPR113 can be measured in a manner similar to using cells with GPR120/GPR113. In such cases,
The space bounded by the membrane can be considered the interior of the cell (also referred to as the "internal space"). Specifically, one of these two spaces can be regarded as the interior of the cell (also referred to as the "internal space"), and the other can be regarded as the exterior of the cell (also referred to as the "external space"). Of those spaces, the one containing the test substance can be considered the external space. In such cases, the terms "intracellular calcium concentration" and "intracellular cAMP concentration" in the description of measuring activation of GPR120/GPR113 using GPR120/GPR113-bearing cells are respectively referred to as "intracellular calcium concentration ” and “cAMP concentration in the inner space”.

In either case, measurable parameters can be selected according to the mode of use of GPR120/GPR113.

It should be noted that "measure a certain parameter and use it as an index for measuring the response of GPR120/GPR113 to a test substance" means that the response can be measured (i.e., it can be determined whether the response is observed). , it is sufficient to obtain and use the data that reflects the parameter, and it is not necessary to obtain the value of the parameter itself. That is, when data reflecting a certain parameter is acquired, it is not necessary to calculate the value of the parameter itself from the data. Specifically, for example, when measuring the intracellular calcium concentration by calcium imaging and using it as an index for measuring the activation of GPR120/GPR113 by a test substance, the activation can be measured (i.e., the activity As long as it is possible to determine whether or not the calcium concentration is recognized), it is sufficient to obtain and use data that reflects the intracellular calcium concentration (e.g., fluorescence intensity), and it is not possible to calculate the intracellular calcium concentration itself from the data. do not need.

Thus, the target substance can be identified. The method of the present invention may further comprise a step of evaluating the ability of the identified target substance to enhance the oral sensation of fats and oils. That is, by evaluating the function of the identified target substance to enhance the oral sensation of fats and oils, it is possible to confirm whether the target substance actually enhances the oral sensation of fats and oils. There is no particular limitation on the method for evaluating the function of the identified target substance to enhance the sensation in the oral cavity of fats and oils. The function of the identified target substance to enhance the sensation of the fat in the oral cavity can be evaluated, for example, by a known method for evaluating the taste of the substance. Sensory evaluation (evaluation by sensory test) is mentioned as such a method. Specifically, the function of the identified target substance to enhance the oral sensation of fats and oils is, for example, the oral sensation of fats and oils in the presence of the target substance and the oral sensation of fats and oils in the absence of the target substance. It can be evaluated by comparing the feeling.

The use of the screened target substance is not particularly limited. The target substance can be used, for example, by blending it with an object containing fats and oils (for example, food and drink). By blending the target substance, it is possible to enhance the sensation of fat in the oral cavity in a fat-containing object. In addition, by blending the target substance, even if the content of fats and oils is reduced in an object containing fats and oils, it is possible to maintain the feeling of fats and oils in the oral cavity. Fats and oils include triglycerides (ie, triacylglycerols). Specific examples of fats and oils include animal-derived fats and oils (animal fats and oils) and plant-derived fats and oils (vegetable fats and oils). Examples of food and drink containing fat include food and drink containing 0.5% by weight or more, preferably 2.0% by weight or more of fat. Specific examples of foods and drinks containing fats and oils include dressings, cheese, butter, margarine, mayonnaise, lard, soup, curry, yogurt, fried foods, snacks, cream, and chocolate. The amount of the target substance to be used is not particularly limited as long as it is an amount effective for enhancing the sensation of fat in the oral cavity. The amount of the target substance to be used can be appropriately set according to various conditions such as the type of object containing fats and oils. The amount of the target substance used may be, for example, 0.0001 ppm to 1000 ppm by weight, preferably 0.0002 ppm to 500 ppm by weight, in terms of concentration in a fat-containing object (eg, food and drink). The target substance can also be used, for example, as a raw material for the development of a new target substance.

EXAMPLES Non-limiting examples are given below to more specifically describe the present invention.

<1> Construction of Human GPR120 Protein Expression Vector The sequence of full-length cDNA encoding human GPR120 protein is registered in GenBank of NCBI (accession No. NM — 001195755) (SEQ ID NO: 1). Using human mRNA as a template, RT-PCR reaction was performed using the primers shown in SEQ ID NOs: 5 and 6 to amplify a DNA fragment containing full-length cDNA encoding human GPR120 protein. The resulting DNA fragment was subcloned into the HindIII-XbaI site of the plasmid pcDNA3.1(+) (Life Technologies) using GENEART Seamless cloning and Assembly kit (A13288, Life Technologies) to generate a human GPR120 protein expression vector. It was constructed. The plasmid pcDNA3.1(+) has a cytomegalovirus-derived promoter sequence and can be used to express the polypeptide encoded by the cloned fragment in animal cells.

<2> Construction of human GPR113 protein expression vector The sequence of full-length cDNA encoding human GPR113 protein is registered in GenBank of NCBI (accession No. NM_001145168) (SEQ ID NO: 3). Using human mRNA as a template, RT-PCR reaction was performed using the primers shown in SEQ ID NOS: 7 and 8 to amplify a DNA fragment containing full-length cDNA encoding human GPR113 protein. The resulting DNA fragment was subcloned into plasmid pcDNA3.1(+) (Life Technologies) using GENEART Seamless cloning and assembly kit (A13288, Life Technologies) to construct a human GPR113 protein expression vector.

<3> Construction of human mGluR4 protein expression vector The sequence of full-length cDNA encoding human mGluR4 protein is registered in GenBank of NCBI (accession No. NM_000841) (SEQ ID NO: 11). Using human mRNA as a template, RT-PCR reaction was performed using the primers shown in SEQ ID NOs: 9 and 10 to amplify a DNA fragment containing full-length cDNA encoding human mGluR4 protein. The resulting DNA fragment was subcloned into plasmid pcDNA3.1(+) (Life Technologies) using restriction enzymes HindIII (New England Biolabs) and NotI (New England Biolabs) to construct a human mGluR4 protein expression vector. In addition, mGluR4 was used as a receptor (negative control) unrelated to the function of GPR120.

<4> Gene introduction into cultured cells and response evaluation to GPR120 agonist
Expression vector pGα15-trans48LD of Gα15-trans48LD in PEAKrapid cells (ATCC CRL-2828)
(JP-A-2015-192664) was introduced by a conventional method to obtain cells that constitutively express Gα15-trans48LD. The same cells maintained in DMEM/Ham's F-12 (Nacalai Tesque) medium containing 10% Fetal bovine serum (Nichirei) and 1% Pen Strep (GIBCO) were washed with D-PBS(-) (Nacalai Tesque). , cells were harvested from flasks using 0.25% Trypsin EDTA (GIBCO). After centrifugation (1,200 rpm, 3 min), the supernatant was removed and suspended in 5% FBS DMEM/Ham's F-12 at 0.75×10 ⁷ cells/ml. 10 ml of this was seeded in a 150 cm ² flask (IWAKI) and cultured overnight (37°C, 5% CO ₂ ). The next day, the medium was replaced with 30 ml of Opti-MEM (Invitrogen, Life Technologies), and four expression vector mixture solutions (1. GPR113 expression vector and pcDNA3.1(+), 2. GPR120 expression vector and pcDNA3.1(+), 3. GPR120 expression vector and GPR113 expression vector, and 4. GPR113 expression vector and mGluR4 expression vector; A total of 60.0 μg of the expression vector mixed with ) was slowly added to the cell suspension and cultured (37° C., 5% CO ₂ ) for 6 hours to introduce genes.

After the gene transfer, the cells were washed with D-PBS(-), and the cells were detached from the flask using 0.25% Trypsin EDTA (GIBCO) and collected. After counting the number of cells, the cells were suspended in 5% FBS DMEM (containing 2.78 mM glucose, GIBCO) medium at 0.5×10 ⁶ cells/ml. This cell suspension was seeded into each well of a D-Lysine coat 96-well plate (BD Biosciences) at 7.0×10 ⁴ cells and cultured overnight.

After culturing, discard all the medium in the 96 wells and add assay buffer (20 mM HEPES, 146 mM NaCl, 1
200 μl of a staining solution for intracellular calcium ion measurement (Calcium assay kit Express (Molecular Device) diluted 80-fold with mM MgSO ₄ , 1.39 mM glucose, 1 mM CaCl ₂ , 2.5 mM Probenecid) was added and incubated at 37° C. for 30 minutes. After resting at room temperature for 45 minutes, staining was performed. Assay buffer after staining
Using FDSS μCELL (Hamamatsu Photonics), the test substance solution prepared in 1. was added to the plate at 50 μl/well, and fluorescence values were measured for up to 120 seconds after stimulation. By measuring the fluorescence values (Ex480:Em540) before and after stimulation, the change in intracellular free calcium ion concentration induced by the addition of stimulants via GPR120 and G protein was quantitatively measured. Measurement and analysis of fluorescence values were performed using software attached to FDSSμCELL (FDSS7000EX), and ΔF/F values were calculated by the following formula and used for evaluation. The ΔF/F value indicates the strength of the cell's response to the added test substance.
ΔF/F = (maximum fluorescence value after stimulation - minimum fluorescence value after stimulation)/fluorescence value before stimulation

Using the obtained ΔF/F values, _EC50 values for each test substance were calculated using analysis software Graphpad Prism 7 (Graphpad). The _EC50 value is the concentration of test substance that produces 50% of the maximal response. That is, the lower the _EC50 value, the higher the sensitivity for detecting the test substance, indicating that the test substance can be detected in a lower concentration range.

As test substances, the following three GPR120 agonists were used: linoleic acid (WAKO), compound 1 (4-[(4-Fluoro-4'-methyl[1,1'-biphenyl]-2 -yl)methoxy]-benzenepropanoic acid: TUG-891, Mol Pharmacol.84(5):710-25.), compound 2 (2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1,2 -benzisothiazol-3(2H)-one-1,1-dioxide; Example 9 of WO2010104195). Compound 1 and compound 2 were synthesized in-house and used.

Cellular responses (ΔF/F values) to GPR120 agonists are shown in FIGS. _EC50 values are shown in Table 1. In the case of GPR113-single-expressing cells, almost no increase in ΔF/F value with increasing concentration of GPR120 agonist was observed for any of the GPR120 agonists. i.e. GPR113
Single-expressing cells showed no response to either GPR120 agonist. On the other hand, in the case of GPR120 single-expressing cells, GPR120/GPR113 co-expressing cells, and GPR120/mGluR4 co-expressing cells, an increase in the ΔF/F value was observed with increasing concentration of the GPR120 agonist for all GPR120 agonists. That is, GPR120 single-expressing cells, GPR120/GPR113 co-expressing cells, and GPR120/mGluR4 co-expressing cells responded to any GPR120 agonist. GPR120/mGluR4 co-expressing cells responded to any GPR120 agonist to the same extent as GPR120 single-expressing cells. On the other hand, GPR120/GPR113 co-expressing cells responded to any GPR120 agonist at lower concentrations compared to GPR120 alone and GPR120/mGluR4 co-expressing cells, i.e. lower _EC50 values. showed that. Thus, although GPR113 alone does not respond to GPR120 agonists, by combining GPR120 and GPR113, it is possible to detect GPR120 agonists with higher sensitivity than when GPR120 is used alone. was done. GPR120 agonists may function as substances that enhance the oral sensation of fats and oils. In other words, it was shown that by combining GPR120 and GPR113, it is possible to detect a substance that enhances the sensation of fat in the oral cavity with high sensitivity.

[Description of Sequence Listing]
SEQ ID NO: 1: base sequence of human GPR120 gene (cDNA) SEQ ID NO: 2: amino acid sequence of human GPR120 protein SEQ ID NO: 3: base sequence of human GPR113 gene (cDNA) SEQ ID NO: 4: amino acid sequence of human GPR113 protein SEQ ID NOs: 5 to 10: primers SEQ ID NO: 11: base sequence of human mGluR4 gene (cDNA)

Claims (20)

A method for screening a target substance,
The following steps (A) to (C):
(A) contacting GPR120/GPR113 with a test substance;
(B) measuring the response of said GPR120/GPR113 to said test substance; and (C) identifying said test substance as a target substance based on said response;
including
identifying the test substance as a target substance if the response is observed;
The target substance is a substance that enhances the oral sensation of fats and oils,
A method, wherein said GPR120/GPR113 is a combination of GPR120 and GPR113 proteins. 2. The method of claim 1, wherein said response is binding of said GPR120/GPR113 to said test substance or activation of said GPR120/GPR113 by said test substance. 3. The method according to claim 1 or 2, wherein said GPR120/GPR113 is used in the form carried by cells, cell membranes, artificial lipid bilayer vesicles, or artificial lipid bilayer membranes. The method according to any one of claims 1 to 3, wherein said GPR120/GPR113 is used in a cell-loaded form. 5. The method of claim 3 or 4, wherein said cells are animal cells. 6. The method of any one of claims 3-5, wherein the cell has the alpha subunit of the G protein. 7. The method of claim 6, wherein the alpha subunit is Gas or Gaq. The method of any one of claims 1 to 7, wherein steps (B) and (C) are performed by steps (B1) and (C1), respectively:
(B1) the step of measuring the degree of activation D1 of the GPR120/GPR113 when the step (A) is carried out;
(C1) A step of identifying the test substance as a target substance based on the degree of activation D1. 9. The method of claim 8, wherein step (C1) is performed by step (C2):
(C2) the degree of activation D1 and the degree of activation of GPR120/GPR113 in control conditions D
identifying the test substance as a target substance based on the difference from 2; The method according to claim 9, wherein the control condition is the following condition (C2-1) or (C2-2):
(C2-1) Conditions under which the GPR120/GPR113 and the test substance are not brought into contact;
(C2-2) Conditions for contacting the GPR120/GPR113 with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in the step (A). 11. The method of claim 9 or 10, further comprising measuring the degree of activation D2. A method according to any one of claims 9 to 11, wherein said test substance is identified as a target substance if said degree of activation D1 is higher than said degree of activation D2. The method according to any one of claims 3 to 12, wherein the response is measured using intracellular calcium concentration or intracellular cAMP concentration as an indicator. The method according to any one of claims 1 to 13, wherein the GPR120 protein is a protein according to (A) or (B) below:
(A) a mammalian GPR120 protein;
(B) Chimeric proteins of two or more mammalian GPR120 proteins. The method according to any one of claims 1 to 14, wherein the GPR120 protein is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(b) an amino acid sequence comprising substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and linoleic acid in combination with GPR113 protein; 4-[(4-Fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid or 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1 a protein responsive to ,2-benzisothiazol-3(2H)-one-1,1-dioxide ;
(c) 4-[(4-Fluoro- 4' -methyl[ linoleic acid, 4-[(4-Fluoro-4'-methyl[ 1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid or 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1,2-benzisothiazol-3(2H)-one-1 A protein responsive to ,1-dioxide . The method according to any one of claims 1 to 15, wherein the GPR113 protein is a protein according to (A) or (B) below:
(A) a mammalian GPR113 protein;
(B) Chimeric proteins of two or more mammalian GPR113 proteins. The method according to any one of claims 1 to 16, wherein the GPR113 protein is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4;
(b) an amino acid sequence comprising substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4, and linoleic acid in combination with GPR120 protein; 4-[(4-Fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid or 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1 a protein responsive to ,2-benzisothiazol-3(2H)-one-1,1-dioxide ;
(c) contains an amino acid sequence having 90 % or more identity to the amino acid sequence shown in SEQ ID NO: 4, and in combination with GPR120 protein linoleic acid, 4-[(4-Fluoro-4'-methyl[ 1,1'-biphenyl]-2-yl)methoxy]-benzenepropanoic acid or 2-[3-fluoro-(pyridin-3-yloxy)phenyl]-1,2-benzisothiazol-3(2H)-one-1 A protein responsive to ,1-dioxide . The method according to any one of claims 1 to 17, wherein the target substance is a GPR120 agonist. The method according to any one of claims 1 to 18, further comprising a step of evaluating the function of the identified target substance to enhance the oral sensation of fats and oils. 20. The method of claim 19, wherein said evaluation is performed by sensory evaluation.

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