Method for evaluating and / or selecting agents for inhibiting odors caused by amines

Abstract

To search for substances capable of selectively inhibiting odors caused by amines.SOLUTION: A method for evaluating and / or selecting agents for inhibiting odors caused by amines includes measuring the response of at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8, and their functionally equivalent polypeptides after the addition of a test substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for discovering perfume materials that suppress odors caused by amines. [Background technology]

[0002] In our living environment, there are many malodorous molecules with different polarities and molecular weights, and amines are one of them. Amines include compounds known to be the causative agents of various malodorous odors, such as urine odor, tobacco odor, cooking odor, body odor, fecal odor, and the unpleasant odor of skin tanning agents, as well as specific malodorous substances designated under the Offensive Odor Prevention Law (e.g., Patent Documents 1-3). Technology that can suppress odors caused by amines will lead to the solution of these malodorous issues.

[0003] As a countermeasure against odors caused by amines, a deodorizing technology based on a chemical neutralization reaction has been proposed, but it is not immediately effective because it takes time for the odorous substances to decrease. In addition, a technology that masks the odors using fragrances that have a stronger odor than the amines has been proposed, but such a technology does not deodorize the odors caused by amines and cannot fundamentally eliminate the odors caused by amines. In addition, the odor of the fragrance may cause discomfort.

[0004] In mammals such as humans, odors are recognized when odor molecules bind to olfactory receptors on olfactory nerve cells present in the olfactory epithelium in the upper part of the nasal cavity, and the receptor response to this is transmitted to the central nervous system. In humans, it has been reported that there are about 400 types of olfactory receptors, and the genes that code for these account for about 2% of all human genes. In general, olfactory receptors and odor molecules correspond in a multiple-to-multiple combination. That is, each olfactory receptor receives multiple odor molecules with similar structures with different affinities, while each odor molecule is received by multiple types of olfactory receptors. In addition, it has been reported that an odor molecule that activates a certain olfactory receptor acts as an antagonist that inhibits the activation of another olfactory receptor. The combination of responses of these multiple types of olfactory receptors results in the recognition of each odor.

[0005] In the past, in the development of fragrance substances, the odor of candidate substances was evaluated by sensory testing by experts. However, sensory testing has problems such as the need to train experts who can evaluate odors and low throughput. In recent years, a method for searching for fragrance substances using the response of olfactory receptors to candidate substances as an index has been developed (Patent Document 4). Since the response of olfactory receptors is selective to odor substances, it is essential to first find an olfactory receptor that has selectivity for the target odor. If such an olfactory receptor is identified, for example, when the purpose is to deodorize a bad odor, an approach based on olfactory receptor suppression by an antagonist or cross-adaptation by an agonist becomes possible. In other words, it becomes possible to more effectively suppress odors caused by amines, which are bad odors. In fact, several olfactory receptors that respond to amines have been identified so far. For example, OR5K1 (Patent Document 1) has been identified as an olfactory receptor that responds to pyrazine derivatives, OR2W1, OR4S2, OR5K1, and OR5P3 (Patent Document 2) as olfactory receptors that respond to 3-ethylpyridine and 2-ethylpyrazine, and TAAR5 (Patent Document 3) as a receptor that responds to trimethylamine. However, existing receptor analysis methods have only succeeded in functional analysis of about 12% of all human receptors (Non-Patent Document 1). Thus, the olfactory system has not yet been elucidated, and therefore, there has been a problem in that it is not easy to find all olfactory receptors that are sensitive to a specific odor using a molecular biological approach.

[0006] The reason why most olfactory receptors have not been successfully analyzed is that even if the olfactory receptor polypeptide of interest is produced in cultured cells, it does not migrate to the surface of the cell (membrane expression) but remains in the endoplasmic reticulum. Therefore, it is not possible to evaluate the binding to odorants administered from outside the cell. The reason why olfactory receptors are not expressed on the membrane has long been unknown, but in recent years, mouse olfactory receptors that are expressed on the membrane in cultured cells and mouse olfactory receptors that are not expressed on the membrane have been comparatively analyzed (Non-Patent Document 2). As a result, it was found that olfactory receptors that are not expressed on the membrane may have low stability of their three-dimensional structure. And, importantly, it was shown that there are amino acid positions in the primary amino acid sequence of the protein that are statistically significantly different in Grantham distance between olfactory receptors that can be expressed on the membrane and those that cannot, and that these amino acid positions are amino acids that are highly common in all approximately 1,000 types of mouse olfactory receptors. In other words, it was suggested that olfactory receptors that are not expressed on the membrane lack stability of their three-dimensional structure due to a mutation to a different amino acid at a position in the polypeptide where a common amino acid should be used, and that a decision was made not to migrate to the cell membrane in cultured cells. Taking this into consideration, it is believed that by using the "consensus method," which introduces amino acids that are highly common between olfactory receptors, it will be possible to obtain the desired olfactory receptor as a stable protein and efficiently express it on the cell membrane.

[0007] Based on this idea, in Non-Patent Document 1, a consensus method was used to enable analysis of specific olfactory receptors. Specifically, for the three types of human olfactory receptors OR6Y1, OR6B2, and OR56A4, consensus olfactory receptors OR6Y1, OR6B2, and OR56A4 were produced by introducing amino acids with high commonality between the homologous genes of 10 types of mammals (gorilla, bonobo, chimpanzee, Sumatran orangutan, rhesus monkey, drill, common marmoset, gray mouse lemur, rat, and mouse) into each human olfactory receptor. As a result, it has been disclosed that, of the three types of olfactory receptors, OR6Y1 has become possible to measure the response to odorants using cultured cells. Therefore, it is suggested that although there are receptors for which consensus analysis is possible, the proportion is about 1 / 3.

[0008] The consensus method has been used for a long time to design industrially useful enzymes to be stable. However, according to Non-Patent Document 3, a review of this method, the probability of introducing consensus into a certain amino acid sequence to obtain an improvement effect is about 50%, and the remaining 40% has the risk of worsening the protein, which makes it difficult to use the consensus method. In addition, there is a view that the application of the consensus method is not appropriate in research aimed at estimating the original function of olfactory receptors. In other words, there is a concern that the approach of introducing amino acid substitutions will change the ligand binding site of the original olfactory receptor, and as a result, the original odor responsiveness will not be observed. From the above, consensus is predicted to have two problems: a low success rate and a risk of changing the original function of the target olfactory receptor, so there have been no examples of its effectiveness against a wide range of olfactory receptors.

[0009] Under these circumstances, the present inventors have conducted extensive research into methods for expressing and functioning olfactory receptors on the membrane of cultured cells, and have found that by modifying the amino acid sequence of a target olfactory receptor based on a consensus amino acid sequence derived from an alignment of the amino acid sequence of the target olfactory receptor with the amino acid sequence of an olfactory receptor encoded by a specific ortholog or specific orthologs and paralogs of the target olfactory receptor according to criteria other than those of the conventional methods, it is possible to improve the membrane expression of the modified olfactory receptor in cultured cells compared to the original olfactory receptor, improve the odor responsiveness of the modified olfactory receptor compared to the original olfactory receptor, and even if the membrane expression of the modified olfactory receptor in cultured cells is not improved compared to the original olfactory receptor, the odor responsiveness of the modified olfactory receptor can be improved, the modified olfactory receptor can maintain the ligand selectivity of the original olfactory receptor before modification, and the analytical results obtained using the modified olfactory receptor closely reflect the human sense of smell (Patent Document 5).

[0010] On the other hand, trace amine associated receptors (TAARs) are G protein-coupled receptors (GPCRs) like olfactory receptors, and six types are known in humans. Of these, TAARs other than TAAR1 are highly expressed in the olfactory epithelium, and have been shown to play a role in selectively and sensitively recognizing volatile amines and generating the sense of smell (Non-Patent Document 4). There have been no known examples of consensus-based trace amine associated receptors. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 6061955 [Patent Document 2] Special Publication No. 2017-176134 [Patent Document 3] International Publication No. 2016 / 126903 [Patent Document 4] JP 2018-74944 A [Patent Document 5] JP 2023-2484 A [Non-patent literature]

[0012] [Non-Patent Document 1] Trimmer C et al. PNAS 116:9475-9480 (2019) [Non-Patent Document 2] Ikegami K et al. PNAS 117:2957-2967 (2020) [Non-Patent Document 3] Porebski TB et al. Protein Engineering, Design & Selection 29:245-251 (2016) [Non-Patent Document 4] Liberles SD Curr Opin Neurobiol. 34:1-7 (2015) Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides a method for efficiently evaluating and / or selecting an agent for suppressing odor caused by amines, using the response of olfactory receptors or trace amine-associated receptors as an index. [Means for solving the problem]

[0014] The present inventors have discovered that membrane expression in cultured cells can also be improved for trace amine-related receptors by applying a consensus method similar to that described in Patent Document 5, and have previously filed a patent application (Patent Application No. 2022-205847).

[0015] By using such a consensus method, the present inventors have succeeded in identifying new olfactory receptors and trace amine-related receptors that selectively respond to amines. Furthermore, the present inventors have found that by using the responses of the olfactory receptors, the trace amine-related receptors, or polypeptides having similar functions as indicators, it is possible to efficiently search for substances that suppress odors caused by amines and to detect odors caused by amines in a sample.

[0016] Therefore, the present invention provides the following 1) and 2). 1) A method for evaluating and / or selecting an inhibitor of amine-induced odor, comprising measuring the response of at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8 and polypeptides having equivalent functions thereto after addition of a test substance. 2) A method for detecting an odor caused by an amine, comprising contacting a sample with at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8 and polypeptides having equivalent functions thereto, and measuring the response of the olfactory receptor polypeptide to the sample. Effect of the Invention

[0017] According to the present invention, it is possible to efficiently evaluate or select a substance capable of selectively suppressing an odor caused by an amine, and also to detect an odor caused by an amine in a sample. [Brief description of the drawings]

[0018] [Figure 1] Luciferase assay of receptors. Bar graphs show the response magnitude to varying concentrations of piperidine, pyridine, cadaverine or trimethylamine for each consensus receptor. EC50 values ​​obtained by sigmoidal curve fitting of the data are shown. Error bars represent SEM (from three replicates in one experiment). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0020] As used herein, the term "agonist" refers to a substance that binds to and activates a receptor, whereas the term "antagonist" refers to a substance that binds to a receptor but does not activate the receptor or suppresses the response of the receptor to an agonist.

[0021] As used herein, "receptor agonism" refers to binding to a receptor and activating the receptor.

[0022] In this specification, "odor cross-adaptation (or olfactory cross-adaptation)" with respect to a target odor refers to a phenomenon in which olfactory sensitivity to a target odor causative substance is reduced or changed by receiving the odor of a substance other than the target odor causative substance in advance and becoming accustomed to the odor. The present inventors have previously revealed that "odor cross-adaptation" is a phenomenon based on receptor agonism (International Publication No. 2016 / 194788). That is, in "odor cross-adaptation", a receptor for a target odor causative substance responds to a different odor causative substance prior to responding to the target odor causative substance, and then desensitizes, so that even if it is later exposed to the target odor causative substance, it can only respond poorly, resulting in a reduction or alteration of the intensity of the target odor recognized by the individual. The mechanism of odor cross-adaptation caused by such receptor behavior is also referred to as "odor cross-adaptation due to receptor agonism" in this specification.

[0023] As used herein, "suppression by receptor antagonism" of a target odor refers to suppressing the receptor response to a substance having the target odor by an antagonist, thereby suppressing the target odor recognized by an individual.

[0024] As used herein, the term "olfactory receptor polypeptide" refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and the term "polypeptide having a function equivalent to that of an olfactory receptor" refers to a polypeptide that can be expressed on the cell membrane, like an olfactory receptor, is activated by the binding of odor molecules, and, upon activation, has the function of increasing the amount of intracellular cAMP by coupling with intracellular Gαs to activate adenylate cyclase (Nat. Neurosci., 2004, 5:263-278).

[0025] As used herein, the term "trace amine related receptor polypeptide" refers to a trace amine related receptor or a polypeptide having a function equivalent thereto, and the term "polypeptide having a function equivalent to that of a trace amine related receptor" refers to a polypeptide which, like the trace amine related receptor, can be expressed on the cell membrane, is activated by binding to a ligand, and, upon activation, has the function of increasing the amount of intracellular cAMP by coupling with the intracellular Gs type G protein α subunit to activate adenylate cyclase (Non-Patent Document 4).

[0026] In the present specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, the identity is calculated by performing analysis with a unit size to compare (ktup) of 2 using the search homology program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development).

[0027] As used herein, "at least 80% identity" with respect to a nucleotide sequence and an amino acid sequence means 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identity. Also, "at least 90% identity" with respect to a nucleotide sequence and an amino acid sequence means 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity.

[0028] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0029] As used herein, amino acid modifications may be designated by the accepted IUPAC one-letter amino acid abbreviations: [original amino acid, position, modified amino acid].

[0030] In the present specification, the "corresponding position" on an amino acid sequence can be determined by aligning a target sequence with a reference sequence (the amino acid sequence shown in SEQ ID NO: 1, 4 or 7 in the present invention) so as to give maximum homology. Alignment of amino acid sequences can be performed using a known algorithm, and the procedure is known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin ([www.clustal.org]), the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0031] In this specification, the term "amine" refers to a compound in which the hydrogen atom of ammonia is replaced with a hydrocarbon group. Preferably, the amine is a cyclic amine or an aliphatic amine. More preferred examples of the amine include cyclic amines such as piperidine and pyridine, and aliphatic amines such as cadaverine and trimethylamine.

[0032] The "odor caused by amines" suppressed by the present invention is an odor caused by the above-mentioned amines, preferably an odor caused by a cyclic amine or an aliphatic amine, and more preferably an odor caused by at least one selected from the group consisting of piperidine, pyridine, cadaverine, and trimethylamine. For example, the odor of piperidine is an animal-like odor, and is included in urine odor, etc. The odor of pyridine is a fish-like odor, and is included in tobacco odor, cooking odor, body odor, defecation odor, etc. The odor of cadaverine is a sperm-like or animal corpse-like odor, and is included in body odor, corpse odor, semen odor, etc. The odor of trimethylamine is a fish-like odor, and is included in cooking odor, body odor, pet odor, garbage odor, etc. Therefore, typically, the "odor caused by amines" suppressed by the present invention may be urine odor, tobacco odor, cooking odor, body odor, defecation odor, corpse odor, semen odor, pet odor, or garbage odor. More preferably, it is body odor.

[0033] The present inventors have previously discovered that by modifying the amino acid sequence of a target olfactory receptor based on a consensus amino acid sequence derived from an alignment of the amino acid sequence of the target olfactory receptor with the amino acid sequence of an olfactory receptor encoded by a specific ortholog or specific ortholog and paralog of the target olfactory receptor, according to criteria different from conventional standards, it is possible to improve the membrane expression of the modified olfactory receptor in cultured cells compared to the original olfactory receptor, improve the odor responsiveness of the modified olfactory receptor compared to the original olfactory receptor, and even if the membrane expression of the modified olfactory receptor in cultured cells is not improved compared to the original olfactory receptor, it is possible to improve the odor responsiveness of the modified olfactory receptor, furthermore, the modified olfactory receptor can well maintain the ligand selectivity of the original olfactory receptor before modification, and the analytical results obtained using the modified olfactory receptor well reflect the human sense of smell (Patent Document 5). In this specification, the olfactory receptor before modification is referred to as the "original olfactory receptor," modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence is referred to as "consensusization," and the consensus-based olfactory receptor is sometimes referred to as the "consensus olfactory receptor."

[0034] Here, the "consensus amino acid sequence" of an olfactory receptor refers to an amino acid sequence consisting of consensus residues identified from an alignment of the amino acid sequence of a target olfactory receptor and an olfactory receptor encoded by a specific ortholog or a specific ortholog and paralog of the target olfactory receptor (e.g., at least 11 types of olfactory receptors selected from the group consisting of olfactory receptors encoded by orthologs of the target olfactory receptor in mammals) according to the following criteria (i) to (iii). (i) at each amino acid position of the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target olfactory receptor and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) when there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target olfactory receptor is identified as a consensus residue; (i-iii) if an amino acid residue is present in the olfactory receptor of interest and no amino acid residue is present with an occurrence frequency of 40% or more, it is identified as having no consensus residue; (i-iv) if no amino acid residue is present in the target olfactory receptor and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and if two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue; (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target olfactory receptor as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or the C-terminus of the olfactory receptor of interest and is not a methionine residue, the consensus residue N-terminal to the consensus residue consisting of the methionine residue at the position closest to the N-terminus is changed to no consensus residue; (iii) When a consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the olfactory receptor of interest and is not a methionine residue, the alignment is traced back one amino acid position at a time toward the N-terminus from the position of the consensus residue until a methionine residue appears, and the most frequently occurring amino acid residue is identified as the consensus residue. If there are two or more types of most frequently occurring amino acid residues, the amino acid residue with the smallest molecular weight among those amino acid residues is identified as the consensus residue. Additionally, herein, "modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence," i.e., "creating a consensus," refers to modifying at least one amino acid residue in the amino acid sequence of the olfactory receptor of interest that differs from the consensus amino acid sequence to an amino acid residue of the consensus amino acid sequence at the corresponding position.

[0035] The above-mentioned consensus olfactory receptor involves the introduction of amino acid mutations into the olfactory receptor, which may at first glance raise concerns about the possibility of providing measurement results that do not accurately reflect the original functions of the original olfactory receptor and human olfactory sensation. However, as shown in Patent Document 5, the consensus olfactory receptor maintains a high degree of ligand response selectivity of the original olfactory receptor and provides measurement results that best explain human olfactory sensation.

[0036] Therefore, although the amino acid sequence of the consensus olfactory receptor has been modified, its responsiveness reflects the responsiveness of the original olfactory receptor in olfactory cells, and its function as an olfactory receptor is equivalent to that of the original olfactory receptor.

[0037] The present inventors have found that consensus OR5AR1 and consensus OR4X2 respond to amines among olfactory receptors that have been subjected to the above-mentioned consensus synthesis. As described above, consensus olfactory receptors maintain the ligand selectivity of the original olfactory receptors to a high degree. That is, the response of the consensus olfactory receptor to a certain odorant reflects the response of the original olfactory receptor to the odorant in the olfactory cell. Therefore, OR5AR1 and OR4X2 can be identified as olfactory receptors that respond to amines.

[0038] In addition, the present inventor has previously found that the membrane expression of the modified trace amine-related receptor in cultured cells can be improved compared to the original trace amine-related receptor by modifying the amino acid sequence of the target trace amine-related receptor based on a consensus amino acid sequence derived from the alignment of the amino acid sequence of the target trace amine-related receptor and the amino acid sequence of the trace amine-related receptor encoded by a specific ortholog of the target trace amine-related receptor according to the same criteria as in the case of the olfactory receptor, and filed a patent application (Patent Application No. 2022-205847). In this specification, the trace amine-related receptor before modification is referred to as the "original trace amine-related receptor", modifying the amino acid sequence of the trace amine-related receptor based on the consensus amino acid sequence is referred to as "consensusization", and the consensus-made trace amine-related receptor is sometimes referred to as the "consensus trace amine-related receptor".

[0039] Here, the "consensus amino acid sequence" for a trace amine related receptor is an amino acid sequence consisting of consensus residues identified from an alignment of the amino acid sequence of a target trace amine related receptor and the amino acid sequence of a trace amine related receptor encoded by a specific ortholog of the target trace amine related receptor (for example, at least two types of trace amine related receptors selected from the group consisting of trace amine related receptors encoded by orthologs of the target trace amine related receptor in mammals) according to the following criteria (i) to (iii). (i) at each amino acid position of the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target trace amine-related receptor and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) when there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target trace amine-related receptor is identified as a consensus residue; (i-iii) if an amino acid residue is present in the target trace amine-related receptor and no amino acid residue is present with an occurrence frequency of 40% or more, it is identified as having no consensus residue; (i-iv) when no amino acid residue is present in the target trace amine-related receptor and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and when two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue. (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target trace amine-related receptor as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or the C-terminus of the target trace amine-related receptor and is not a methionine residue, change the consensus residue N-terminal to no consensus residue, the consensus residue consisting of the methionine residue at the position closest to the N-terminus. (iii) When a consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the target trace amine-related receptor and is not a methionine residue, the amino acid residue that occurs most frequently is identified as the consensus residue by going back one amino acid position at a time N-terminally from the position of the consensus residue in the alignment until a methionine residue appears, and if there are two or more types of most frequently occurring amino acid residues, the amino acid residue with the smallest molecular weight among those amino acid residues is identified as the consensus residue. Furthermore, herein, "modifying the amino acid sequence of a trace amine related receptor based on a consensus amino acid sequence", i.e., "creating a consensus", refers to modifying at least one amino acid residue in the amino acid sequence of a target trace amine related receptor that differs from the consensus amino acid sequence to an amino acid residue of the consensus amino acid sequence at a corresponding position.

[0040] The present inventors found that the consensus TAAR8 responds to amines among the trace amine-related receptors obtained by applying the above-mentioned consensus method. Therefore, TAAR8 can be identified as a trace amine-related receptor that responds to amines.

[0041] It has not previously been recognized that OR5AR1, OR4X2 and TAAR8 are or may be responsive to amines.

[0042] As shown in FIG. 1, consensus OR5AR1 responds to piperidine in a concentration-dependent manner. Consensus OR4X2 responds to pyridine in a concentration-dependent manner. Consensus OR5AR1 responds to cadaverine in a concentration-dependent manner. Consensus TAAR8 responds to trimethylamine. Thus, OR5AR1, OR4X2 and TAAR8 are newly discovered amine receptors. Substances that suppress the response of OR5AR1, OR4X2, TAAR8 or polypeptides having equivalent functions to these can cause changes in the recognition of odors caused by amines in the central nervous system based on odor suppression by receptor antagonism, and as a result, can selectively suppress odors caused by amines. On the other hand, substances that enhance the response of OR5AR1, OR4X2, TAAR8 or polypeptides having equivalent functions to these can cause changes in the recognition of odors caused by amines in the central nervous system based on odor cross-adaptation by receptor agonism, and as a result, can selectively suppress odors caused by amines. Therefore, by using substances that suppress or enhance the response of these polypeptides, it is possible to deodorize odors caused by amines without causing problems such as discomfort due to the strong odor of the fragrance, which occurred in conventional deodorizing methods using deodorants or fragrances, or the problem of suppressing other odors.

[0043] Therefore, the present invention provides a method for evaluating and / or selecting an inhibitor of an amine-induced odor (hereinafter referred to as the method for evaluating and / or selecting an odor inhibitor of the present invention). The method includes measuring the response of at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8, and polypeptides having equivalent functions thereto, after addition of a test substance. Based on the measured response, a test substance that enhances or inhibits the response of the receptor polypeptide is detected. The detected test substance is selected as an inhibitor of an amine-induced odor. That is, a test substance that enhances the response of the receptor polypeptide is selected as an inhibitor of an amine-induced odor based on odor cross-adaptation due to receptor agonism, and a test substance that inhibits the response of the receptor polypeptide is selected as an inhibitor of an amine-induced odor based on receptor antagonism.

[0044] The above-mentioned method for evaluating and / or selecting an odor suppressant of the present invention can be carried out in vitro or ex vivo.

[0045] The test substance used in the odor suppressant evaluation and / or selection method of the present invention is not particularly limited as long as it is a substance desired to be used as an odor suppressant caused by amines. The test substance may be a naturally occurring substance, or a substance artificially synthesized by a chemical or biological method, or may be a compound, composition, or mixture.

[0046] The receptor polypeptide used in the odor suppressant evaluation and / or selection method of the present invention is at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8, and polypeptides having equivalent functions thereto. OR5AR1 and OR4X2 are olfactory receptors expressed in human olfactory cells, and TAAR8 is a trace amine-related receptor expressed in human olfactory cells. The accession number and amino acid sequence of each receptor in the NCBI database are shown in Table 1.

[0047] [Table 1]

[0048] An example of a polypeptide having a function equivalent to OR5AR1 includes a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:1 and having responsiveness to at least one selected from the group consisting of piperidine and cadaverine. Another example of a polypeptide having a function equivalent to OR5AR1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 2 below, and having responsiveness to at least one selected from the group consisting of piperidine and cadaverine. Yet another example of a polypeptide having a function equivalent to OR5AR1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 2 below, and having responsiveness to at least one selected from the group consisting of piperidine and cadaverine. Here, having an SVL (2) at the amino acid position corresponding to the C-terminus of (1) in Table 2 below means that in a certain amino acid sequence, an SVL is added to the C-terminus side of the position corresponding to the last amino acid position of the amino acid sequence shown in SEQ ID NO: 1. Note that in the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having amino acid residues (2) at all of the amino acid positions of (1) in Table 2 below is consensus OR5AR1 consisting of the amino acid sequence shown in SEQ ID NO: 3. In other words, Table 2 below shows the amino acid residues that differ between OR5AR1 and consensus OR5AR1.

[0049] Yet another example of a polypeptide having a function equivalent to OR5AR1 includes a polypeptide consisting of consensus OR5AR1 consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of piperidine and cadaverine. The consensus OR5AR1 is composed of the consensus amino acid sequence shown in SEQ ID NO: 3, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR5AR1 shown in SEQ ID NO: 1 and the amino acid sequences of 27 olfactory receptors encoded by orthologues of OR5AR1 in mammals, and is responsive to piperidine and cadaverine. The orthologue is a gene that is highly homologous to OR5AR1 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as OR5AR1. The amino acid sequence identity between the consensus OR5AR1 and OR5AR1 is 77%.

[0050] Examples of polypeptides having functions equivalent to OR4X2 include polypeptides having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 4 and that are responsive to pyridine. Another example of a polypeptide having a function equivalent to OR4X2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (3) in the amino acid sequence shown in SEQ ID NO: 4 in Table 2 below, and having an amino acid residue of (4) at each position, and having a responsiveness to pyridine. Yet another example of a polypeptide having a function equivalent to OR4X2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (3) in the amino acid sequence shown in SEQ ID NO: 4 in Table 2 below, and having an amino acid residue of (4) at each position, and having a responsiveness to pyridine. Here, having MANTHNV (4) at the amino acid position corresponding to position 1 of (3) in Table 2 below means that in a certain amino acid sequence, the amino acid residue at the position corresponding to M at position 1 of the amino acid sequence shown in SEQ ID NO: 4 is replaced with V, and MANTHN is added immediately before it. In addition, in the amino acid sequence shown in SEQ ID NO: 4, a polypeptide having amino acid residues of (4) at all positions of each amino acid position of (3) in Table 2 below is consensus OR4X2 consisting of the amino acid sequence shown in SEQ ID NO: 6. In other words, Table 2 below shows the amino acid residues that differ between OR4X2 and consensus OR4X2.

[0051] Another example of a polypeptide having a function equivalent to OR4X2 includes a polypeptide consisting of consensus OR4X2 consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to pyridine. Consensus OR4X2 consists of the consensus amino acid sequence shown in SEQ ID NO: 6, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR4X2 shown in SEQ ID NO: 4 and the amino acid sequences of 220 olfactory receptors encoded by orthologues of OR4X2 in mammals, and has responsiveness to pyridine. The orthologue is a gene that is highly homologous to OR4X2 among olfactory receptor genes possessed by mammalian species and contains the same name as OR4X2. The amino acid sequence identity between consensus OR4X2 and OR4X2 is 89%.

[0052] An example of a polypeptide having a function equivalent to that of TAAR8 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7 and having responsiveness to trimethylamine. Another example of a polypeptide having a function equivalent to TAAR8 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, having an amino acid residue of (6) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (5) in the amino acid sequence shown in SEQ ID NO: 7 in Table 2 below, and having responsiveness to trimethylamine. Yet another example of a polypeptide having a function equivalent to TAAR8 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, having an amino acid residue of (6) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (5) in the amino acid sequence shown in SEQ ID NO: 7 in Table 2 below, and having responsiveness to trimethylamine. In addition, in the amino acid sequence shown in SEQ ID NO: 7, a polypeptide having amino acid residues of (6) at all of the amino acid positions of (5) in Table 2 below is consensus TAAR8 consisting of the amino acid sequence shown in SEQ ID NO: 9. In other words, Table 2 below shows the amino acid residues that differ between TAAR8 and consensus TAAR8.

[0053] Yet another example of a polypeptide having a function equivalent to TAAR8 includes a consensus TAAR8 consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to trimethylamine. The consensus TAAR8 is composed of the consensus amino acid sequence shown in SEQ ID NO: 9, which is derived by the above-mentioned method from the alignment of the amino acid sequence of TAAR8 shown in SEQ ID NO: 7 and the amino acid sequences of 119 kinds of trace amine-related receptors encoded by orthologues of TAAR8 in mammals, and has responsiveness to trimethylamine. The orthologue is a gene that is highly homologous to TAAR8 among trace amine-related receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as TAAR8. The amino acid sequence identity between the consensus TAAR8 and TAAR8 is 91%.

[0054] [Table 2]

[0055] In the odor suppressant evaluation and / or selection method of the present invention, at least one selected from the above-mentioned receptor polypeptides may be used, but any two or more may be used in combination. Preferably, at least one selected from the group consisting of OR5AR1, OR4X2, TAAR8, consensus OR5AR1, consensus OR4X2, and consensus TAAR8 is used, and more preferably, at least one selected from the group consisting of consensus OR5AR1, consensus OR4X2, and consensus TAAR8 is used.

[0056] In one example, when the amine is piperidine, the receptor polypeptide is preferably at least one selected from the group consisting of OR5AR1 and polypeptides having equivalent functions thereto, more preferably at least one selected from the group consisting of OR5AR1 and consensus OR5AR1, and even more preferably consensus OR5AR1.

[0057] In another example, when the amine is pyridine, the receptor polypeptide is preferably at least one selected from the group consisting of OR4X2 and polypeptides having equivalent functions, more preferably at least one selected from the group consisting of OR4X2 and consensus OR4X2, and even more preferably consensus OR4X2 is used.

[0058] In another example, when the amine is cadaverine, the receptor polypeptide is preferably at least one selected from the group consisting of OR5AR1 and polypeptides having equivalent functions thereto, more preferably at least one selected from the group consisting of OR5AR1 and consensus OR5AR1, and even more preferably consensus OR5AR1.

[0059] In another example, when the amine is trimethylamine, the receptor polypeptide is preferably at least one selected from the group consisting of TAAR8 and polypeptides having equivalent functions thereto, more preferably at least one selected from the group consisting of TAAR8 and consensus TAAR8, and even more preferably consensus TAAR8.

[0060] In the odor suppressant evaluation and / or selection method of the present invention, the receptor polypeptide can be used in any form as long as it does not lose its responsiveness to amines.For example, the receptor polypeptide can be used in the form of tissues or cells that naturally express the receptor polypeptide, such as olfactory receptors or olfactory cells isolated from living organisms, or cultures thereof; the membrane of olfactory cells carrying the receptor polypeptide; recombinant cells genetically engineered to express the receptor polypeptide or cultures thereof; the membrane of the recombinant cell carrying the receptor polypeptide; artificial lipid bilayer membrane carrying the receptor polypeptide, etc. All of these forms are included in the scope of the receptor polypeptide used in the present invention.

[0061] In a preferred embodiment, the receptor polypeptide is a cell that naturally expresses the receptor polypeptide, such as an olfactory cell, or a recombinant cell genetically engineered to express the receptor polypeptide, or a culture of these. The recombinant cell can be prepared by transforming a cell with a vector incorporating a gene encoding the receptor polypeptide.

[0062] Preferably, in order to promote cell membrane expression of the receptor polypeptide, a gene encoding a receptor-transporting protein (RTP) is introduced into the cell together with the gene encoding the receptor polypeptide. Preferably, a gene encoding RTP1S is introduced into the cell together with the gene encoding the receptor polypeptide. An example of RTP1S is human RTP1S. Human RTP1S is registered in GenBank as GI:50234917, and is a polypeptide consisting of the amino acid sequence of SEQ ID NO:11, encoded by a gene having the nucleotide sequence of SEQ ID NO:10.

[0063] Methods for adding a test substance to the receptor polypeptide include, but are not limited to, adding the test substance to the culture medium in which cells expressing the receptor polypeptide are cultured, or directly adding, dripping, sprinkling or spraying the test substance onto the receptor polypeptide or cells or tissues containing it.

[0064] In the method for evaluating and / or selecting an odor suppressant of the present invention, the response of the receptor polypeptide is measured following the addition of a test substance to the receptor polypeptide. The measurement may be performed by any method known in the art as a method for measuring the response of an olfactory receptor or a trace amine-related receptor, such as measuring the amount of intracellular cAMP. For example, it is known that when an olfactory receptor is activated by an odor molecule, it couples with a G protein α subunit classified as the Gαs family in the cell to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP (Nat.Neurosci., 2004, 5:263-278). On the other hand, when an olfactory receptor is activated by an odor molecule, it also couples with a protein belonging to the Gq family, such as Gα15, in the cell, thereby increasing the amount of calcium ions in the cell. For example, it is known that when a trace amine-related receptor is activated by a ligand, it couples with a G protein α subunit classified as the Gs family in the cell to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP. On the other hand, when the trace amine-related receptor is activated by a ligand, it also couples with proteins belonging to the Gq family, such as Gα15, in the cell, and can increase the amount of calcium ions in the cell. Therefore, the response of the olfactory receptor polypeptide or the trace amine-related receptor polypeptide can be measured by using the amount of intracellular cAMP or the amount of calcium ions, or the behavior of downstream molecules activated through them as an index. Methods for measuring the amount of cAMP include ELISA, reporter gene assay, and a method using a cAMP-responsive reporter protein. In the method using a cAMP-responsive reporter protein, for example, a firefly luciferase genetically modified to contain a part of a cAMP-binding protein is used, and the amount of cAMP is measured by monitoring the luminescence value of the luciferase corresponding to the amount of cAMP (GloSensor (trademark) cAMP Assay (Promega)). Methods for measuring calcium ion concentration include calcium imaging and TGFα shedding assay.As an example of a method that uses the behavior of downstream molecules activated via the amount of cAMP as an indicator, the two-electrode voltage clamp method is also effective in measuring the potential changes inside and outside the cell membrane of Xenopus oocytes mediated by the cystic fibrosis transmembrane conductance regulator CFTR, which is activated by cAMP signals.

[0065] In a first embodiment of the present invention, the method for evaluating and / or selecting an inhibitor of amine-induced odor according to the present invention comprises adding a test substance to the above-mentioned receptor polypeptide; and measuring the response of the receptor polypeptide to the test substance. Then, based on the measured response, a test substance that enhances the response of the receptor polypeptide is detected. The detected test substance is selected as an inhibitor of amine-induced odor.

[0066] The test substance that enhances the response of the receptor polypeptide can weaken the response of the receptor when it is exposed to amine later by enhancing the response of the receptor first.As a result, it can suppress the recognition of the odor caused by amine by an individual based on odor cross-adaptation.Therefore, in the first embodiment, the suppressor of the odor caused by amine is selected based on the odor cross-adaptation by receptor agonism.

[0067] The effect of a test substance on the receptor polypeptide can be evaluated, for example, by comparing the response of the receptor polypeptide (test group) to which a test substance has been added with the response in a control group. Examples of the control group include the receptor polypeptide to which no test substance has been added, the receptor polypeptide to which a control substance has been added, the receptor polypeptide to which a lower concentration of a test substance has been added, the receptor polypeptide before the addition of a test substance, and cells in which the receptor polypeptide is not expressed. Preferably, the method for evaluating and / or selecting an odor suppressor of the present invention in the first embodiment includes measuring the activity of the receptor polypeptide in the presence and absence of a test substance. Also preferably, the method for evaluating and / or selecting an odor suppressor of the present invention in the first embodiment includes measuring the response of cells in which the receptor polypeptide is expressed and cells in which the receptor polypeptide is not expressed to the test substance in the presence of a test substance.

[0068] For example, if the response in the test group is enhanced compared to the control group, the test substance can be selected as a substance that inhibits the response of the receptor polypeptide to amines.For example, if the response of the receptor polypeptide in the test group is enhanced preferably by 120% or more, more preferably by 150% or more, and even more preferably by 200% compared to the control group, the test substance can be selected as a substance that inhibits the response of the receptor polypeptide to amines.Alternatively, if the response of the receptor polypeptide in the test group is statistically significantly enhanced compared to the control group, the test substance can be selected as a substance that inhibits the response of the receptor polypeptide to amines.

[0069] Examples of odors caused by amines that are suppressed by the amine-related odor inhibitor selected according to the first embodiment include urine odor, cigarette odor, cooking odor, body odor, defecation odor, corpse odor, semen odor, pet odor, garbage odor, piperidine odor, pyridine odor, cadaverine odor, and trimethylamine odor. Preferably, when OR5AR1 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the olfactory receptor, examples include urine odor, body odor, corpse odor, semen odor, piperidine odor, and cadaverine odor. When OR4X2 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the olfactory receptor, examples include cigarette odor, cooking odor, body odor, defecation odor, and pyridine odor. When TAAR8 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the trace amine-related receptor, examples include cooking odor, body odor, pet odor, garbage odor, and trimethylamine odor. More preferably, it is body odor.

[0070] An embodiment of the use of the amine-induced odor suppressant selected according to the first embodiment is as follows: First, a subject who desires to suppress an amine-induced odor is made to smell the suppressant before the subject is exposed to the odor. Alternatively, the suppressant is applied to the subject so that the odor is stronger than the amine-induced odor. As a result, even if the subject is exposed to the amine-induced odor, the odor is perceived as weak or not felt due to the reduced olfactory sensitivity to the odor.

[0071] In a second embodiment of the present invention, the method for evaluating and / or selecting an inhibitor of odor caused by amines according to the present invention comprises adding a test substance and an agonist of the receptor polypeptide to the above-mentioned receptor polypeptide; and measuring the response of the receptor polypeptide to the agonist. The agonist is not limited to, but is preferably at least one selected from amines, more preferably at least one selected from cyclic amines and aliphatic amines, and even more preferably at least one selected from the group consisting of piperidine, pyridine, cadaverine, and trimethylamine. More preferably, when the receptor is specifically OR5AR1 or a polypeptide having an equivalent function thereto as the olfactory receptor, at least one selected from the group consisting of piperidine and cadaverine is used; when the receptor is specifically OR4X2 or a polypeptide having an equivalent function thereto as the olfactory receptor, pyridine is used; and when the receptor is specifically TAAR8 or a polypeptide having an equivalent function thereto as the trace amine-related receptor, trimethylamine is used. Then, based on the measured response, a test substance that inhibits the response of the receptor polypeptide to the agonist is detected. The test substances detected are selected as inhibitors of odor caused by amines.

[0072] In the second embodiment, a test substance that inhibits the response of the receptor polypeptide to an agonist of the receptor polypeptide is selected as an inhibitor of amine-based odors based on receptor antagonism.

[0073] The effect of the test substance on the response of the receptor polypeptide to the agonist can be measured, for example, by comparing the response of the receptor polypeptide to which the test substance has been added (test group) to the agonist with the response of the control group to the agonist. Examples of the control group include those mentioned above. Preferably, the method for evaluating and / or selecting an odor suppressor of the present invention in the second embodiment includes measuring the activity of the receptor polypeptide to the agonist in the presence and absence of a test substance.

[0074] For example, if the response in the test group is more suppressed than that in the control group, the test substance can be selected as a substance that suppresses the response of the receptor polypeptide to an amine. For example, if the response of the receptor polypeptide in the test group is suppressed preferably by 60% or less, more preferably by 50% or less, and even more preferably by 25% or less compared to the control group, the test substance can be selected as a substance that suppresses the response of the receptor polypeptide to an amine. Alternatively, if the response of the receptor polypeptide in the test group is statistically significantly suppressed compared to the control group, the test substance can be selected as a substance that suppresses the response of the receptor polypeptide to an amine.

[0075] The amine-based odors suppressed by the amine-based odor suppressor selected according to the second embodiment include the same amine-based odors suppressed by the amine-based odor suppressor selected according to the first embodiment described above.

[0076] One embodiment of the use of the amine-based odor suppressant selected according to the second embodiment is as follows: First, a subject who desires to suppress an amine-based odor is made to smell the suppressant before or at the same time that the subject is exposed to the odor. As a result, even when the subject is exposed to the amine-based odor, the odor is perceived as weak or not detected due to the reduced olfactory sensitivity to the odor.

[0077] By the above procedure, an inhibitor of an amine-induced odor can be obtained based on the response activity of the receptor polypeptide responsive to an amine-induced odor. If necessary, the ability of the above-selected test substance to inhibit an amine-induced odor may be further evaluated by a sensory test. That is, in one embodiment of the odor inhibitor evaluation and / or selection method of the present invention, the test substance selected by the above procedure is obtained as a candidate substance for an inhibitor of an amine-induced odor. Then, the inhibitory effect of the candidate substance on an amine-induced odor is evaluated by a sensory test. The candidate substance that is evaluated better in the sensory test is selected as an inhibitor of an amine-induced odor.

[0078] The sensory test of the candidate substance can be performed according to the evaluation procedure of deodorants that is usually performed in the field. If the candidate substance is an inducer of cross-adaptation of odor, the order of application of the candidate substance and the substance causing the target odor to the evaluator can be adjusted. For example, when the test substance selected in the first embodiment described above is used as a candidate substance for sensory test, the evaluator first smells the candidate substance and adapts to the odor. Then, the evaluator smells the target odor (preferably the odor caused by amine) and evaluates its intensity. The obtained evaluation result is compared with the intensity of the target odor when the candidate substance is not adapted. Also, for example, when the test substance selected in the second embodiment described above is used as a candidate substance for sensory test, the evaluator smells the target odor (preferably the odor caused by amine) at the same time as the candidate substance, and evaluates the intensity of the target odor. The obtained evaluation result is compared with the intensity of the target odor alone. Candidate substances that are evaluated as reducing the intensity of the target odor as a result of the sensory test are selected as inhibitors of odors caused by amines.

[0079] The amine-induced odor suppressant obtained by the odor suppressant evaluation and / or selection method of the present invention can be used as an active ingredient for suppressing amine-induced odor.For example, the suppressant can be contained in a composition or article for suppressing amine-induced odor as an active ingredient for suppressing amine-induced odor.Alternatively, the suppressant can be used for the manufacture of a composition or article for suppressing amine-induced odor. Examples of applications of the amine-caused odor suppressant obtained by the odor suppressant evaluation and / or selection method of the present invention include sanitary products such as sanitary products and disposable diapers containing the agent; clothing accessories such as underwear, undergarments, and linens, cloth products, or textiles containing the agent; laundry detergents or fabric softeners containing the agent; cosmetics, cleaning agents, external preparations such as deodorants, medicines, foods, etc. containing the agent; placing or spraying the agent in the kitchen: placing or spraying the agent in a refrigerator; placing or spraying the agent in front of or in a trash can; placing or spraying the agent in front of or in a toilet; placing or spraying the agent in a hospital ward or care facility, etc.; a method in which a person involved in excretion treatment in a hospital ward or care facility, etc., carries the agent or is exposed to the agent before the treatment; application to environments in which odors caused by amines are generated, but are not limited to these.

[0080] As described above, OR5AR1, OR4X2, and TAAR8 are newly discovered amine receptors, and therefore, by using OR5AR1, OR4X2, TAAR8, or a polypeptide having a function equivalent thereto, an odor caused by an amine can be detected. Thus, the present invention provides a method for detecting an odor caused by an amine (hereinafter, referred to as the detection method of the present invention). The method includes contacting a sample with at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8, and a polypeptide having a function equivalent thereto; and measuring the response of the receptor polypeptide to the sample. Based on the measured response, an odor caused by an amine contained in the sample is detected.

[0081] Here, the term "detection" can be replaced with "evaluation" or "measurement", and "detection of an odor caused by an amine" includes proving the presence or absence of an odor caused by an amine and measuring the degree of the odor caused by an amine.

[0082] The sample used in the detection method of the present invention is not particularly limited as long as it is a sample for which confirmation of the presence or absence or the level of odor caused by amine is desired. The sample may be a compound, a composition, or a mixture, and may be a gas, a liquid, or a solid. The sample is preferably a real sample, and examples of the real sample include samples derived from body fluids (e.g., saliva, sweat, urine), wastewater, food, fragrances, cosmetics, medicines, detergents, daily necessities, and laundry.

[0083] The receptor polypeptide and its form used in the detection method of the present invention are the same as the receptor polypeptide and its form used in the odor suppressor evaluation and / or selection method of the present invention.

[0084] Methods for contacting a sample with the receptor polypeptide include, but are not limited to, adding the sample to a culture medium in which cells expressing the receptor polypeptide are cultured, and directly adding, dripping, scattering or spraying the sample onto the receptor polypeptide or cells or tissues containing it.

[0085] The response of the receptor polypeptide can be measured using the same techniques as those used in the evaluation and / or selection of odor suppressants of the present invention.

[0086] Based on the measured response, the odor caused by the amine in the sample is detected.The sample that causes the response of the receptor polypeptide can be evaluated as a sample that exhibits an odor caused by amine, in other words, a sample that contains amine.The stronger the response intensity caused by the sample, the stronger the odor caused by the amine exhibited by the sample, in other words, the more amines are contained in the sample.

[0087] Preferably, the response of the receptor polypeptide to a sample can be evaluated by comparing the response of the receptor polypeptide (test group) contacted with a sample with the response of the receptor polypeptide in a control group. The control group can include the receptor polypeptide to which no sample has been added, the receptor polypeptide to which a control substance has been added, the receptor polypeptide contacted with a lower concentration of a sample, the receptor polypeptide before contacting with a sample, etc. If the response in the test group is higher than that in the control group, the sample is evaluated as a sample exhibiting an odor caused by an amine.

[0088] Therefore, in one embodiment of the detection method of the present invention, the response of the receptor polypeptide in the presence and absence of a sample is measured, and then it is determined whether the response in the presence of a sample is higher than the response in the absence of a sample.If the response in the presence of a sample is higher, the sample is evaluated as a sample that exhibits an odor caused by an amine.In a preferred embodiment, if the response intensity of the receptor polypeptide in the presence of a sample is preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more compared to the absence of a sample, the sample is evaluated as a sample that exhibits an odor caused by an amine.In another preferred embodiment, if the response intensity of the receptor polypeptide in the presence of a sample is statistically significantly increased compared to the absence of a sample, the sample is evaluated as a sample that exhibits an odor caused by an amine.

[0089] Alternatively, the response of the receptor polypeptide to a sample can be evaluated by comparing the response of the receptor polypeptide (test group) to which the sample has been contacted with a calibration curve previously prepared from the response when a standard sample containing an amine of known concentration is used as the sample. If the response in the test group is within the quantitative range of the calibration curve, the sample is evaluated as one that exhibits an odor caused by an amine, and the intensity of the odor can be calculated based on the calibration curve.

[0090] Examples of odors caused by amines detected by the detection method of the present invention include urine odor, cigarette odor, cooking odor, body odor, defecation odor, corpse odor, semen odor, pet odor, food waste odor, piperidine odor, pyridine odor, cadaverine odor, trimethylamine odor, etc. Preferably, when OR5AR1 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the olfactory receptor, urine odor, body odor, corpse odor, semen odor, piperidine odor, cadaverine odor are included; when OR4X2 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the olfactory receptor, cigarette odor, cooking odor, body odor, defecation odor, pyridine odor are included; and when TAAR8 or a polypeptide having a function equivalent thereto is used as the receptor, specifically as the trace amine-related receptor, cooking odor, body odor, pet odor, food waste odor, trimethylamine odor are included. More preferably, it is body odor. EXAMPLES

[0091] The present invention will be described more specifically below with reference to examples.

[0092] Example 1 Identification of olfactory receptors or trace amine-associated receptors that respond to amines 1) Preparation of cells expressing olfactory receptors or trace amine-related receptors The consensus olfactory receptor or consensus trace amine-related receptor was designed in the same manner as described in the examples of Patent Document 5 and Japanese Patent Application No. 2022-205847. In designing the consensus olfactory receptor or consensus trace amine-related receptor, a search for homologous gene candidates of the target olfactory receptor gene or the target trace amine-related receptor gene was performed using NCBI BLAST. An ortholog group was identified for the obtained gene group. Specifically, genes with the same name as the target olfactory receptor were selected as orthologs from the top homologous genes searched by BLAST. For example, in the case of human OR5AR1, the amino acid sequence of human OR5AR1 (NP_001004730.1) was used as the query sequence, and 27 genes with OR5AR1 in their names were selected from the top 250 homologous genes searched by BLAST. These 27 genes were identified as orthologs. The amino acid sequences of these 27 genes and human OR5AR1 were added to the total of 28 genes, and alignment analysis and identification of consensus amino acids were performed as described below. Consensus amino acids were also identified for another human olfactory receptor, OR4X2. In addition, for the genes of Mus musculus and Rattus norvegicus, which use different receptor nomenclature systems, these genes were included in the top 250 genes in the search results, so the most homologous gene of each was also selected as an ortholog. Specifically, genes with the same name as the target trace amine-related receptor were selected as orthologs from the top homologous genes searched by BLAST. For example, in the case of human TAAR8, the amino acid sequence of human TAAR8 (NP_444508.1) was used as the query sequence, and 119 genes with TAAR8 in their name were selected from the top 250 homologous genes searched by BLAST. These 119 genes were identified as orthologs. For the amino acid sequences of these 119 genes plus human TAAR8, a total of 120 genes, we performed alignment analysis and identified consensus amino acids as described below.

[0093] The alignment analysis of the identified gene group was performed using ClustalW, and further adjustment was made to optimize the amino acid or amino acid motif highly conserved between olfactory receptors or trace amine-related receptors. Based on the alignment results, a consensus olfactory receptor or a consensus trace amine-related receptor was designed using Jalview. In the alignment, when there is one amino acid residue different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% or more at a position corresponding to each amino acid position of the original human receptor amino acid sequence serving as a reference, the amino acid residue of the reference amino acid sequence was modified to the amino acid residue. Even if there is one amino acid residue different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% at a position corresponding to each amino acid position of the original human receptor amino acid sequence serving as a reference, if the occurrence frequency of the amino acid residue of the reference amino acid sequence is also 50%, the amino acid residue of the reference amino acid sequence was not modified. In the alignment, when there is a deletion with an occurrence frequency of 40% or more at a position corresponding to each amino acid position of the original human receptor amino acid sequence serving as a reference, the amino acid residue of the reference amino acid sequence was modified to a deletion. On the other hand, when an amino acid with an occurrence frequency of 60% or more exists at a position corresponding to the deletion position of the original reference human receptor amino acid sequence, the most conserved amino acid was inserted at the deletion position of the reference amino acid sequence. When there are two or more types of the most conserved amino acid, the amino acid with the smallest molecular weight was inserted. The topology of the receptor in the design was confirmed using TMHMM (Transmembrane Hidden Markov Model). The DNA sequences encoding the various designed receptor polypeptides were obtained by DNA synthesis after optimizing the base sequence codons corresponding to the amino acid sequences for expression in human cultured cells. The base sequences with optimized codons encoding consensus OR5AR1, consensus OR4X2, and consensus TAAR8 consisting of the amino acid sequences shown in SEQ ID NOs: 3, 6, and 9 are shown in SEQ ID NOs: 2, 5, and 8, respectively. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined with EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector. In addition, a gene encoding human RTP1S, which transfers receptor proteins made in cultured cells onto the cell membrane, was inserted into the EcoRI and XhoI sites of another pME18S vector to create the pME18S-RTP1S vector.

[0094] A reaction solution with the composition shown in Table 3 was prepared, left to stand in a clean bench for 20 minutes, and then added to each well of a 96-well plate (BD). For the olfactory receptor OR4X2, the composition shown in Table 4 was used. Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well at 2 × 10 5 cells / cm 2 The cells were seeded at 100°C and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that do not express the receptor (Mock) were prepared.

[0095] [Table 3]

[0096] [Table 4]

[0097] 2) Luciferase assay The receptor expressed in HEK293 cells increases the amount of intracellular cAMP by coupling with endogenous GαS and activating adenylate cyclase. To measure odor responses, a luciferase reporter gene assay was used to monitor the increase in intracellular cAMP as luminescence values ​​derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a Renilla luciferase gene fused downstream of the CMV promoter (hRluc2P-CMV-hygro) was simultaneously transfected and used as an internal standard to correct errors in gene transfection efficiency and cell number.

[0098] The medium was removed from the culture prepared in 1) above, and 75 μL of test substance solution (piperidine (CAS: 110-89-4, Fujifilm Wako Pure Chemical Industries, Ltd.), pyridine (CAS: 110-86-1, Fujifilm Wako Pure Chemical Industries, Ltd.), cadaverine (CAS: 462-94-2, Tokyo Chemical Industry Co., Ltd.) or trimethylamine (CAS: 75-50-3, Tokyo Chemical Industry Co., Ltd.), concentration range 0 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM) prepared in new medium was added. The cells were cultured in a CO2 incubator for 4 hours to allow the luciferase gene to be fully expressed in the cells. Luciferase activity was measured using Dual-Glo TM The measurements were performed using a luciferase assay system (Promega) according to the product's operating manual. In each well of a 96-well plate, the luminescence value derived from firefly luciferase induced by stimulation with the test substance was divided by the luminescence value derived from Renilla luciferase to calculate the signal (fluc / hRluc) and used for analysis. For the signals under each transfection condition, the signal value (fluc / hRluc) under the condition without stimulation with odorant was set to 0%, and the signal value (fluc / hRluc) under stimulation with 10 μM forskolin was set to 100%, and the signal was used for analysis as the response (%).

[0099] 3) Results Among the consensus olfactory receptors or consensus trace amine-related receptors tested, the receptors that responded to piperidine, pyridine, cadaverine, or trimethylamine are shown in Figure 1. Figure 1 shows the response values ​​of each receptor to each concentration of piperidine, pyridine, cadaverine, or trimethylamine. A statistically significant difference was observed between the response value (Response (%)) of each receptor to the highest concentration tested, 300 μM, and the response value (Response (%)) of cells under mock conditions in which the receptor is not expressed (Student's t-test, P<0.05). In addition, a significant difference was observed by the same statistical method when comparing the signal value (fluc / hRluc) under conditions in which no stimulation with odorants was performed and the signal value (fluc / hRluc) under conditions in which stimulation was performed with the highest concentration of 300 μM for each receptor. The values ​​in Tables 5 to 7 are EC values ​​calculated by regressing the Response (%) value onto a sigmoid curve. 50 (μM). The EC 50 TAAR8, for which no μM is listed, is a receptor that can respond to trimethylamine, but in the concentration range up to 300 μM tested in this study, there was insufficient data to allow regression to a sigmoid curve, and the EC 50 It means that the ratio was not calculated. Therefore, the consensus OR5AR1 responded to piperidine in a dose-dependent manner. The consensus OR4X2 responded to pyridine in a dose-dependent manner. The consensus OR5AR1 responded to cadaverine in a dose-dependent manner. The consensus TAAR8 responded to trimethylamine. Therefore, it was found that the consensus OR5AR1, the consensus OR4X2, and the consensus TAAR8 responded to at least one amine selected from the group consisting of piperidine, pyridine, cadaverine, and trimethylamine.

[0100] [Table 5]

[0101] [Table 6]

[0102] [Table 7]

[0103] From the above results, the original OR5AR1, OR4X2 and TAAR8 were identified as amine receptors.

Claims

1. A method for evaluating and / or selecting an inhibitor of amine-induced odor, comprising measuring the response of at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8 and polypeptides having equivalent functions thereto after addition of a test substance.

2. 2. The method of claim 1, wherein the amine is at least one selected from the group consisting of piperidine, pyridine, cadaverine, and trimethylamine.

3. The method according to claim 1 or 2, wherein the amine is at least one selected from the group consisting of piperidine and cadaverine when the receptor polypeptide is OR5AR1 or a polypeptide having a function equivalent thereto, pyridine when the receptor polypeptide is OR4X2 or a polypeptide having a function equivalent thereto, and trimethylamine when the receptor polypeptide is TAAR8 or a polypeptide having a function equivalent thereto.

4. The method according to any one of claims 1 to 3, wherein the OR5AR1 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the OR4X2 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, and the TAAR8 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

7.

5. the polypeptide having a function equivalent to OR5AR1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1 and having responsiveness to at least one selected from the group consisting of piperidine and cadaverine; the polypeptide having a function equivalent to OR4X2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4 and having responsiveness to pyridine; The polypeptide having a function equivalent to that of TAAR8 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7 and having responsiveness to trimethylamine. The method according to any one of claims 1 to 4.

6. the polypeptide having a function equivalent to OR5AR1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at at least one position corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of piperidine and cadaverine; The polypeptide having a function equivalent to OR4X2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, having an amino acid residue of (4) at at least one position corresponding to each amino acid position of (3) in the amino acid sequence shown in SEQ ID NO: 4 in Table 1 below, and having responsiveness to pyridine, The polypeptide having a function equivalent to that of TAAR8 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, having an amino acid residue of (6) at at least one position corresponding to each amino acid position of (5) in the amino acid sequence shown in SEQ ID NO: 7 in Table 1 below, and having responsiveness to trimethylamine. The method according to any one of claims 1 to 5. 【Table 1】

7. the polypeptide having a function equivalent to OR5AR1 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 3 and having responsiveness to at least one selected from the group consisting of piperidine and cadaverine; the polypeptide having a function equivalent to OR4X2 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:6 and having responsiveness to pyridine; The polypeptide having a function equivalent to that of TAAR8 is a polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9 and having responsiveness to trimethylamine. The method according to any one of claims 1 to 4.

8. adding a test substance to the receptor polypeptide; and measuring the response of said receptor polypeptide to said test substance; The method according to any one of claims 1 to 7, comprising:

9. The method of claim 8, further comprising selecting a test substance that enhances the response of said receptor polypeptide to said test substance as an inhibitor of amine-induced odor.

10. The method of claim 8, further comprising measuring the response of said receptor polypeptide in a control group.

11. The method of claim 10, further comprising selecting a test substance that enhances the response of the receptor polypeptide to the test substance compared to the control group as an inhibitor of amine-induced odor.

12. adding a test substance and an agonist of the receptor polypeptide to the receptor polypeptide; and measuring the response of said receptor polypeptide to said agonist; The method according to any one of claims 1 to 7, comprising:

13. The method of claim 12, further comprising selecting a test substance that inhibits the response of said receptor polypeptide to said agonist as an inhibitor of amine-induced odor.

14. The method of claim 12, further comprising measuring the response of said receptor polypeptide to said agonist in a control group.

15. The method of claim 14, further comprising selecting a test substance that inhibits the response of the receptor polypeptide to the agonist compared to the control group as an inhibitor of amine-induced odor.

16. contacting a sample with at least one receptor polypeptide selected from the group consisting of OR5AR1, OR4X2, TAAR8, and polypeptides having equivalent functions thereto; and measuring the response of said receptor polypeptide to said sample; A method for detecting an odor caused by an amine, comprising:

17. The method according to any one of claims 1 to 16, wherein the response of the receptor polypeptide is measured by ELISA, measurement of intracellular cAMP level using a reporter gene assay or a cAMP-responsive reporter protein, measurement of calcium ion level using calcium imaging or a TGFα shedding assay, or measurement of potential changes inside and outside the cell membrane using a two-electrode voltage clamp method using Xenopus oocytes.

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