Evaluation and / or selection method for odor suppressants caused by carboxylic acids

Abstract

To discover a substance capable of selectively suppressing odors caused by a carboxylic acid.SOLUTION: A method for evaluating and / or selecting an agent for suppressing odors caused by a carboxylic acid includes measuring the response of at least one odorant receptor polypeptide selected from the group consisting of test substance-added OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, and OR2A12 and polypeptides having equivalent functions to them.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for searching for a fragrance material that suppresses odors caused by carboxylic acids.

Background Art

[0002] In our living environment, there are a large number of malodorous molecules with different polarities and molecular weights, and carboxylic acid is one of them. Carboxylic acids include compounds known as causative substances of various malodors such as body odor, axillary odor, foot odor, fecal odor, cooking odor, tobacco odor, pet odor, and garbage odor, as well as specific malodorous substances designated based on malodor prevention methods (for example, Patent Documents 1 and 2). Technologies that can suppress odors caused by carboxylic acids lead to the solution of these malodor problems.

[0003] As a countermeasure against odors caused by carboxylic acids, a deodorizing technology that adsorbs and decomposes the causative odor substances to reduce their abundance has been proposed, but it lacks immediate effectiveness because it takes time to reduce the odor substances. In addition, a technology for masking with a fragrance having a stronger odor than carboxylic acid has been proposed, but such a technology does not deodorize the odor caused by carboxylic acid and cannot fundamentally eliminate the odor caused by carboxylic acid. In addition, discomfort may be caused by the odor of the air freshener.

[0004] In mammals such as humans, odors are perceived when odor molecules bind to olfactory receptors on olfactory nerve cells located in the olfactory epithelium of the upper nasal cavity, and the receptor's response is transmitted to the central nervous system. In humans, approximately 400 types of olfactory receptors have been reported, and the genes encoding them account for about 2% of all human genes. Generally, olfactory receptors and odor molecules are associated in multiple-to-multiple combinations. That is, individual olfactory receptors receive multiple odor molecules with similar structures at different affinities, while individual odor molecules are received by multiple types of olfactory receptors. Furthermore, it has been reported that odor molecules that activate one olfactory receptor can act as antagonists, inhibiting the activation of another olfactory receptor. The combination of responses from these multiple types of olfactory receptors leads to the recognition of individual odors.

[0005] Traditionally, in the development of fragrance substances, the odor of candidate substances has been evaluated by sensory tests conducted by experts. However, sensory tests have 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 that uses the response of olfactory receptors to candidate substances as an indicator has been developed (Patent Document 3). Since the response of olfactory receptors is selective for odor substances, it is crucial to first find olfactory receptors that are selective for the target odor. Once such olfactory receptors are identified, for example, when the purpose is to deodorize malodors, an approach based on olfactory receptor inhibition by antagonists or cross-adaptation by agonists becomes possible. In other words, it becomes possible to more effectively suppress odors caused by carboxylic acids, which are malodorous. In fact, several olfactory receptors that respond to carboxylic acids have been identified to date. For example, OR2W1, OR10A6, OR51E1, OR51I2, and OR51L1 have been identified as olfactory receptors that respond to hexanoic acid, OR2W1, OR10A6, and OR51E1 as olfactory receptors that respond to nonanoic acid, and OR51I2 and OR51E1 as olfactory receptors that respond to isovaleric acid (Patent Document 1). OR1A1, OR2W1, OR10A6, OR51B2, OR51E1, and OR51I2 have been identified as olfactory receptors that respond to 3-methyl-2-hexenoic acid, and OR10A6 has been identified as an olfactory receptor that responds to 3-hydroxy-3-methylhexanoic acid (Patent Document 2). OR51I2 has been identified as an olfactory receptor that responds to valeric acid, OR51I2, OR51L1, and OR51V1 as olfactory receptors that respond to hexanoic acid, and OR2AK2, OR2W1, OR51L1, and OR51V1 as olfactory receptors that respond to 4-methyl-3-hexenoic acid (Patent Document 4). In addition, OR51E1 has been identified as an olfactory receptor that responds to butyric acid, OR51I2 and OR2W1 as olfactory receptors that respond to 3-methyl-2-hexenoic acid, and OR2W1 as an olfactory receptor that responds to 3-hydroxy-3-methylhexanoic acid (Patent Document 5). However, existing receptor analysis methods have only succeeded in functionally analyzing about 12% of all human receptors (Non-Patent Document 1).Thus, the olfactory system remains largely unknown, and therefore, it is not easy to identify all olfactory receptors sensitive to specific odors using molecular biological approaches.

[0006] The reason why most olfactory receptors have not been successfully analyzed is that even when the target olfactory receptor polypeptide is produced in cultured cells, it does not migrate to the cell surface (membrane expression) but remains in the endoplasmic reticulum. Therefore, it is not possible to evaluate its binding affinity to odor substances administered from outside the cell. The reason why olfactory receptors do not undergo membrane expression remained unknown for a long time, but in recent years, mouse olfactory receptors that undergo membrane expression and mouse olfactory receptors that do not undergo membrane expression have been comparatively analyzed in cultured cells (Non-Patent Literature 2). As a result, it was found that olfactory receptors that do not undergo membrane expression may have low three-dimensional stability. Importantly, there is an amino acid site in the primary amino acid sequence of the protein where the Grantham distance differs statistically significantly between olfactory receptors that can undergo membrane expression and those that cannot, and it was shown that this amino acid site is an amino acid that is highly common to all approximately 1,000 types of mouse olfactory receptors. In other words, it was suggested that olfactory receptors that do not undergo membrane expression lack three-dimensional stability because a mutation to a different amino acid has occurred at the position in the polypeptide where a common amino acid should be used, and the cell cultured cells decide not to migrate them to the cell membrane. Based on this, it is thought that a "consensus method," which involves introducing amino acids with high commonality among olfactory receptors, could make it possible to obtain the target olfactory receptor as a stable protein and to efficiently express it on the cell membrane.

[0007] Based on this idea, Non-Patent Document 1 uses a consensus method to enable the analysis of specific olfactory receptors. Specifically, for three types of human olfactory receptors, OR6Y1, OR6B2, and OR56A4, consensus olfactory receptors OR6Y1, OR6B2, and OR56A4 were created by introducing amino acids with high commonality among homologous genes of 10 different 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 was disclosed that it became possible to measure the response to odor substances using cultured cells for OR6Y1, one of the three olfactory receptors. Therefore, it is suggested that although there are receptors for which response analysis can be performed through consensus, the proportion is only about one-third.

[0008] Consensus synthesis has long been used to design stable enzymes for industrial use. However, according to Non-Patent Literature 3, a review of this method, the probability of obtaining improvement by introducing consensus synthesis to a single amino acid sequence is only about 50%, and the remaining 40% carries the risk of degrading the protein, which makes the use of consensus synthesis difficult. Furthermore, there is a view that the application of consensus synthesis is not appropriate in research aimed at estimating the original function of olfactory receptors. In other words, the approach of introducing amino acid substitutions alters the ligand binding site of the original olfactory receptor, and as a result, there is a concern that the original odor responsiveness will not be observed. For these reasons, consensus synthesis is predicted to have two drawbacks: a low success rate and the risk of altering the original function of the target olfactory receptor. Therefore, there are no examples of its effectiveness being verified for a wide range of olfactory receptors. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 5697383 [Patent Document 2] Patent No. 6422665 [Patent Document 3] Japanese Patent Publication No. 2018-74944 [Patent Document 4] Japanese Patent Publication No. 2020-108370 [Patent Document 5] Special Publication No. 2021-504673 [Non-patent literature]

[0010] [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) [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention provides a method for efficiently evaluating and / or selecting an odor inhibitor that suppresses carboxylic acid-induced odors using the response of olfactory receptors as an indicator. [Means for solving the problem]

[0012] The inventors investigated various previously unconsidered consensus-building methods, such as performing consensus-building of a target olfactory receptor based on the amino acid sequences of olfactory receptors from more species than those described in Non-Patent Document 1. As a result, they discovered a method that can improve cell membrane expression levels and response measurement efficiency for olfactory receptors, and have filed a patent application for this method (Japanese Patent Application No. 2021-103675).

[0013] By using such a consensus method, the present inventors have successfully newly identified an olfactory receptor that selectively responds to carboxylic acids. Further, the present inventors have found that by using the response of the olfactory receptor or a polypeptide having a similar function thereto as an index, it is possible to efficiently search for a substance that suppresses an odor caused by carboxylic acids.

[0014] Therefore, the present invention provides a method for evaluating and / or selecting an inhibitor of an odor caused by carboxylic acids, which comprises measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12 and polypeptides having equivalent functions thereto after addition of a test substance.

Effects of the Invention

[0015] According to the present invention, a substance that can selectively suppress an odor caused by carboxylic acids can be efficiently evaluated or selected.

Brief Description of the Drawings

[0016] [Figure 1]Cell membrane expression levels and odor responsiveness of human olfactory receptors and consensus olfactory receptors designed for the olfactory receptors. The two histograms and one bar graph from left represent the amounts of receptor proteins on the HEK293 cell membrane determined by the Flow cytometry method. In the Flow cytometry method, an anti-FLAG mouse IgG antibody that recognizes the FLAG tag added to the N-terminus of the olfactory receptor was used as the primary antibody, and a phycoerythrin (PE)-conjugated anti-mouse IgG antibody was used as the secondary antibody to measure the PE signal. As controls, cells that did not express the receptor (Mock) and cells that expressed the M2 acetylcholine receptor (M2AcR), which efficiently expresses on the membrane, were analyzed. Normalization was performed with the PE signal of Mock set as 0% and the PE signal of M2AcR set as 100%, and the PE signal of each olfactory receptor was calculated. On the right is shown the result of measuring the ligand responsiveness of the receptor by luciferase assay. Error bars represent SEM (n = 3). [Figure 2] Ligand selectivity of two types of ligands for human olfactory receptors and consensus olfactory receptors designed for the olfactory receptors. The results of the luciferase assay are shown. Error bars represent SEM (n = 3). [Figure 3] Ligand selectivity of human olfactory receptors and consensus olfactory receptors designed for the olfactory receptors. The results of the luciferase assay are shown. Error bars represent SEM (n = 3). The numbers shown in the lower part correspond to the numbers of the odor substances shown in Figure 4, respectively. [Figure 4] Odor substances used in Figure 3. [Figure 5] Luciferase assay of olfactory receptors. For three types of human olfactory receptors and consensus olfactory receptors designed for the olfactory receptors, odor substances were administered at varying concentrations and the responses were measured. Error bars represent SEM (n = 3). [Figure 6]This study involved luciferase assays of olfactory receptors. For each of the four consensus olfactory receptors, modified versions with amino acid substitutions that have been reported to cause individual variability in odor responsiveness were expressed in HEK293 cells. Odor substances were administered at varying concentrations, and the response was measured. Error bars represent SEM (n=3). [Figure 7] Luciferase assay of olfactory receptors. The bar graph shows the response intensity of each odor substance administered at varying concentrations to each consensus olfactory receptor. The EC50 obtained when the data was curve-fitted to a sigmoid curve is shown in the figure. Error bars represent SEM (from 3 copies in one experiment). A: propionic acid, B: butyric acid, C: isobutyric acid, D: valeric acid, E: isovaleric acid, F: hexanoic acid, G: acetic acid, H: 3-hydroxy-3-methylhexanoic acid, I: nonanoic acid. [Modes for carrying out the invention]

[0017] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0018] In this specification, "agonist" means a substance that binds to and activates a receptor. On the other hand, in this specification, "antagonist" means a substance that binds to a receptor but does not activate the receptor or inhibits the receptor's response to the agonist.

[0019] In this specification, "olfactory receptor agonism" means binding to a receptor and activating that receptor.

[0020] In this specification, "odor cross-adaptation (or olfactory cross-adaptation)" with respect to a target odor refers to the phenomenon in which olfactory sensitivity to the target odor-causing substance decreases or changes as a result of prior exposure to the odor of a substance other than the target odor-causing substance and becoming accustomed to that odor. The inventors previously revealed that "odor cross-adaptation" is a phenomenon based on olfactory receptor agonism (International Publication No. 2016 / 194788). That is, in "odor cross-adaptation," the olfactory receptors for the target odor-causing substance respond to a different odor-causing substance before responding to the target odor-causing substance, and then desensitize, so that even when later exposed to the target odor-causing substance, they can only give a low response, and as a result, the intensity of the target odor recognized by the individual decreases or changes. In this specification, the mechanism of odor cross-adaptation caused by this behavior of olfactory receptors is also called "odor cross-adaptation by olfactory receptor agonism."

[0021] In this specification, "inhibition of target odor by olfactory receptor antagonism" means suppressing the response of olfactory receptors to a substance having the target odor using an antagonist, thereby suppressing the target odor that is recognized by the individual.

[0022] In this specification, "olfactory receptor polypeptide" means an olfactory receptor or a polypeptide having equivalent function, and "polypeptide having equivalent function to an olfactory receptor" means a polypeptide that, like an olfactory receptor, can be expressed on the cell membrane, is activated by the binding of odor molecules, and, once activated, increases the amount of intracellular cAMP by activating adenylyl cyclase in conjunction with intracellular Gαs (Nat. Neurosci., 2004, 5:263-278).

[0023] In this specification, the identity of nucleotide and amino acid sequences is calculated using the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, it is calculated by performing the homology analysis (Search homology) using the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a unit size to compare (ktup) of 2.

[0024] In this specification, "at least 80% identity" with respect to nucleotide sequences and amino acid sequences means identity of 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 still more preferably 99% or more.

[0025] In this specification, "amino acid residue" means the 20 amino acid residues that make up 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).

[0026] In this specification, amino acid modifications may be indicated using the recognized IUPAC single-letter amino acid abbreviation as [original amino acid, position, modified amino acid].

[0027] In this specification, the "corresponding position" on the amino acid sequence can be determined by aligning the target sequence with the reference sequence (in this invention, the amino acid sequence indicated by SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, or 22) to give the greatest possible homology. The alignment of amino acid sequences can be performed using known algorithms, and the procedure is known to those skilled in the art. For example, the alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 or Clustal Omega, can be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Databank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]), operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above alignment is considered to be the "corresponding position" to that arbitrary position.

[0028] In this specification, "carboxylic acid" is not particularly limited and may be any of the following: straight-chain short-chain carboxylic acid (1 to 5 carbon atoms), branched short-chain carboxylic acid (1 to 5 carbon atoms), or polymeric carboxylic acid (6 or more carbon atoms). Examples of more preferred carboxylic acids include acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid (n-hexanoic acid), 3-hydroxy-3-methylhexanoic acid, nonanoic acid, etc.

[0029] The "odor caused by carboxylic acids" suppressed by the present invention refers to the odor produced by the carboxylic acids described above, preferably the odor produced by linear short-chain carboxylic acids, branched short-chain carboxylic acids, or polymeric carboxylic acids, and more preferably the odor produced by at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid, and nonanoic acid. For example, the odor of acetic acid is included in body odor, shoe odor, fecal odor, cooking odor, cigarette odor, pet odor, and garbage odor. Propionic acid is a specified malodorous substance, and its odor is included in fecal odor, etc. Butyric acid is a specified malodorous substance, and its odor is included in shoe odor, fecal odor, pet odor, and garbage odor, etc. Valeric acid is a specified malodorous substance, and its odor is included in body odor, shoe odor, fecal odor, and garbage odor, etc. The odor of isobutyric acid is included in body odor, shoe odor, fecal odor, etc. Isovaleric acid is a specific malodorous substance, and its odor is found in body odor, shoe odor, fecal odor, pet odor, etc. Hexanoic acid odor is found in body odor, etc. 3-hydroxy-3-methylhexanoic acid odor is found in underarm odor, etc. Nonanoic acid odor is found in body odor, etc. Therefore, typically, the "odors caused by carboxylic acids" that are suppressed by the present invention may be body odor, underarm odor, shoe odor, fecal odor, cooking odor, cigarette odor, pet odor, and garbage odor. More preferably, it is body odor.

[0030] The inventors have previously found that by modifying the amino acid sequence of a target olfactory receptor based on a consensus amino acid sequence derived from the alignment of the amino acid sequence of the target olfactory receptor with a specific ortholog or specific ortholog and paralog of the target olfactory receptor, membrane expression of the olfactory receptor in cultured cells can be improved, the odor responsiveness of the olfactory receptor can be improved, the modified olfactory receptor can maintain good ligand selectivity of the original olfactory receptor, and the analytical results obtained from the modified olfactory receptor accurately reflect human olfaction. A patent application for this finding has been filed (Japanese Patent Application No. 2021-103675). In this specification, the original olfactory receptor is referred to as the "original olfactory receptor," the modification of the amino acid sequence of the olfactory receptor based on a consensus amino acid sequence is referred to as "consensing," and the consensused olfactory receptor is sometimes referred to as the "consensus olfactory receptor."

[0031] Here, "consensus amino acid sequence" refers to an amino acid sequence consisting of consensus residues identified from the amino acid sequence of the target olfactory receptor and the amino acid sequence alignment of a specific ortholog or specific ortholog and paralog of the target olfactory receptor (for example, at least 11 olfactory receptors selected from the group consisting of olfactory receptors that encode 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 residues of the target olfactory receptor and has an occurrence frequency of 50% or more, that amino acid residue is identified as the consensus residue. (i-ii) If two amino acid residues with an occurrence frequency of 50% are present, the amino acid residue of the target olfactory receptor is identified as the consensus residue. (i-iii) If amino acid residues are present in the olfactory receptor of the target and are absent at an occurrence frequency of 40% or more, it is identified as having no consensus residue. (i-iv) If no amino acid residues are present in the target olfactory receptor, and amino acid residues are present with an occurrence frequency of 60% or more, the amino acid residue with the highest occurrence frequency is identified as the consensus residue. If there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among them is identified as the consensus residue. (iv) If none of the above (ii) to (i-iv) apply, identify the amino acid residue of the olfactory receptor of the subject as the consensus residue. (ii) When consensus residues are identified according to the criteria in (i) above, if the N-terminal consensus residue is located at the N-terminus or C-terminus of the olfactory receptor of interest and is not a methionine residue, the consensus residues N-terminal to the consensus residue consisting of the methionine residue closest to the N-terminus are replaced with no consensus residues. (iii) When a consensus residue is identified according to the criteria in (i) above, if the N-terminal consensus residue is located at a position N-terminal to the N-terminus of the target olfactory receptor and is not a methionine residue, the amino acid position is traced back one amino acid at a time from the position of the consensus residue in the alignment toward the N-terminus until a methionine residue appears, and the amino acid residue with the highest frequency is identified as the consensus residue. If there are two or more amino acid residues with the highest frequency, the amino acid residue with the smallest molecular weight among them is identified as the consensus residue. Furthermore, "modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence," or "consensing," here means changing at least one amino acid residue in the amino acid sequence of the target olfactory receptor that differs from the consensus amino acid sequence to an amino acid residue of the consensus amino acid sequence at a corresponding position.

[0032] The consensus-building of olfactory receptors described above, which involves introducing amino acid mutations into the olfactory receptors, may at first glance raise concerns that it could yield measurement results that do not accurately reflect the original function of the olfactory receptors and the human sense of smell. However, as will be discussed later, consensus-building olfactory receptors maintain a high degree of ligand response selectivity of the original olfactory receptors and, moreover, provide measurement results that best explain the human sense of smell.

[0033] As shown in the previous patent application, for human olfactory receptors with known ligands, a consensus olfactory receptor was created by modifying the amino acid sequence of the olfactory receptor based on a consensus amino acid sequence derived from, for example, an alignment of the amino acid sequences of at least 11 olfactory receptors selected from the group consisting of the amino acid sequence of the olfactory receptor and olfactory receptors that encode orthologs of the olfactory receptor in mammals. When the original olfactory receptor and the consensus olfactory receptor were expressed in cultured cells and the membrane expression level and responsiveness to the ligand were measured, the membrane expression level increased and the responsiveness improved after consensus formulation (Figure 1).

[0034] Furthermore, as shown in the previous patent application, when the response selectivity to ligands was examined using human olfactory receptors with known ligands and consensus olfactory receptors prepared in the same manner as above for said olfactory receptors, no significant changes in response selectivity due to consensus were observed (Figure 2). Moreover, as shown in the previous patent application, when the response selectivity to ligands and their structural analogs (Figure 4) was examined using human olfactory receptors with known ligands and consensus olfactory receptors prepared in the same manner as above for said olfactory receptors, no significant changes in response selectivity due to consensus were observed (Figure 3). In addition, while some substances became detectable due to the overall increase in measurement sensitivity due to consensus, none of the substances that clearly responded with the original olfactory receptors became undetectable with the consensus olfactory receptors. Thus, the consensus olfactory receptors maintain a high degree of ligand response selectivity of the original olfactory receptors.

[0035] Non-patent document 1 reports candidate olfactory receptors responsible for sensitivity to a particular odor by conducting comparative genomic analysis on populations with different individual odor perceptions. For example, OR6B2 has been reported as an olfactory receptor associated with sensitivity to isobutyraldehyde, OR5C1 as an olfactory receptor associated with sensitivity to citral, and OR10D3 as an olfactory receptor associated with sensitivity to isoeugenol. However, it had not been successful to express these original olfactory receptors in cultured cells and demonstrate their responsiveness to the corresponding odor substances. Therefore, as shown in the previous patent application, we applied the same consensus methodology as described above to these olfactory receptors and performed response analysis, which made it possible to measure the response to the corresponding odor substances (Figure 5). Furthermore, a gene locus has been reported that explains individual differences in the perception of odors derived from cilantro (Eriksson N et al. Flavour 1:22 (2012)). Although there are eight olfactory receptors at this locus, functional analysis had not been successful in determining which receptors actually receive odors derived from cilantro. Therefore, as shown in the previous patent application, the same consensus model described above was applied to the eight receptors, and a response analysis was performed to the main aroma components of cilantro ((E)-2-decenal, (E)-2-dodecenal). It was revealed that OR10A2 and OR10A4 can recognize these odors. Furthermore, as shown in the previous patent application, the same consensus-building process described above was applied to each of OR10A2, OR10A4, OR6B2, and OR5C1, and amino acid sequences with reported individual differences were introduced to verify whether a responsiveness corresponding to individual differences in the perception of odor in sensory evaluation could be obtained. For example, for OR10A2, the amino acid sequence registered in NP_001004460.1 occurs in 68% of the population, while the amino acid sequence with the three mutations H43R, H207R, and K258T occurs in 32% of the population (frequency data from the 1000 genomes project phase 3 allele frequencies). Of the three mutations, H207R is also a consensus residue. These two amino acid sequence types found in the population were expected to alter the responsiveness to the main aroma components of cilantro ((E)-2-decenal, (E)-2-dodecenal), resulting in differences in whether or not humans perceive the aroma of cilantro as an unpleasant, soapy smell. Therefore, when consensus formulation was applied to OR10A2 and these two amino acid sequence types were introduced, it was found, as expected, that the H43R, H207R, and K258T mutants showed significantly lower responsiveness to the above aroma components (10A2 in Figure 6). Although the olfactory receptor 10A4, which showed lower responsiveness to the above aroma components than 10A2, also has an R262Q mutant that appears in 31% of the population, this mutant did not alter the responsiveness to the above aroma components (10A4 in Figure 6). Therefore, only through the consensus-building of olfactory receptors has it become possible to identify OR10A2 as the receptor that explains individual differences in how people perceive the scent of cilantro. Regarding OR6B2, the R122C and C179R variants were found in 1% of the population (test participants in Non-Patent Literature 1), and according to Non-Patent Literature 1, this population was unable to strongly perceive the odor of low concentrations of isobutyraldehyde. As shown in the previous patent application, in line with these individual differences in sensory evaluation, it was confirmed that introducing the R122C and C179R variants into consensus OR6B2 significantly reduced its responsiveness to isobutyraldehyde (6B2 in Figure 6). Regarding OR5C1, the N5K variant was found in 1% of the population (frequency from the 1000 genomes project phase 3 allele frequencies), and according to Non-Patent Literature 1, this population is unable to strongly perceive the odor of low concentrations of citral. As shown in the previous application, consistent with this individual difference in sensory evaluation, it was confirmed that introducing the N5K mutation into consensus OR5C1 significantly reduced responsiveness to citral (5C1 in Figure 6). Thus, response analysis using consensus olfactory receptors provides measurement results that best explain olfactory phenomena measured by sensory evaluation, i.e., human olfactory perception.

[0036] Therefore, although the amino acid sequence of the consensus olfactory receptor has been modified to improve membrane expression, 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 inventors have found that, among olfactory receptors to which the same consensus-forming process described above has been applied, consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, consensus OR2L5, consensus OR2AE1, consensus OR2A12, and consensus OR2AK2 respond to carboxylic acids. As described above, consensus olfactory receptors maintain a high degree of ligand selectivity of the original olfactory receptors. That is, the response of consensus olfactory receptors to a certain odor substance reflects the response of the olfactory cells of the original olfactory receptors to that odor substance. Therefore, OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, and OR2AK2 can be identified as olfactory receptors that respond to carboxylic acids.

[0038] It had not been previously recognized that OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, and OR2A12 respond to or potentially respond to carboxylic acids. On the other hand, while OR2AK2 was known to respond to 4-methyl-3-hexenoic acid, it was not previously known to respond to other carboxylic acids.

[0039] As shown in Figure 7, consensus OR2L13, consensus OR2L8, and consensus OR2L2 respond to acetic acid, with consensus OR2L13 responding in a concentration-dependent manner to acetic acid. Consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5 respond to propionic acid, with consensus OR2L13, consensus OR2L8, and consensus OR2L2 responding in a concentration-dependent manner to propionic acid. Consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5 respond in a concentration-dependent manner to butyric acid. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 respond to valeric acid in a concentration-dependent manner. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 respond to isobutyric acid, and among them, Consensus OR2L13, Consensus OR2L8, and Consensus OR2L2 respond to isobutyric acid in a concentration-dependent manner. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 respond to isovaleric acid, and among them, Consensus OR2L8 and Consensus OR2L2 respond to isovaleric acid in a concentration-dependent manner. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, Consensus OR2AE1, and Consensus OR2AK2 respond to hexanoic acid, and among them, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2AE1 respond to hexanoic acid in a concentration-dependent manner. Consensus OR2L13 responds to 3-hydroxy-3-methylhexanoic acid. Consensus OR2AE1 and Consensus OR2A12 respond to nonanoic acid, and among them, Consensus OR2A12 responds to nonanoic acid in a concentration-dependent manner.

[0040] Therefore, OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, and OR2A12 are newly discovered carboxylic acid receptors, and OR2AK2 is a carboxylic acid receptor. Substances that inhibit the response of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, OR2AK2, or polypeptides with equivalent functions can selectively suppress carboxylic acid-induced odors by altering the central nervous system's perception of carboxylic acid-induced odors based on odor suppression through olfactory receptor antagonism. On the other hand, substances that enhance the response of polypeptides such as OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, OR2AK2, or polypeptides having similar functions, cause changes in the recognition of carboxylic acid-caused odors in the central nervous system based on cross-adaptation of odors through olfactory receptor agonism, and as a result, selective suppression of carboxylic acid-caused odors can be achieved. Therefore, substances that suppress or enhance the response of these polypeptides can deodorize carboxylic acid-caused odors without causing discomfort due to the strong odor of fragrances or suppressing other odors, which occurred in conventional deodorizing methods using deodorants or fragrances.

[0041] Furthermore, OR2AK2 is a newly discovered hexanoic acid receptor. Substances that inhibit the response of OR2AK2 or polypeptides with equivalent function can selectively suppress hexanoic acid odor by altering the central nervous system's perception of hexanoic acid odor based on odor suppression through olfactory receptor agonism. On the other hand, substances that enhance the response of OR2AK2 or polypeptides with similar function can selectively suppress hexanoic acid odor by altering the central nervous system's perception of hexanoic acid odor based on cross-adaptation of odors through olfactory receptor agonism. Therefore, by using substances that inhibit or enhance the response of these polypeptides, it is possible to deodorize hexanoic acid odor, which is an odor caused by carboxylic acids, without causing discomfort due to the strong odor of the fragrance or the suppression of other odors, which occurred in conventional deodorizing methods using deodorants or fragrances.

[0042] Accordingly, the present invention provides a method for evaluating and / or selecting an odor inhibitor for carboxylic acid-induced odors. The method includes measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12 and polypeptides having equivalent functions, after the addition of a test substance. The method may further include measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent functions, after the addition of a test substance. Based on the measured response, a test substance that enhances or inhibits the response of the olfactory receptor polypeptide is detected. The detected test substance is selected as an odor inhibitor for carboxylic acid-induced odors. That is, a test substance that enhances the response of the olfactory receptor polypeptide is selected as an odor inhibitor for carboxylic acid-induced odors based on cross-adaptation of odors due to olfactory receptor agonism, and a test substance that inhibits the response of the olfactory receptor polypeptide is selected as an odor inhibitor for carboxylic acid-induced odors based on olfactory receptor antagonism.

[0043] The present invention also provides a method for evaluating and / or selecting hexanoic acid odor inhibitors. The method comprises measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent function after the addition of a test substance. Based on the measured response, a test substance that enhances or inhibits the response of the olfactory receptor polypeptide is detected. The detected test substance is selected as a hexanoic acid odor inhibitor. That is, a test substance that enhances the response of the olfactory receptor polypeptide is selected as a hexanoic acid odor inhibitor based on cross-adaptation of odors by olfactory receptor agonism, and a test substance that inhibits the response of the olfactory receptor polypeptide is selected as a hexanoic acid odor inhibitor based on olfactory receptor antagonism.

[0044] The method of the present invention described above can be performed in vitro or ex vivo.

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

[0046] OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, and OR2AK2 are olfactory receptors expressed in human olfactory cells. Table 1 shows the Accession No. and amino acid sequence of each olfactory receptor in the NCBI database.

[0047] [Table 1]

[0048] Examples of polypeptides having equivalent functionality to OR2L13 include polypeptides having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in Sequence ID No. 1, and that are responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, and 3-hydroxy-3-methylhexanoic acid. Another example of a polypeptide having equivalent functionality to OR2L13 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 1, having amino acid residues of (2) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position in (1) of Table 2 below in the amino acid sequence shown in SEQ ID NO: 1, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, and 3-hydroxy-3-methylhexanoic acid. Note that the polypeptide having amino acid residues of (2) at all of the amino acid positions in (1) of Table 2 below in the amino acid sequence shown in SEQ ID NO: 1 is consensus OR2L13, which consists 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 OR2L13 and consensus OR2L13.

[0049] Another example of a polypeptide having equivalent functionality to OR2L13 is a polypeptide consisting of the consensus OR2L13, which comprises the amino acid sequence shown in Sequence ID No. 3, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, and 3-hydroxy-3-methylhexanoic acid. Consensus OR2L13 consists of the consensus amino acid sequence shown in Sequence ID No. 3, which is derived by the above method from the alignment of the amino acid sequences of 214 olfactory receptors encoded by orthologs of OR2L13 in mammals, and is responsive to acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, and 3-hydroxy-3-methylhexanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2L13 and contains the same name as OR2L13. For olfactory receptor genes of Mus musculus (house mouse) and Rattus norvegicus (brown rat), which use different olfactory receptor naming systems, the one with the highest homology was selected as the ortholog. The amino acid sequence identity between consensus OR2L13 and OR2L13 is 92%.

[0050] Examples of polypeptides having equivalent functionality to OR2L8 include polypeptides that have an amino acid sequence having at least 80% identity with the amino acid sequence shown in Sequence ID No. 4, and that are responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Another example of a polypeptide having equivalent functionality to OR2L8 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 4, having amino acid residues of (4) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position (3) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 4, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Note that the polypeptide having amino acid residues of (4) at all positions of each amino acid position (3) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 4 is consensus OR2L8, which consists 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 OR2L8 and consensus OR2L8.

[0051] Another example of a polypeptide having equivalent functionality to OR2L8 is a polypeptide consisting of the consensus OR2L8, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Consensus OR2L8 consists of the consensus amino acid sequence shown in SEQ ID NO: 6, which is derived by the above method from the amino acid sequence of OR2L8 shown in SEQ ID NO: 4 and the alignment of the amino acid sequences of 112 olfactory receptors encoded by orthologs of OR2L8 in mammals, and is responsive to acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2L8 and contains the same name as OR2L8. The amino acid sequence identity between consensus OR2L8 and OR2L8 is 92%.

[0052] Examples of polypeptides having equivalent functionality to OR2L2 include polypeptides that have an amino acid sequence having at least 80% identity with the amino acid sequence shown in Sequence ID No. 7, and that are responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Another example of a polypeptide having equivalent functionality to OR2L2 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7, having amino acid residue (6) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position (5) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 7, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Note that the polypeptide having amino acid residue (6) at all positions of each amino acid position (5) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 7 is consensus OR2L2, which consists 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 OR2L2 and consensus OR2L2.

[0053] Another example of a polypeptide having equivalent functionality to OR2L2 is a polypeptide consisting of the consensus OR2L2, which comprises the amino acid sequence shown in Sequence ID No. 9, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Consensus OR2L2 consists of the consensus amino acid sequence shown in Sequence ID No. 9, which is derived by the above method from the alignment of the amino acid sequences of 44 olfactory receptors encoded by orthologs of OR2L2 in mammals, and is responsive to acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2L2 and contains the same name as OR2L2. The amino acid sequence identity between consensus OR2L2 and OR2L2 is 93%.

[0054] Examples of polypeptides having equivalent functionality to OR2L3 include polypeptides that have an amino acid sequence having at least 80% identity with the amino acid sequence shown in Sequence ID No. 10, and that are responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Another example of a polypeptide having equivalent functionality to OR2L3 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 10, having amino acid residue (8) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position (7) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 10, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Note that the polypeptide having amino acid residue (8) at all of the amino acid positions (7) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 10 is consensus OR2L3, which consists of the amino acid sequence shown in SEQ ID NO: 12. In other words, Table 2 below shows the amino acid residues that differ between OR2L3 and consensus OR2L3.

[0055] Another example of a polypeptide having equivalent functionality to OR2L3 is a polypeptide consisting of the consensus OR2L3, which comprises the amino acid sequence shown in Sequence ID No. 12, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Consensus OR2L3 consists of the consensus amino acid sequence shown in SEQ ID NO: 12, which is derived by the above method from the alignment of the amino acid sequence of OR2L3 shown in SEQ ID NO: 10 and the amino acid sequences of 37 olfactory receptors encoded by orthologs of OR2L3 in mammals, and is responsive to propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2L3 and contains the same name as OR2L3. The amino acid sequence identity between consensus OR2L3 and OR2L3 is 92%.

[0056] Examples of polypeptides having equivalent functionality to OR2L5 include polypeptides that have an amino acid sequence having at least 80% identity with the amino acid sequence shown in Sequence ID No. 13, and that are responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. Another example of a polypeptide having equivalent functionality to OR2L5 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 13, having at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, amino acid residue (10) at each amino acid position corresponding to (9) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 13, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. Note that the polypeptide having amino acid residue (10) at all amino acid positions of (9) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 13 is consensus OR2L5, which consists of the amino acid sequence shown in SEQ ID NO: 15. In other words, Table 2 below shows the amino acid residues that differ between OR2L5 and consensus OR2L5.

[0057] Another example of a polypeptide having equivalent functionality to OR2L5 is a polypeptide consisting of the consensus OR2L5, which comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. Consensus OR2L5 consists of the consensus amino acid sequence shown in SEQ ID NO: 15, which is derived by the above method from the alignment of the amino acid sequence of OR2L5 shown in SEQ ID NO: 13 and the amino acid sequences of 55 olfactory receptors encoded by orthologs of OR2L5 in mammals, and is responsive to propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2L5 and contains the same name as OR2L5. The amino acid sequence identity between consensus OR2L5 and OR2L5 is 96%.

[0058] Examples of polypeptides having equivalent functionality to OR2AE1 include polypeptides that consist of an amino acid sequence having at least 80% identity with the amino acid sequence shown in Sequence ID No. 16, and that are responsive to at least one selected from the group consisting of hexanoic acid and nonanoic acid. Another example of a polypeptide having equivalent functionality to OR2AE1 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 16, having amino acid residues of (2) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position in (1) of Table 3 below in the amino acid sequence shown in SEQ ID NO: 16, and being responsive to at least one selected from the group consisting of hexanoic acid and nonanoic acid. Here, having LL of (2) at the amino acid position corresponding to position 208 of (1) in Table 3 below means that in a given amino acid sequence, L is inserted immediately after the amino acid residue at the position corresponding to L at position 208 of the amino acid sequence shown in SEQ ID NO: 16. Note that the polypeptide having amino acid residues of (2) at all of the amino acid positions in (1) of Table 3 below in the amino acid sequence shown in SEQ ID NO: 16 is consensus OR2AE1, which consists of the amino acid sequence shown in SEQ ID NO: 18. In other words, Table 3 below shows the amino acid residues that differ between OR2AE1 and consensus OR2AE1.

[0059] Another example of a polypeptide having equivalent functionality to OR2AE1 is a polypeptide consisting of the consensus OR2AE1 amino acid sequence shown in Sequence ID No. 18, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one selected from the group consisting of hexanoic acid and nonanoic acid. Consensus OR2AE1 consists of the consensus amino acid sequence shown in SEQ ID NO: 18, which is derived by the above method from the amino acid sequence of OR2AE1 shown in SEQ ID NO: 16 and the amino acid sequences of 171 olfactory receptors encoded by orthologs of OR2AE1 in mammals, and is responsive to hexanoic acid and nonanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2AE1 and contains the same name as OR2AE1. The amino acid sequence identity between consensus OR2AE1 and OR2AE1 is 89%.

[0060] Examples of polypeptides with equivalent functionality to OR2A12 include polypeptides that have an amino acid sequence with at least 80% identity to the amino acid sequence shown in Sequence ID No. 19 and that are responsive to nonanoic acid. Another example of a polypeptide having equivalent functionality to OR2A12 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 19, having amino acid residue (4) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position (3) in Table 3 below in the amino acid sequence shown in SEQ ID NO: 19, and being responsive to nonanoic acid. Note that the polypeptide having amino acid residue (4) at all positions of each amino acid position (3) in Table 3 below in the amino acid sequence shown in SEQ ID NO: 19 is consensus OR2A12, which consists of the amino acid sequence shown in SEQ ID NO: 21. In other words, Table 3 below shows the amino acid residues that differ between OR2A12 and consensus OR2A12.

[0061] Another example of a polypeptide having equivalent functionality to OR2A12 is a polypeptide consisting of the consensus OR2A12 amino acid sequence shown in Sequence ID No. 21, or an amino acid sequence having at least 90% identity thereto, and which is responsive to nonanoic acid. Consensus OR2A12 consists of the consensus amino acid sequence shown in SEQ ID NO: 21, which is derived by the above method from the amino acid sequence of OR2A12 shown in SEQ ID NO: 19 and the amino acid sequences of 97 olfactory receptors encoded by orthologs of OR2A12 in mammals, and is responsive to nonanoic acid. The ortholog is a gene among olfactory receptor genes belonging to mammalian species that has a high homology to OR2A12 and contains the same name as OR2A12. The amino acid sequence identity between consensus OR2A12 and OR2A12 is 95%.

[0062] Examples of polypeptides having equivalent functionality to OR2AK2 include polypeptides that have an amino acid sequence with at least 80% identity to the amino acid sequence shown in Sequence ID No. 22, and that are responsive to hexanoic acid. Another example of a polypeptide having equivalent functionality to OR2AK2 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 22, having amino acid residues of (2) at at least one, preferably at least three, more preferably at least five, even more preferably at least ten, and even more preferably all, positions corresponding to each amino acid position (1) in Table 4 below in the amino acid sequence shown in SEQ ID NO: 22, and being responsive to hexanoic acid. Note that the polypeptide having amino acid residues of (2) at all positions of each amino acid position (1) in Table 4 below in the amino acid sequence shown in SEQ ID NO: 22 is consensus OR2AK2, which consists of the amino acid sequence shown in SEQ ID NO: 24. In other words, Table 4 below shows the amino acid residues that differ between OR2AK2 and consensus OR2AK2.

[0063] Another example of a polypeptide having equivalent functionality to OR2AK2 is a polypeptide consisting of the consensus OR2AK2 amino acid sequence shown in Sequence ID No. 24, or an amino acid sequence having at least 90% identity thereto, and which is responsive to hexanoic acid. Consensus OR2AK2 consists of the consensus amino acid sequence shown in SEQ ID NO: 24, which is derived by the above method from the amino acid sequence of OR2AK2 shown in SEQ ID NO: 22 and the amino acid sequences of 184 olfactory receptors encoded by orthologs of OR2AK2 in mammals, and is responsive to hexanoic acid. The ortholog is a gene among the olfactory receptor genes of mammalian species that has a high homology to OR2AK2 and contains the same name as OR2AK2. For the olfactory receptor genes of Mus musculus and Rattus norvegicus, the one with the highest homology was selected as the ortholog. The amino acid sequence identity between consensus OR2AK2 and OR2AK2 is 86%.

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] In the method for evaluating and / or selecting an odor inhibitor caused by carboxylic acids of the present invention, at least one olfactory receptor polypeptide selected from OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, and polypeptides having equivalent functions thereto may be used, but two or more of these may be used in combination. Preferably, at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, consensus OR2L5, consensus OR2AE1, and consensus OR2A12 is used, and more preferably, at least one selected from the group consisting of consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, consensus OR2L5, consensus OR2AE1, and consensus OR2A12 is used. Furthermore, at least one selected from the group consisting of OR2AK2 and polypeptides having equivalent functionality, preferably at least one selected from the group consisting of OR2AK2 and consensus OR2AK2, and more preferably consensus OR2AK2 may be used in combination.

[0068] In one example, when the carboxylic acid is acetic acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L13 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2L13 and consensus OR2L13; and even more preferably consensus OR2L13 is used. In another example, when the carboxylic acid is propionic acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, consensus OR2L13, consensus OR2L8 and consensus OR2L2; and even more preferably at least one selected from the group consisting of consensus OR2L13, consensus OR2L8 and consensus OR2L2. In another example, when the carboxylic acid is butyric acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5; and even more preferably at least one selected from the group consisting of consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L. In another example, when the carboxylic acid is valeric acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5; and even more preferably at least one selected from the group consisting of consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L.

[0069] In another example, when the carboxylic acid is isobutyric acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, consensus OR2L13, consensus OR2L8 and consensus OR2L2; and even more preferably at least one selected from the group consisting of consensus OR2L13, consensus OR2L8 and consensus OR2L2. In another example, when the carboxylic acid is isovaleric acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L8, OR2L2 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2L8, OR2L2, consensus OR2L8 and consensus OR2L2; and even more preferably at least one selected from the group consisting of consensus OR2L8 and consensus OR2L2.

[0070] In another example, when the carboxylic acid is hexanoic acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2AE1 and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2L8, OR2L2, OR2L3, OR2AE1 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2L8, OR2L2, OR2L3, OR2AE1, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2AE1; and even more preferably at least one selected from the group consisting of consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2AE1. Furthermore, at least one polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent functionality may be used in combination, preferably at least one polypeptide selected from the group consisting of OR2AK2 and consensus OR2AK2, and more preferably consensus OR2AK2. In another example, when the carboxylic acid is 3-hydroxy-3-methylhexanoic acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2L13 and polypeptides having equivalent function, more preferably at least one selected from the group consisting of OR2L13 and consensus OR2L13, and even more preferably consensus OR2L13. In another example, when the carboxylic acid is a nonanoic acid, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2AE1, OR2A12, and polypeptides having equivalent functions thereto; more preferably at least one selected from the group consisting of OR2A12 and polypeptides having equivalent functions thereto; even more preferably at least one selected from the group consisting of OR2A12 and consensus OR2A12; and even more preferably consensus OR2A12 is used.

[0071] In the evaluation and / or selection method for hexanoic acid odor inhibitors of the present invention, at least one olfactory receptor polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent function may be used, but two or more of these may be used in combination. Preferably, at least one selected from the group consisting of OR2AK2 and consensus OR2AK2 is used, and more preferably, consensus OR2AK2 is used.

[0072] In the method for evaluating and / or selecting an odor inhibitor caused by a carboxylic acid according to the present invention, the olfactory receptor polypeptide may be used in any form, as long as it does not lose its responsiveness to the carboxylic acid. For example, the olfactory receptor polypeptide may be used in the form of tissues or cells that naturally express the olfactory receptor polypeptide, such as olfactory receptors or olfactory cells isolated from living organisms, or cultures thereof; membranes of olfactory cells carrying the olfactory receptor polypeptide; recombinant cells or cultures thereof genetically engineered to express the olfactory receptor polypeptide; membranes of recombinant cells having the olfactory receptor polypeptide; artificial lipid bilayers having the olfactory receptor polypeptide, and so on. All of these forms are included in the range of olfactory receptor polypeptides used in the present invention.

[0073] In a preferred embodiment, the olfactory receptor polypeptide used may be cells that naturally express the olfactory receptor polypeptide, such as olfactory cells, or recombinant cells genetically engineered to express the olfactory receptor polypeptide, or cultures thereof. The recombinant cells can be prepared by transforming cells using a vector into which the gene encoding the olfactory receptor polypeptide has been incorporated.

[0074] Preferably, in order to promote the cell membrane expression of the olfactory receptor polypeptide, a gene encoding RTP (receptor-transporting protein) is introduced into the cells together with the gene encoding the olfactory receptor polypeptide. Preferably, a gene encoding RTP1S is introduced into the cells together with the gene encoding the olfactory 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: 26, encoded by a gene having the nucleotide sequence of SEQ ID NO: 25.

[0075] Methods for adding a test substance to the olfactory receptor polypeptide include, but are not limited to, adding the test substance to a culture medium for cells expressing the olfactory receptor polypeptide, or directly dropping, spraying, or misting the test substance onto the olfactory receptor polypeptide or cells or tissues containing it.

[0076] In the method for evaluating and / or selecting an odor inhibitor caused by carboxylic acids according to the present invention, the response of the olfactory receptor polypeptide is measured following the addition of a test substance to the olfactory receptor polypeptide. The measurement may be performed by any method known in the art for measuring the response of olfactory receptors, such as intracellular cAMP level measurement. For example, it is known that when olfactory receptors are activated by odor molecules, they increase intracellular cAMP levels by activating adenylyl cyclase in conjunction with the α subunit of G proteins classified as the Gαs family in the cell (Nat. Neurosci., 2004, 5:263-278). On the other hand, when olfactory receptors are activated by odor molecules, they can also increase intracellular calcium ion levels by coupling with proteins belonging to the Gq family, such as Gα15, in the cell. Therefore, the response of olfactory receptor polypeptides can be measured by using the intracellular cAMP level or calcium ion level after the addition of odor molecules, or the behavior of downstream molecules activated via them, as indicators. Methods for measuring cAMP levels include ELISA and reporter gene assay. Other methods for measuring the response of olfactory receptor polypeptides include measuring calcium ion concentration, such as calcium imaging and the TGFα shedding assay. Furthermore, as an example of a method that uses the behavior of downstream molecules activated via cAMP levels as an indicator, the two-electrode membrane potential fixation method, which measures the potential changes across the cell membrane via the cystic fibrosis membrane conductance regulator CFTR activated by cAMP signaling in African clawed frog oocytes, is also effective.

[0077] In a first embodiment of the present invention, a method for evaluating and / or selecting an odor inhibitor for carboxylic acid-induced odors according to the present invention comprises adding a test substance to the olfactory receptor polypeptide described above; and measuring the response of the olfactory receptor polypeptide to the test substance. Then, based on the measured response, a test substance that enhances the response of the olfactory receptor polypeptide is detected. The detected test substance is selected as an odor inhibitor for carboxylic acid-induced odors.

[0078] A test substance that enhances the response of the olfactory receptor polypeptide can weaken the response of the olfactory receptor when it is later exposed to a carboxylic acid by first enhancing the response of the olfactory receptor. As a result, the recognition of odors caused by carboxylic acids by individuals can be suppressed based on odor cross-adaptation. Therefore, in the first embodiment, an odor inhibitor for carboxylic acids based on odor cross-adaptation by olfactory receptor agonism is selected.

[0079] The effect of the test substance on the olfactory receptor polypeptide can be evaluated, for example, by comparing the response of the olfactory receptor polypeptide to which the test substance has been added (test group) with the response of the control group. Examples of the control group include the olfactory receptor polypeptide without the test substance, the olfactory receptor polypeptide with the control substance added, the olfactory receptor polypeptide with a lower concentration of the test substance added, the olfactory receptor polypeptide before the addition of the test substance, and cells that do not express the olfactory receptor polypeptide. Preferably, the method of the present invention in the first embodiment includes measuring the activity of the olfactory receptor polypeptide in the presence and absence of the test substance. Also preferably, the method of the present invention in the first embodiment includes measuring the response of cells expressing and not expressing the olfactory receptor polypeptide to the agonist in the presence of the test substance.

[0080] For example, in the first embodiment, if the response in the test group is enhanced compared to the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to the carboxylic acid. For example, if the response of the olfactory receptor polypeptide in the test group is enhanced by preferably 120% or more, more preferably 150% or more, and even more preferably 200% compared to the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to the carboxylic acid. Alternatively, if the response of the olfactory 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 suppresses the response of the olfactory receptor polypeptide to the carboxylic acid.

[0081] Odors caused by carboxylic acids that are suppressed by the carboxylic acid odor inhibitor selected according to the first embodiment include, for example, body odor, underarm odor, shoe odor, fecal odor, cooking odor, tobacco odor, pet odor, garbage odor, acetic acid odor, propionic acid odor, butyric acid odor, valeric acid odor, isobutyric acid odor, isovaleric acid odor, hexanoic acid odor, 3-hydroxy-3-methylhexanoic acid odor, nonanoic acid odor, and the like. Preferably, when OR2L13 or a polypeptide with equivalent function is used as the olfactory receptor, examples include body odor, underarm odor, shoe odor, fecal odor, cooking odor, tobacco odor, pet odor, garbage odor, acetic acid odor, propionic acid odor, butyric acid odor, valeric acid odor, isobutyric acid odor, isovaleric acid odor, hexanoic acid odor, and 3-hydroxy-3-methylhexanoic acid odor. When OR2L8, OR2L2 or a polypeptide with equivalent function is used, examples include body odor, shoe odor, fecal odor, pet odor, garbage odor, propionic acid odor, butyric acid odor, valeric acid odor, isobutyric acid odor, isovaleric acid odor, and hexanoic acid odor. When OR2L3 or a polypeptide with equivalent function is used, examples include body odor, shoe odor, fecal odor, pet odor, garbage odor, propionic acid odor, butyric acid odor, valeric acid odor, isobutyric acid odor, isovaleric acid odor, and hexanoic acid odor. Preferably, when OR2L5 or a polypeptide with equivalent function is used, body odor, shoe odor, fecal odor, pet odor, garbage odor, propionic acid odor, butyric acid odor, valeric acid odor, isobutyric acid odor, and isovaleric acid odor are examples. When OR2AE1 or a polypeptide with equivalent function is used, body odor, hexanoic acid odor, and nonanoic acid odor are examples. When OR2A12 or a polypeptide with equivalent function is used, body odor and nonanoic acid odor are examples. Preferably, when OR2AK2 or a polypeptide with equivalent function is used, body odor and hexanoic acid odor are examples. More preferably, body odor is used.

[0082] One embodiment of the use of an odor suppressant for carboxylic acid-induced odors selected according to the first embodiment is as follows: First, a person who desires suppression of carboxylic acid-induced odors is allowed to smell the suppressant before being exposed to the odor. Alternatively, the suppressant is applied to the person in such a way that it has a stronger odor than the carboxylic acid-induced odor. As a result, even when the person is exposed to the carboxylic acid-induced odor, their olfactory sensitivity to the odor is reduced, so they perceive the odor as weak or not perceive it at all.

[0083] In a second embodiment of the present invention, a method for evaluating and / or selecting an odor inhibitor caused by a carboxylic acid according to the present invention comprises adding a test substance and an agonist of the olfactory receptor polypeptide to the olfactory receptor polypeptide described above; and measuring the response of the olfactory receptor polypeptide to the agonist. The agonist is not limited to, but preferably at least one selected from the group consisting of carboxylic acids, more preferably linear short-chain carboxylic acids, branched short-chain carboxylic acids and polymeric carboxylic acids, and even more preferably at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid and nonanoic acid. More preferably, when using OR2L13 or a polypeptide with equivalent function as the olfactory receptor, at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid and 3-hydroxy-3-methylhexanoic acid; when using OR2L8, OR2L2 or a polypeptide with equivalent function as these, at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid and hexanoic acid; when using OR2L3 or a polypeptide with equivalent function as these, propionic acid, butyric acid, valeric acid If OR2L5 or a polypeptide with equivalent function is used, at least one selected from the group consisting of isobutyric acid, isovaleric acid, and hexanoic acid is used. If OR2AE1 or a polypeptide with equivalent function is used, at least one selected from the group consisting of hexanoic acid and nonanoic acid is used. If OR2A12 or a polypeptide with equivalent function is used, nonanoic acid is used. If OR2AK2 or a polypeptide with equivalent function is used, hexanoic acid is used. Then, based on the measured response, a test substance that inhibits the response of the olfactory receptor polypeptide to the agonist is detected. The detected test substance is selected as an inhibitor of carboxylic acid-induced odors.

[0084] In the second embodiment, the test substance that inhibits the response of the olfactory receptor polypeptide to the agonist is selected as an inhibitor of carboxylic acid-induced odors based on olfactory receptor antagonism. The effect of the test substance on the response of the olfactory receptor polypeptide to the agonist can be determined, for example, by comparing the response of the olfactory receptor polypeptide to the agonist with that of the control group to that agonist. Examples of the control group include those described above. Preferably, the method of the present invention in the second embodiment includes measuring the activity of the olfactory receptor polypeptide to the agonist in the presence and absence of the test substance.

[0085] For example, if the response in the test group is more suppressed than in the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to the carboxylic acid. For example, if the response of the olfactory receptor polypeptide in the test group is preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to the carboxylic acid. Alternatively, if the response of the olfactory 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 olfactory receptor polypeptide to the carboxylic acid.

[0086] Odors caused by carboxylic acids that are suppressed by the carboxylic acid odor inhibitor selected according to the second embodiment include those similar to the carboxylic acid odors that are suppressed by the carboxylic acid odor inhibitor selected according to the first embodiment described above.

[0087] One embodiment of the use of an odor inhibitor for carboxylic acid-induced odors selected according to the second embodiment is as follows: First, a person who desires to suppress odors caused by carboxylic acids is exposed to the odor of the inhibitor before or at the same time as being exposed to the odor. As a result, even when the person is exposed to odors caused by carboxylic acids, their olfactory sensitivity to the odor is reduced, so they perceive the odor as weak or not perceive it at all.

[0088] By following the above procedure, an odor inhibitor for carboxylic acid-induced odors can be obtained based on the response activity of olfactory receptor polypeptides that are responsive to odors caused by carboxylic acids. If necessary, the odor inhibitory ability of the test substance selected above for carboxylic acid-induced odors may be further evaluated by a sensory test. That is, in one embodiment of the method of the present invention, the test substance selected in the above procedure is obtained as a candidate substance for an odor inhibitor for carboxylic acid-induced odors. Next, the odor inhibitory effect of the candidate substance for carboxylic acid-induced odors is evaluated by a sensory test. The candidate substance that receives a better evaluation in the sensory test is selected as an odor inhibitor for carboxylic acid-induced odors.

[0089] Sensory testing of the candidate substance may be conducted in accordance with the evaluation procedures for deodorants commonly used in the field. If the candidate substance is an inducer of cross-adaptation of odors, the order in which the evaluator is exposed to the candidate substance and the target odor-causing substance may be adjusted. For example, when sensory testing a test substance selected in the first embodiment described above as a candidate substance, the evaluator first smells the candidate substance and adapts to its odor. Then the evaluator smells the target odor (preferably an odor caused by a carboxylic acid) and evaluates its intensity. The evaluation results obtained are compared with the intensity of the target odor without adaptation to the candidate substance. Alternatively, for example, when sensory testing a test substance selected in the second embodiment described above as a candidate substance, the evaluator smells the target odor (preferably an odor caused by a carboxylic acid) simultaneously with the candidate substance and evaluates the intensity of the target odor. The evaluation results obtained are compared with the intensity of the target odor alone. Based on the results of sensory testing, candidate substances that were evaluated as reducing the intensity of the target odor are selected as inhibitors of carboxylic acid-induced odors.

[0090] The carboxylic acid odor inhibitor obtained in the present invention can be used as an active ingredient for inhibiting carboxylic acid odors. For example, the inhibitor can be included as an active ingredient for inhibiting carboxylic acid odors in a composition or article for inhibiting carboxylic acid odors. Alternatively, the inhibitor can be used for the manufacture of a composition or article for inhibiting carboxylic acid odors. Examples of applications for the carboxylic acid odor suppressant obtained in the present invention include, but are not limited to, sanitary products such as sanitary napkins and disposable diapers containing the agent; clothing such as underwear, linens, and other fabric products or textiles containing the agent; laundry detergents or fabric softeners containing the agent; external preparations such as cosmetics, cleaning agents, deodorants, pharmaceuticals, and foods containing the agent; placing or spraying the agent in the kitchen; placing or spraying the agent inside a refrigerator; placing or spraying the agent in front of or inside a trash can; placing or spraying the agent in front of or inside a toilet; placing or spraying the agent in hospital wards or nursing homes; having persons involved in excretion procedures in hospital wards or nursing homes carry the agent or expose them to the agent before such procedures; and application to environments that generate carboxylic acid odors.

[0091] The evaluation and / or selection method for the hexanoic acid odor inhibitor of the present invention can be appropriately applied by replacing carboxylic acid with hexanoic acid and odor caused by carboxylic acid with hexanoic acid odor.

[0092] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc., are further disclosed herein. However, the present invention is not limited to these embodiments.

[0093] [1] A method for evaluating and / or selecting an odor inhibitor caused by a carboxylic acid, comprising measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12 and polypeptides having equivalent functions thereto, after the addition of a test substance. [2] The method according to [1], further comprising measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent function after the addition of a test substance. [3] Preferably, the carboxylic acid is at least one selected from the group consisting of linear short-chain carboxylic acids, branched short-chain carboxylic acids and polymeric carboxylic acids, more preferably at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid and nonanoic acid, and even more preferably, when the olfactory receptor polypeptide is OR2L13 or a polypeptide having equivalent function thereto, at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid and 3-hydroxy-3-methylhexanoic acid, and when it is OR2L8, OR2L2 or a polypeptide having equivalent function thereto, acetic acid, propionic acid The method according to [1] or [2], wherein the at least one selected from the group consisting of acids, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid is selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid when it is OR2L3 or a polypeptide having equivalent function thereto, at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid when it is OR2L5 or a polypeptide having equivalent function thereto, at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid when it is OR2AE1 or a polypeptide having equivalent function thereto, and nonanoic acid is selected from the group consisting of hexanoic acid and nonanoic acid when it is OR2A12 or a polypeptide having equivalent function thereto. [4] The method according to [2] or [3], preferably wherein the carboxylic acid is hexanoic acid when the olfactory receptor polypeptide is OR2AK2 or a polypeptide having equivalent function. [5] Preferably, the method according to any one of [1] to [4], wherein the olfactory receptor polypeptide is a polypeptide consisting of the amino acid sequence shown in the following sequence number. OR2L13; SEQ ID NO: 1 OR2L8; Sequence ID 4 OR2L2; Sequence ID 7 OR2L3; SEQ ID NO: 10 OR2L5; Sequence ID 13 OR2AE1; Sequence ID 16 OR2A12; Sequence ID 19 [6] Preferably, the method according to any one of [2] to [5], wherein OR2AK2 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22. [7] Preferably, the polypeptide having a function equivalent to the olfactory receptor polypeptide below comprises an amino acid sequence having identity with the amino acid sequence shown by the following sequence number, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, and is a polypeptide that responds to at least one selected from the group consisting of the following odor substances. The method according to any one of [1] to [6]. OR2L13; SEQ ID NO: 1; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid OR2L8; SEQ ID NO: 4; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L2; SEQ ID NO: 7; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L3; SEQ ID NO: 10; Propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L5; SEQ ID NO: 13; Propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid OR2AE1; SEQ ID NO: 16; Hexaneic acid, Nonanoic acid OR2A12; SEQ ID NO: 19; Nonanoic acid [8] Preferably, the polypeptide having the same function as OR2AK2 comprises an amino acid sequence having identity with the amino acid sequence shown in SEQ ID NO: 22, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, and is a polypeptide that is responsive to hexanoic acid, according to any one of claims [2] to [7].

[0094] [9] Preferably, the polypeptide having the same function as OR2L13 consists of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, has at least one amino acid residue of (2) at a position corresponding to each amino acid position of (1) in Table 2 above in the amino acid sequence shown in SEQ ID NO: 1, and is a polypeptide that responds to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid and 3-hydroxy-3-methylhexanoic acid. A polypeptide having equivalent functionality to OR2L8 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 4, having at least one amino acid residue (4) at a position corresponding to each amino acid position (3) in Table 2 above in the amino acid sequence shown in SEQ ID NO: 4, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L2 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7, having at least one amino acid residue (6) at a position corresponding to each amino acid position (5) in Table 2 above in the amino acid sequence shown in SEQ ID NO: 7, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L3 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 10, having at least one amino acid residue (8) at a position corresponding to each amino acid position (7) in Table 2 above in the amino acid sequence shown in SEQ ID NO: 10, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L5 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 13, having at least one amino acid residue (10) at a position corresponding to each amino acid position (9) in Table 2 above in the amino acid sequence shown in SEQ ID NO: 13, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. A polypeptide having equivalent functionality to OR2AE1 is a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 16, having at least one amino acid residue of (2) at a position corresponding to each amino acid position of (1) in Table 3 above in the amino acid sequence shown in SEQ ID NO: 16, and being responsive to at least one selected from the group consisting of hexanoic acid and nonanoic acid. A polypeptide having equivalent functionality to OR2A12 consists of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, and has at least one amino acid residue (4) at a position corresponding to each amino acid position (3) in Table 3 above in the amino acid sequence shown in SEQ ID NO: 19, and is a polypeptide that is responsive to nonanoic acid. "At least 80% identity" means preferably 80% or more, more preferably 85% or more, even 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; and "at least one location" means preferably at least one location, more preferably at least three locations, even more preferably at least five locations, even more preferably at least ten locations, and even more preferably all locations. The method described in any one of items [1] to [8].

[10] Preferably, the polypeptide having the same function as OR2AK2 comprises an amino acid sequence having identity with the amino acid sequence shown in SEQ ID NO: 22, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, preferably at least one amino acid residue of (2) at positions corresponding to each amino acid position of (1) in Table 4 above in the amino acid sequence shown in SEQ ID NO: 64, and is a polypeptide that is responsive to hexanoic acid, according to any one of the claims [2] to [9].

[11] Preferably, the polypeptide having a function equivalent to the olfactory receptor polypeptide below comprises an amino acid sequence having an amino acid sequence that is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identical to the amino acid sequence shown by the following sequence number, and is a polypeptide that is responsive to at least one selected from the group consisting of the following odor substances. OR2L13; SEQ ID NO: 3; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid OR2L8; SEQ ID NO: 6; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L2; SEQ ID NO: 9; Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L3; SEQ ID NO: 12; Propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid OR2L5; SEQ ID NO: 15; Propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid OR2AE1; SEQ ID NO: 18; Hexanoic acid, Nonanoic acid OR2A12; SEQ ID NO: 21; Nonanoic acid

[12] Preferably, the polypeptide having the same function as OR2AK2 comprises an amino acid sequence having 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence shown in Sequence ID No. 24, and is a polypeptide that is responsive to hexanoic acid, according to any one of claims [2] to [6].

[13] A method for evaluating and / or selecting a hexanoic acid odor inhibitor, comprising measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AK2 and polypeptides having equivalent function thereto after the addition of a test substance.

[0095]

[14] Preferably, the following: Adding a test substance to the olfactory receptor polypeptide, and To measure the response of the olfactory receptor polypeptide to the test substance, A method described in any one of items [1] to

[13] , including the method described in item [1] to

[13] .

[15] Preferably, the method according to

[14] further comprises selecting a test substance as an inhibitor that enhances the response of the olfactory receptor polypeptide to the test substance.

[16] Preferably, the method according to

[14] further comprising measuring the response of the olfactory receptor polypeptide in a control group.

[17] Preferably, the test substance is selected as an inhibitor that enhances the response of the olfactory receptor polypeptide to the test substance compared to the control group, More preferably, the inhibitor is selected to be a test substance that enhances the response of the olfactory receptor polypeptide to the test substance by 120% or more compared to the control group, or a test substance that statistically significantly enhances the response of the olfactory receptor polypeptide to the test substance compared to the control group. The method described in

[16] .

[18] Preferably, the following: Adding the test substance and the agonist of the olfactory receptor polypeptide to the olfactory receptor polypeptide, and Measuring the response of the olfactory receptor polypeptide to the agonist, A method described in any one of items [1] to

[13] , including the method described in item [1] to

[13] .

[19] Preferably, the method according to

[18] further comprises selecting a test substance as an inhibitor that suppresses the response of the olfactory receptor polypeptide to the agonist.

[20] Preferably, the method according to

[18] further comprising measuring the response of the olfactory receptor polypeptide to the agonist in a control group.

[21] Preferably, the test substance is selected as an inhibitor that suppresses the response of the olfactory receptor polypeptide to the agonist compared to the control group, More preferably, the test substance is selected as an inhibitor that suppresses the response of the olfactory receptor polypeptide to the agonist by 60% or less compared to the control group, or the test substance is selected as an inhibitor that statistically significantly suppresses the response of the olfactory receptor polypeptide to the agonist compared to the control group. The method described in

[20] .

[22] Preferably, the agonist is a carboxylic acid, more preferably at least one selected from the group consisting of linear short-chain carboxylic acids, branched short-chain carboxylic acids and polymeric carboxylic acids, even more preferably at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid and nonanoic acid, and even more preferably at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid and 3-hydroxy-3-methylhexanoic acid when the olfactory receptor polypeptide is OR2L13 or a polypeptide having equivalent function thereto, and when it is OR2L8, OR2L2 or a polypeptide having equivalent function thereto, acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, The method according to any one of the claims

[18] to

[21] , wherein the at least one selected from the group consisting of isovaleric acid and hexanoic acid is selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid and hexanoic acid when it is OR2L3 or a polypeptide having equivalent function thereto, at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid and isovaleric acid when it is OR2L5 or a polypeptide having equivalent function thereto, at least one selected from the group consisting of hexanoic acid and nonanoic acid when it is OR2AE1 or a polypeptide having equivalent function thereto, nonanoic acid when it is OR2A12 or a polypeptide having equivalent function thereto, and hexanoic acid when it is OR2AK2 or a polypeptide having equivalent function thereto.

[23] Preferably, the olfactory receptor polypeptide is expressed on recombinant cells that have been genetically engineered to express the olfactory receptor polypeptide, according to any one of [1] to

[22] .

[24] Preferably, the response of the olfactory receptor polypeptide is measured by measuring the amount of intracellular cAMP by ELISA or a reporter gene assay, measuring the amount of calcium ions by calcium imaging or a TGFα shedding assay, or measuring the change in potential across the cell membrane by a two-electrode membrane potential fixation method using African clawed frog oocytes, according to any one of the methods in any one of [1] to [3].

[25] Preferably, the method according to any one of [1] to

[24] , further comprising evaluating the test substance by a sensory test. [Examples]

[0096] The present invention will be described in more detail below with reference to examples.

[0097] Example 1: Identification of olfactory receptors that respond to carboxylic acids 1) Creation of olfactory receptor-expressing cells A consensus olfactory receptor was designed in the same manner as described in the examples of Japanese Patent Application No. 2021-103675. In designing the consensus olfactory receptor, homologous gene candidates for the target olfactory receptor gene were searched using NCBI BLAST. Orthologs were identified from the obtained gene group. Specifically, from the genes with the highest homology, searched using BLAST, genes with the same name as the target olfactory receptor were selected as mammalian orthologues. For example, in the case of human OR2L13, the amino acid sequence of human OR2L13 (NP_001291464.1) was used as the query sequence, and from the 250 genes with the highest homology, searched using BLAST with the target organism name set to mammal, 212 genes containing OR2L13 in their name were selected. In addition, for the genes of Mus musculus and Rattus norvegicus, which use different olfactory receptor naming systems, one gene with the highest homology was selected from the genes included in the top 250 search results for each species. These 214 genes were identified as mammalian orthologues. Alignment analysis and consensus amino acid identification were performed on the amino acid sequences of these 214 genes plus human OR2L13, for a total of 215 genes, as described below. Similarly, consensus amino acids were identified for other human olfactory receptors, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, and OR2AK2. For the genes of Mus musculus and Rattus norvegicus, if these genes were included in the top 250 search results, the gene with the highest homology was selected for each.

[0098] Alignment analysis of the identified gene groups was performed using ClustalW, and further adjustments were made to optimize the design based on highly conserved amino acids or amino acid motifs among olfactory receptors. Based on the alignment results, a consensus olfactory receptor was designed using Jalview. In this alignment, if there was one amino acid residue at a position corresponding to each amino acid position in the original human olfactory receptor amino acid sequence that differed from the amino acid residue in the reference amino acid sequence and had an occurrence frequency of 50% or more, the amino acid residue in the reference amino acid sequence was modified to that amino acid residue. However, even if there was one amino acid residue at a position corresponding to each amino acid position in the original human olfactory receptor amino acid sequence that differed from the amino acid residue in the reference amino acid sequence and had an occurrence frequency of 50%, if the occurrence frequency of the amino acid residue in the reference amino acid sequence was also 50%, the amino acid residue in the reference amino acid sequence was not modified. In this alignment, if there was a deletion at a position corresponding to each amino acid position in the original human olfactory receptor amino acid sequence with an occurrence frequency of 40% or more, the amino acid residue in the reference amino acid sequence was modified to a deletion. On the other hand, if an amino acid exists at a location corresponding to the deletion site in the original human olfactory receptor amino acid sequence with an occurrence frequency of 60% or more, the sequence was modified to insert the most conserved amino acid at the deletion site in the reference amino acid sequence. If there are two or more amino acids that are most conserved, the sequence was modified to insert the amino acid with the smallest molecular weight. The topology of olfactory receptors in the design was confirmed using the TMHMM (Transmembrane Hidden Markov Model). The DNA sequences encoding the various olfactory receptor polypeptides designed were obtained by DNA synthesis after optimizing the base sequence codons corresponding to their amino acid sequences for expression in human cultured cells. The optimized base sequences encoding the codons of consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, consensus OR2L5, consensus OR2AE1, consensus OR2A12, and consensus OR2AK2 are shown in SEQ ID NOs. 2, 5, 8, 11, 14, 17, 20, and 23, respectively. EcoRI and XhoI sites were added to both ends of these base sequences and recombined into the EcoRI and XhoI sites constructed downstream of the Flag-Rho tag sequence on the pME18S vector. Furthermore, a gene encoding human RTP1S, which translocates olfactory receptor proteins produced in cultured cells to the cell membrane, was incorporated into the EcoRI and XhoI sites of another pME18S vector to create the pME18S-RTP1S vector.

[0099] Reaction solutions with the compositions shown in Table 5 were prepared and allowed to stand in a clean bench for 20 minutes before being added to each well of a 96-well plate (BD). For olfactory receptors OR2L2 and OR2L3, the compositions shown in Table 6 were used. Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well in a 2 × 10⁶ solution. 5 cells / cm 2 The cells were seeded and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that did not express olfactory receptors (mock cells) were prepared.

[0100] [Table 5]

[0101] [Table 6]

[0102] 2) Luciferase assay Olfactory receptors expressed in HEK293 cells increase intracellular cAMP levels by activating adenylate cyclase in conjunction with intrinsic GαS. For odor response measurement, a luciferase reporter gene assay was used, monitoring the increase in intracellular cAMP levels as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a gene fused with the sea urchin luciferase gene (hRluc2P-CMV-hygro) downstream of the CMV promoter was simultaneously introduced and used as an internal standard to correct for errors in gene transfer efficiency and cell number.

[0103] From the culture prepared in 1) above, the culture medium is removed, and test substance solutions are prepared in a new culture medium (acetic acid (CAS: 64-19-7, Kanto Chemical), propionic acid (CAS: 79-09-4, Fujifilm Wako Pure Chemical Industries), butyric acid (CAS: 107-92-6, Tokyo Chemical Industries), valeric acid (CAS: 109-52-4, Tokyo Chemical Industries), isobutyric acid (CAS: 79-31-2, Tokyo Chemical Industries), isovaleric acid (CA 75 μL of luciferase (CAS: 503-74-2, Fujifilm Wako Pure Chemical Industries), hexanoic acid (CAS: 142-62-1, Tokyo Chemical Industries), 3-hydroxy-3-methylhexanoic acid (CAS: 58888-76-9, Fujifilm Wako Pure Chemical Industries), or nonanoic acid (CAS: 112-05-0, Fujifilm Wako Pure Chemical Industries), in concentrations of 0 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, and 300 μM, was added. Cells were cultured in a CO2 incubator for 4 hours to allow sufficient expression of the luciferase gene within the cells. Luciferase activity was measured using Dual-Glo TMMeasurements 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 sea urchin luciferase (fluc / hRluc), and this value was used as the signal for analysis. For each transfection condition, the signal value under no odor substance stimulation (fluc / hRluc) was set to 0%, and the signal value when stimulated with 10 μM forskolin (fluc / hRluc) was set to 100%, and this was normalized and used as Response (%) for analysis.

[0104] 3) Results Figure 7 shows the olfactory receptors that responded to acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid, or nonanoic acid among the consensus olfactory receptors tested. Figure 7 shows the response values ​​for each olfactory receptor to each concentration of odor substance. A statistically significant difference was observed between the response value (Response (%)) of each olfactory receptor to the highest tested concentration of 300 μM and the response value (Response (%)) of cells under mock conditions where the receptor was not expressed (Student's t- (test, P<0.05). Furthermore, for each olfactory receptor, a statistically significant difference was observed when comparing the signal value (fluc / hRluc) under conditions without stimulation by odor substances with the signal value (fluc / hRluc) under conditions stimulated with the maximum concentration of 300 μM, using the same statistical method. The values ​​in Tables 7-14 represent the EC calculated by regressing the Response (%) value onto a sigmoid curve. 50 (μM). As shown in Figure 7, Tables 7-14 show EC 50 Olfactory receptors without a (μM) indication are receptors that can respond to acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid, or nonanoic acid. However, in the concentration range up to 300 μM tested in this study, sufficient data was not obtained to revert to a sigmoid curve. 50This means that the result could not be calculated. Therefore, consensus OR2L13, consensus OR2L8, and consensus OR2L2 responded to acetic acid. In particular, consensus OR2L13 showed a dose-dependent response to acetic acid. Consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5 responded to propionic acid. In particular, consensus OR2L13, consensus OR2L8, and consensus OR2L2 showed a dose-dependent response to propionic acid. Consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, and consensus OR2L5 responded in a dose-dependent manner to butyric acid. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 responded in a dose-dependent manner to valeric acid. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 responded to isobutyric acid. In particular, Consensus OR2L13, Consensus OR2L8, and Consensus OR2L2 showed dose-dependent responses to isobutyric acid. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2L5 responded to isovaleric acid. In particular, Consensus OR2L8 and Consensus OR2L2 showed dose-dependent responses to isovaleric acid. Consensus OR2L13, Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, Consensus OR2AE1, and Consensus OR2AK2 responded to hexanoic acid. In particular, dose-dependent responses to hexanoic acid were observed for Consensus OR2L8, Consensus OR2L2, Consensus OR2L3, and Consensus OR2AE1. Consensus OR2L13 responded to 3-hydroxy-3-methylhexanoic acid. Furthermore, Consensus OR2AE1 and Consensus OR2A12 responded to nonanoic acid. In particular, a dose-dependent response to nonanoic acid was observed for Consensus OR2A12.Therefore, it was found that consensus OR2L13, consensus OR2L8, consensus OR2L2, consensus OR2L3, consensus OR2L5, consensus OR2AE1, consensus OR2A12, and consensus OR2AK2 respond to at least one carboxylic acid selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, hexanoic acid, 3-hydroxy-3-methylhexanoic acid, and nonanoic acid.

[0105] [Table 7]

[0106] [Table 8]

[0107] [Table 9]

[0108] [Table 10]

[0109] [Table 11]

[0110] [Table 12]

[0111] [Table 13]

[0112] [Table 14]

[0113] The response of consensus olfactory receptors to odor molecules reflects the response of the original olfactory receptors in olfactory cells. Therefore, from the above results, the original OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, OR2AE1, OR2A12, and OR2AK2 were identified as carboxylic acid receptors.

Claims

1. This includes measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2L13, OR2L8, OR2L2, OR2L3, OR2L5, and polypeptides having equivalent functions thereto, after the addition of a test substance. OR2L13 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, OR2L8 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, OR2L2 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, OR2L3 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, and OR2L5 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

13. A polypeptide having equivalent functionality to OR2L13 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or 3, and is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L8 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4 or 6, and is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L2 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7 or 9, and is responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L3 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 10 or 12, and is responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having equivalent functionality to OR2L5 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 13 or 15, and is responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. A method for evaluating and / or selecting an odor suppressant that contains at least one carboxylic acid selected from the group consisting of linear or branched carboxylic acids having 1 to 6 carbon atoms.

2. The method according to claim 1, wherein the carboxylic acid is at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid.

3. The method according to claim 1 or 2, wherein the carboxylic acid is at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid when the olfactory receptor polypeptide is OR2L13, OR2L8, OR2L2, or a polypeptide having equivalent function thereto; at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid when the polypeptide is OR2L3 or a polypeptide having equivalent function thereto; and at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid when the polypeptide is OR2L5 or a polypeptide having equivalent function thereto.

4. A polypeptide having the same function as OR2L13 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1, having at least one amino acid residue of (2) at a position corresponding to each amino acid position of (1) in Table 1 below in the amino acid sequence shown in SEQ ID NO: 1, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having the same function as OR2L8 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, having at least one amino acid residue of (4) at a position corresponding to each amino acid position of (3) in Table 1 below in the amino acid sequence shown in SEQ ID NO: 4, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having the same function as OR2L2 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7, having at least one amino acid residue of (6) at a position corresponding to each amino acid position of (5) in Table 1 below in the amino acid sequence shown in SEQ ID NO: 7, and being responsive to at least one selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having the same function as OR2L3 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 10, having at least one amino acid residue (8) at a position corresponding to each amino acid position (7) in Table 1 below of the amino acid sequence shown in SEQ ID NO: 10, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and hexanoic acid. A polypeptide having the same function as OR2L5 is a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 13, having at least one amino acid residue (10) at a position corresponding to each amino acid position (9) in Table 1 below of the amino acid sequence shown in SEQ ID NO: 13, and being responsive to at least one selected from the group consisting of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid. The method according to any one of claims 1 to 3. Table 1

5. Adding a test substance to the olfactory receptor polypeptide, and To measure the response of the olfactory receptor polypeptide to the test substance, A method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

6. The method according to claim 5, further comprising selecting a test substance as an inhibitor that enhances the response of the olfactory receptor polypeptide to the test substance.

7. The method according to claim 5, further comprising measuring the response of the olfactory receptor polypeptide in a control group.

8. The method according to claim 7, further comprising selecting a test substance as an inhibitor that enhances the response of the olfactory receptor polypeptide to the test substance compared to the control group.

9. Adding the test substance and the agonist of the olfactory receptor polypeptide to the olfactory receptor polypeptide, and Measuring the response of the olfactory receptor polypeptide to the agonist, A method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

10. The method according to claim 9, further comprising selecting a test substance as an inhibitor that suppresses the response of the olfactory receptor polypeptide to the agonist.

11. The method according to claim 9, further comprising measuring the response of the olfactory receptor polypeptide to the agonist in a control group.

12. The method according to claim 11, further comprising selecting a test substance as an inhibitor that suppresses the response of the olfactory receptor polypeptide to the agonist compared to the control group.

13. The method according to any one of claims 1 to 12, wherein the response of the olfactory receptor polypeptide is measured by measuring the amount of intracellular cAMP by ELISA or a reporter gene assay, measuring the amount of calcium ions by calcium imaging or a TGFα shedding assay, or measuring the change in potential across the cell membrane by a two-electrode membrane potential fixation method using African clawed frog oocytes.

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