Method for evaluating and / or selecting acetoin odor suppressants

Abstract

To search for a substance that can inhibit selectively an acetoin odor.SOLUTION: The present invention provides an evaluation and / or selection method for an acetoin odor inhibitor including measuring a response of at least one odorant receptor polypeptide selected from a group consisting of OR6Y1 or OR6B2 after the addition of a test substance and polypeptides having equivalent functions to them.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for searching for perfume materials that suppress malodor caused by acetoin. [Background technology]

[0002] Acetoin is a causative agent of various odor problems, such as "menstrual odor" (Patent Document 1), "vaginal odor" (non-menstrual odor) (Patent Document 2), and "breast cancer skin ulcer odor" (Patent Document 3). Furthermore, recent reports have shown that resident skin bacteria produce acetoin from organic acids contained in sweat (Non-Patent Document 1), suggesting that acetoin may be involved in sweat odor. Technology that can suppress acetoin odor could lead to solutions to these odor problems. For example, according to Patent Document 3, breast cancer skin ulcers exhibit a distinctive milky fermentation odor, which can cause psychological difficulties for patients in going out or working on a daily basis, significantly reducing their quality of life. Patent Document 3 therefore provides a method for deodorizing the acetoin-based breast cancer skin ulcer odor that arises from breast cancer skin ulcer sites using a deodorizing product made of a sheet-like covering. Specifically, the acetoin-based breast cancer skin ulcer odor can be reduced by utilizing the nucleophilic chemical reaction between a polyhydroxyamine compound and acetoin, followed by adsorption or encapsulation by porous particles. However, conventional techniques have not yet succeeded in completely eliminating the acetoin odor.

[0003] In mammals, including humans, odors are recognized when odorant molecules bind to olfactory receptors on olfactory neurons in the olfactory epithelium in the upper nasal cavity, and the receptor response is transmitted to the central nervous system. Approximately 400 types of olfactory receptors have been reported in humans, and the genes encoding these receptors account for approximately 2% of all human genes. Generally, olfactory receptors and odorant molecules correspond in a multi-to-multiple manner. That is, each olfactory receptor perceives multiple structurally similar odorant molecules with different affinities, while each odorant molecule is perceived by multiple types of olfactory receptors. Furthermore, it has been reported that odorant molecules that activate one olfactory receptor act as antagonists, inhibiting the activation of other olfactory receptors. The combination of responses from these multiple olfactory receptors results in the recognition of individual odors.

[0004] Traditionally, in the development of fragrance substances, the odor of candidate substances has been evaluated by sensory testing conducted by experts. However, sensory testing has problems such as the need to train experts capable of evaluating odors and low throughput. Therefore, in recent years, methods for discovering fragrance substances using the response of olfactory receptors to candidate substances as an indicator have been developed (Patent Document 4). Because olfactory receptor responses are selective to odorants, it is essential to first identify olfactory receptors that are selective for the target odor. Identifying such olfactory receptors enables approaches based on olfactory receptor inhibition using antagonists or cross-adaptation using agonists, for example, when aiming to eliminate malodors. In other words, it becomes possible to more effectively suppress the malodor of acetoin. However, existing receptor analysis methods have only succeeded in functionally analyzing approximately 12% of all human receptors (Non-Patent Document 2). As such, the olfactory system has yet to be fully elucidated, and therefore, it has been difficult to identify olfactory receptors sensitive to specific odors using molecular biological approaches.

[0005] 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 within the endoplasmic reticulum. This makes it impossible to evaluate its binding to extracellularly administered odorants. The reason for the lack of membrane expression of olfactory receptors remained unknown for a long time, but in recent years, a comparative analysis of mouse olfactory receptors that are membrane-expressed in cultured cells and those that are not membrane-expressed was conducted (Non-Patent Document 3). The results revealed that non-membrane-expressed olfactory receptors may have lower structural stability. Importantly, it was shown that there are statistically significant differences in amino acid properties in the primary amino acid sequence of the protein between membrane-expressed and non-membrane-expressed olfactory receptors, and that these amino acid positions are highly common among all approximately 1,000 mouse olfactory receptors. This suggests that non-membrane-expressed olfactory receptors lack structural stability due to a mutation of a different amino acid at a polypeptide position where a common amino acid would normally be used, resulting in a decision not to migrate to the cell membrane in cultured cells. Taking this into consideration, it is thought that the "consensus method," which introduces amino acids that are highly common among olfactory receptors, will make it possible to obtain the desired olfactory receptor as a stable protein and efficiently express it on the cell membrane.

[0006] Based on this idea, Non-Patent Document 2 used a consensus method to enable the analysis of specific olfactory receptors. Specifically, for the three human olfactory receptors OR6Y1, OR6B2, and OR56A4, amino acids highly common among the homologous genes of 10 mammalian species (gorilla, bonobo, chimpanzee, Sumatran orangutan, rhesus monkey, drill, common marmoset, gray mouse lemur, rat, and mouse) were introduced into each human olfactory receptor to create consensus olfactory receptors OR6Y1, OR6B2, and OR56A4. As a result, it was disclosed that, of the three olfactory receptors, OR6Y1 was able to measure responses to odorants using cultured cells. Therefore, it is suggested that although there are receptors for which consensus analysis is possible, the proportion of such receptors is approximately one-third.

[0007] The consensus method has long been used to design stable, industrially useful enzymes. However, according to a review of this method (Non-Patent Document 4), the probability of improving a single amino acid sequence by introducing consensus is approximately 50%, while the remaining 40% carries the risk of adversely affecting the protein, making the consensus method difficult to use. Furthermore, some believe that the application of consensus methods is inappropriate for research aimed at predicting the intrinsic function of olfactory receptors. Specifically, the approach of introducing amino acid substitutions raises concerns that the ligand-binding site of the original olfactory receptor may be altered, resulting in the inability to observe the original odor response. Therefore, consensus methods are expected to have a low success rate and the risk of altering the intrinsic function of the target olfactory receptor, and therefore their effectiveness has not been verified for a wide range of olfactory receptors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-198200 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-198829 [Patent Document 3] Japanese Patent Publication No. 2020-39655 [Patent Document 4] Japanese Patent Application Publication No. 2018-74944 [Non-patent literature]

[0009] [Non-Patent Document 1] :Hara T et al. PLOS ONE 9(11): e111833 (2014) [Non-patent document 2] :Trimmer C et al. PNAS 116:9475-9480 (2019) [Non-patent document 3] :Ikegami K et al. PNAS 117: 2957-2967 (2020) [Non-patent document 4] :Porebski TB et al. Protein Engineering, Design & Selection 29:245-251 (2016) Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides a method for efficiently evaluating and / or selecting acetoin odor suppressants using the response of olfactory receptors as an indicator. [Means for solving the problem]

[0011] The inventors have investigated various previously unexplored methods of consensus generation, such as generating consensus for the target olfactory receptor based on the amino acid sequences of olfactory receptors from more biological species than those described in Non-Patent Document 2. As a result, they have discovered a method that can improve the cell membrane expression level and response measurement efficiency of olfactory receptors, and have previously filed a patent application (Patent Application No. 2021-103675).

[0012] By using this consensus method, the present inventors have succeeded in identifying a new olfactory receptor that selectively responds to acetoin. Furthermore, the present inventors have found that by using the response of this olfactory receptor or a polypeptide with a similar function as an indicator, it is possible to efficiently search for substances that suppress the acetoin odor.

[0013] Therefore, the present invention provides a method for evaluating and / or selecting an acetoin odor suppressant, which comprises measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR6Y1, OR6B2 and polypeptides having equivalent functions thereto after the addition of a test substance. [Effects of the Invention]

[0014] According to the present invention, substances that can selectively suppress acetoin odor can be efficiently evaluated or selected. [Brief explanation of the drawings]

[0015] [Figure 1] The cell membrane expression levels and odor responsiveness of human olfactory receptors and consensus olfactory receptors designed based on these olfactory receptors are shown. The two histograms and one bar graph from the left represent the receptor protein levels on the HEK293 cell membrane as determined by flow cytometry. Flow cytometry measured PE signals using an anti-FLAG mouse IgG antibody, which recognizes the FLAG tag attached to the N-terminus of the olfactory receptor, as the primary antibody and a phycoerythrin (PE)-conjugated anti-mouse IgG antibody as the secondary antibody. As controls, cells that do not express receptors (mock) and cells expressing the M2 acetylcholine receptor (M2AcR), a receptor that is efficiently expressed on the membrane, were analyzed. The PE signal for each olfactory receptor was calculated by normalizing the PE signal of the mock to 0% and the PE signal of the M2AcR to 100%. The right panel shows the results of measuring the receptor's ligand responsiveness by luciferase assay. Error bars represent SEM (n = 3). [Figure 2]Ligand selectivity for two types of ligands of human olfactory receptors and consensus olfactory receptors designed for the olfactory receptors. Results of luciferase assays are shown. Error bars represent SEM (n=3). [Figure 3] Ligand selectivity of human olfactory receptors and consensus olfactory receptors designed for these olfactory receptors. Results of luciferase assays are shown. Error bars represent SEM (n=3). The numbers shown in the bottom row correspond to the odorant numbers shown in Figure 4. [Figure 4] Odorants used in Figure 3. [Figure 5] Luciferase assay of olfactory receptors. Odorants were administered at varying concentrations to three types of human olfactory receptors and consensus olfactory receptors designed for these olfactory receptors, and responses were measured. Error bars represent SEM (n=3). [Figure 6] Luciferase assay of olfactory receptors. Four types of consensus olfactory receptors were expressed in HEK293 cells with amino acid substitutions that have been reported to cause individual differences in odor response, and odorants were administered at various concentrations to measure the response. Error bars represent SEM (n=3). [Figure 7] Responsiveness of various olfactory receptors and consensus olfactory receptors to acetoin. The vertical axis shows the response intensity of each olfactory receptor. Mock refers to the condition in which an empty vector containing no olfactory receptor was used. The average response intensity of three replicates from one experiment is shown. [Figure 8] Dose-dependent responsiveness of OR6Y1 and OR6B2 to acetoin. The vertical axis shows the response intensity of each olfactory receptor, and the horizontal axis shows the concentration of acetoin added. Mock refers to the condition in which an empty vector containing no olfactory receptor was used. Error bars represent SEM (n=3). DETAILED DESCRIPTION OF THE INVENTION

[0016] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0017] As used herein, the term "agonist" refers to a substance that binds to and activates a receptor, whereas the term "antagonist" refers to a substance that binds to a receptor but does not activate the receptor or suppresses the receptor's response to an agonist.

[0018] As used herein, "olfactory receptor agonism" refers to binding to a receptor and activating that receptor.

[0019] As used herein, "odor cross-adaptation (or olfactory cross-adaptation)" with respect to a target odor refers to a phenomenon in which olfactory sensitivity to a target odor is reduced or altered by prior exposure to the odor of a substance other than the target odor and habituation to that odor. The inventors previously demonstrated that "odor cross-adaptation" is a phenomenon based on olfactory receptor agonism (International Publication No. 2016 / 194788). Specifically, in "odor cross-adaptation," olfactory receptors for a target odor respond to a different odorant prior to responding to the target odorant, and then desensitize, resulting in a low response even when later exposed to the target odorant, resulting in a reduction or alteration of the intensity of the target odor perceived by the individual. This mechanism of odor cross-adaptation caused by the behavior of olfactory receptors is also referred to herein as "odor cross-adaptation due to olfactory receptor agonism."

[0020] As used herein, "suppression of a target odor by olfactory receptor antagonism" refers to the suppression of the response of an olfactory receptor to a substance having the target odor by an antagonist, thereby suppressing the target odor recognized by an individual.

[0021] As used herein, the term "olfactory receptor polypeptide" refers to an olfactory receptor or a polypeptide with a function equivalent thereto, and the term "polypeptide with a function equivalent to an olfactory receptor" refers to a polypeptide that, like an olfactory receptor, can be expressed on the cell membrane, is activated by the binding of odorant molecules, and, upon activation, has the function of increasing the amount of intracellular cAMP by coupling with intracellular Gαs to activate adenylate cyclase (Nat. Neurosci., 2004, 5:263-278).

[0022] Herein, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, the identity is calculated by performing analysis using the search homology program in the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a unit size to compare (ktup) of 2.

[0023] As used herein, "at least 80% identity" with respect to nucleotide and amino acid sequences refers to 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identity. Also, "at least 90% identity" with respect to nucleotide and amino acid sequences refers to 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity.

[0024] As used herein, the term "amino acid residue" refers to 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).

[0025] As used herein, amino acid modifications may be designated by the accepted IUPAC single-letter amino acid abbreviations: [original amino acid, position, modified amino acid].

[0026] As used herein, a "corresponding position" in an amino acid sequence can be determined by aligning a target sequence with a reference sequence (the amino acid sequence shown in SEQ ID NO: 2 or 6 in this invention) to maximize homology. Alignment of amino acid sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank 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-mentioned alignment is considered to be the "position corresponding to" that position.

[0027] The "acetoin odor" suppressed by the present invention is an odor produced by acetoin, and includes, for example, menstrual odor, vaginal discharge odor, odor caused by breast cancer skin ulcers, sweat odor, etc.

[0028] The present inventors have previously discovered that modifying the amino acid sequence of a target olfactory receptor based on a consensus amino acid sequence derived from an alignment of the amino acid sequence of the target olfactory receptor with the amino acid sequence of an olfactory receptor encoded by a specific ortholog or specific orthologs and paralogs of the target olfactory receptor can improve membrane expression of the olfactory receptor in cultured cells, improve the odor responsiveness of the olfactory receptor, and also that the modified olfactory receptor can closely maintain the ligand selectivity of the unmodified olfactory receptor, and that analytical results obtained with the modified olfactory receptor closely reflect human olfactory sense. Based on this discovery, a patent application was filed (Japanese Patent Application No. 2021-103675). In this specification, the unmodified olfactory receptor is referred to as the "original olfactory receptor," modifying the amino acid sequence of an olfactory receptor based on the consensus amino acid sequence is referred to as "consensusization," and the consensus-based olfactory receptor is sometimes referred to as the "consensus olfactory receptor."

[0029] Here, the "consensus amino acid sequence" refers to an amino acid sequence consisting of consensus residues identified from an alignment of the amino acid sequence of a target olfactory receptor and the amino acid sequence of an olfactory receptor encoded by a specific ortholog or a specific ortholog and paralog of the target olfactory receptor (e.g., at least 11 types of olfactory receptors selected from the group consisting of olfactory receptors encoded by orthologs of the target olfactory receptor in mammals) according to the following criteria (i) to (iii): (i) at each amino acid position in the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target olfactory receptor and that occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) if there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target olfactory receptor is identified as the consensus residue; (i-iii) If an amino acid residue is present in the target olfactory receptor and no amino acid residue is present with an occurrence frequency of 40% or more, the amino acid residue is identified as not having a consensus residue. (i-iv) If the target olfactory receptor does not contain an amino acid residue but contains an amino acid residue with an occurrence frequency of 60% or more, the amino acid residue with the highest occurrence frequency is identified as the consensus residue, and if two or more types of amino acid residues with the highest occurrence frequency are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue. (iv) If none of the above (ii) to (i-iv) applies, identify the amino acid residues of the target olfactory receptor as consensus residues. (ii) When the consensus residue is identified according to the above criterion (i), if the consensus residue closest to the N-terminus is a consensus residue at a position corresponding to the N-terminus or further C-terminus of the olfactory receptor of interest and is not a methionine residue, change the consensus residue N-terminal to the methionine residue closest to the N-terminus to no consensus residue; (iii) When a consensus residue is identified according to the criteria in (i) above, if the consensus residue closest to the N-terminus is a consensus residue at a position closer to the N-terminus than the N-terminus of the olfactory receptor of interest and is not a methionine residue, the amino acid residue that appears most frequently in the alignment is identified as the consensus residue by going back one amino acid position at a time toward the N-terminus from the position of the consensus residue until a methionine residue appears, and if there are two or more types of amino acid residues that appear most frequently, the amino acid residue with the smallest molecular weight among those amino acid residues is identified as the consensus residue. Furthermore, herein, "modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence," i.e., "creating a consensus," means modifying 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 in the consensus amino acid sequence at the corresponding position.

[0030] The above-mentioned consensus olfactory receptors involve the introduction of amino acid mutations into the olfactory receptors, which at first glance raises concerns that the resulting measurement results may not accurately reflect the original functions of the original olfactory receptors and human olfactory sensations. However, as will be described later, the consensus olfactory receptors maintain the ligand response selectivity of the original olfactory receptors to a high degree and provide measurement results that best describe human olfactory sensations.

[0031] As disclosed in the previous patent application, for a human olfactory receptor with a known ligand, the amino acid sequence of the olfactory receptor was modified based on a consensus amino acid sequence derived from, for example, an alignment of the amino acid sequence of the olfactory receptor with the amino acid sequences of at least 11 olfactory receptors selected from the group consisting of olfactory receptors encoded by orthologs of the olfactory receptor in mammals, to create a consensus olfactory receptor. The original olfactory receptor and the consensus olfactory receptor were then expressed in cultured cells, and the membrane expression level and responsiveness to the ligand were measured. The consensus modification resulted in an increased membrane expression level and improved responsiveness (Figure 1).

[0032] Furthermore, as shown in the previous patent application, when response selectivity to ligands was examined using human olfactory receptors with known ligands and consensus olfactory receptors prepared in the same manner as described above, no significant change in response selectivity was observed due to consensus formation (Figure 2). Furthermore, as shown in the previous patent application, when 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 described above, no significant change in response selectivity was observed due to consensus formation (Figure 3). Furthermore, while the overall increase in measurement sensitivity due to consensus formation resulted in the identification of substances whose responses became detectable, none of the substances that clearly responded to the original olfactory receptors were found to result in a loss of response to the consensus olfactory receptor. In this way, the consensus olfactory receptor maintains a high degree of the ligand response selectivity of the original olfactory receptor.

[0033] In Non-Patent Document 2, comparative genomic analysis of populations with different odor perceptions was performed to identify candidate olfactory receptors responsible for the sensitivity of the odors. 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, attempts to express these original olfactory receptors in cultured cells and demonstrate their responsiveness to the corresponding odorants have not been successful. Therefore, as shown in the previous patent application, the same consensus synthesis as described above was applied to these olfactory receptors, and response analysis was performed, making it possible to measure responses to the corresponding odorants (Figure 5). Additionally, a genetic locus that explains individual differences in perception of coriander-derived odors has been reported (Eriksson N et al. Flavor 1:22 (2012)). This locus contains eight olfactory receptors, but functional analysis to determine which receptors actually recognize coriander-derived odorants has not been successful. Therefore, as shown in the previous patent application, we applied the same consensus method described above to the eight receptors and performed response analysis to the major aroma components of coriander ((E)-2-decenal, (E)-2-dodecenal). This revealed that OR10A2 and OR10A4 can recognize these odorants. Furthermore, as described in the previous patent application, we applied the same consensus methodology to OR10A2, OR10A4, OR6B2, and OR5C1, and then introduced amino acid sequences for which individual differences have been reported. We then examined whether we could obtain responsiveness that corresponds to individual differences in odor perception in sensory evaluations. For example, for OR10A2, the amino acid sequence registered under NP_001004460.1 occurs at a frequency of 68% of the population, while the amino acid sequence containing the three mutations H43R, H207R, and K258T occurs at a frequency of 32% (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 variants found in the population were predicted to alter the responsiveness to the major aroma compounds of cilantro ((E)-2-decenal and (E)-2-dodecenal), resulting in differences in whether humans perceive the cilantro aroma as unpleasantly soapy. We then applied consensus matching to OR10A2 and introduced these two amino acid sequence variants. As expected, the H43R, H207R, and K258T variants significantly reduced the responsiveness to these aroma compounds (Fig. 6, 10A2). The R262Q variant of olfactory receptor 10A4, which was less responsive to the aroma compounds than 10A2, was also reported to occur in 31% of the population, but this variant did not alter the responsiveness to these aroma compounds (Fig. 6, 10A4). Therefore, by reaching a consensus on olfactory receptors, it has become possible for the first time to identify OR10A2 as the receptor that explains the individual differences in how cilantro's aroma is perceived. Regarding OR6B2, the R122C and C179R mutations were found in 1% of the group (test participants in Non-Patent Document 2), and according to Non-Patent Document 2, this group was unable to strongly detect the odor of low concentrations of isobutyraldehyde. As shown in the previous patent application, in line with the individual differences in this sensory evaluation, it was confirmed that introducing the R122C and C179R mutations into consensus OR6B2 significantly reduced the responsiveness to isobutyraldehyde (6B2 in Figure 6). Regarding OR5C1, the N5K mutation was found in 1% of the population (occurrence frequency based on 1000 genomes project phase 3 allele frequencies), and according to Non-Patent Document 2, this population is unable to strongly detect the odor of low concentrations of citral. As shown in the previous application, in line with the individual differences in sensory evaluation, it was confirmed that introducing the N5K mutation into consensus OR5C1 significantly reduced the responsiveness to citral (5C1 in Figure 6). In this way, response analysis using consensus olfactory receptors provides measurement results that best explain the olfactory phenomenon measured by sensory evaluation, i.e., human olfactory sensation.

[0034] Therefore, although the amino acid sequence of the consensus olfactory receptor has been modified to improve membrane expression, its responsiveness reflects that of the original olfactory receptor in olfactory cells, and its function as an olfactory receptor is equivalent to that of the original olfactory receptor.

[0035] As shown in Figure 7, the present inventors discovered that consensus OR6Y1 and consensus OR6B2 respond to acetoin among the olfactory receptors that were subjected to the same consensus synthesis. As described above, consensus olfactory receptors maintain the ligand selectivity of the original olfactory receptors to a high degree. That is, the response of the consensus olfactory receptor to a certain odorant reflects the response of the olfactory receptor to that odorant in the olfactory cells of the original olfactory receptor. Therefore, OR6Y1 and OR6B2 can be identified as olfactory receptors that respond to acetoin.

[0036] OR6Y1 was predicted to be an olfactory receptor involved in the recognition of the odor of diacetyl (Non-Patent Document 2). Furthermore, OR6B2 was predicted to be an olfactory receptor involved in the recognition of the odor of isobutyraldehyde (Non-Patent Document 2). However, it was not previously recognized that OR6Y1 and OR6B2 respond to acetoin or that this could be the case.

[0037] As shown in Figure 8, consensus OR6Y1 and consensus OR6B2 respond to acetoin in a concentration-dependent manner. Therefore, OR6Y1 and OR6B2 are newly discovered acetoin receptors. Substances that suppress the response of OR6Y1, OR6B2, or polypeptides with equivalent functions, cause changes in the central nervous system's perception of acetoin odor based on odor suppression by olfactory receptor antagonism, resulting in selective suppression of acetoin odor. On the other hand, substances that enhance the response of OR6Y1, OR6B2, or polypeptides with equivalent functions cause changes in the central nervous system's perception of acetoin odor based on odor cross-adaptation by olfactory receptor agonism, resulting in selective suppression of acetoin odor. These substances can deodorize acetoin odor without causing the unpleasant feeling caused by the strong odor of the fragrance, which occurs in conventional deodorizing methods using deodorants or fragrances, or the problem of suppressing other odors.

[0038] Therefore, the present invention provides a method for evaluating and / or selecting an acetoin odor inhibitor. The method comprises measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR6Y1, OR6B2, and polypeptides functionally equivalent thereto 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 acetoin odor inhibitor. That is, a test substance that enhances the response of the olfactory receptor polypeptide is selected as an acetoin odor inhibitor based on odor cross-adaptation due to olfactory receptor agonism, and a test substance that inhibits the response of the olfactory receptor polypeptide is selected as an acetoin odor inhibitor based on olfactory receptor antagonism.

[0039] The methods of the present invention described above can be carried out in vitro or ex vivo.

[0040] 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 acetoin odor suppressant. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, or may be a compound, composition, or mixture.

[0041] The olfactory receptor polypeptide used in the method of the present invention is at least one olfactory receptor polypeptide selected from the group consisting of OR6Y1, OR6B2, and polypeptides having equivalent functions thereto. OR6Y1 and OR6B2 are olfactory receptors expressed in human olfactory cells. OR6Y1 is registered in the NCBI database as NP_001005189.1 and is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2, encoded by a gene having the nucleotide sequence shown in SEQ ID NO:1. OR6B2 is registered in the NCBI database as NP_001005853.1 and is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:6, encoded by a gene having the nucleotide sequence shown in SEQ ID NO:5.

[0042] Examples of polypeptides having functions equivalent to OR6Y1 include polypeptides consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2 and having responsiveness to acetoin. Another example of a polypeptide with equivalent function to OR6Y1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, containing the amino acid residue (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, of the positions corresponding to each amino acid position (1) in Table 1 below in the amino acid sequence shown in SEQ ID NO: 2, and having acetoin-responsiveness. Furthermore, a polypeptide having the amino acid residue (2) at all of the amino acid positions (1) in Table 1 below in the amino acid sequence shown in SEQ ID NO: 2 is consensus OR6Y1 consisting of the amino acid sequence shown in SEQ ID NO: 4. In other words, Table 1 below shows the amino acid residues that differ between OR6Y1 and consensus OR6Y1.

[0043] [Table 1]

[0044] Another example of a polypeptide having a function equivalent to OR6Y1 is a polypeptide consisting of consensus OR6Y1, which consists of the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90% identity thereto, and which is responsive to acetoin. Consensus OR6Y1 consists of the consensus amino acid sequence shown in SEQ ID NO: 4, derived by the above-mentioned method from the alignment of the amino acid sequence of OR6Y1 shown in SEQ ID NO: 2 with the amino acid sequences of 176 olfactory receptors encoded by mammalian OR6Y1 orthologs, and is responsive to acetoin. The ortholog is a gene highly homologous to OR6Y1 among olfactory receptor genes possessed by mammalian species, and is a gene containing the same name as 6Y1. Regarding mouse olfactory receptor genes and rat olfactory receptor genes, the most homologous species was selected as the ortholog. The amino acid sequence identity between consensus OR6Y1 and OR6Y1 is 96%.

[0045] Examples of polypeptides having functions equivalent to OR6B2 include polypeptides consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 6 and having responsiveness to acetoin. Another example of a polypeptide with equivalent function to OR6B2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 6, containing the amino acid residue (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, of the positions corresponding to each amino acid position (1) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 6, and having acetoin-responsiveness. Furthermore, a polypeptide having the amino acid residue (2) at all of the amino acid positions (1) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 6 is consensus OR6B2 consisting of the amino acid sequence shown in SEQ ID NO: 8. In other words, Table 2 below shows the amino acid residues that differ between OR6B2 and consensus OR6B2.

[0046] [Table 2]

[0047] Another example of a polypeptide having a function equivalent to OR6B2 is a polypeptide consisting of consensus OR6B2, which consists of the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 90% identity thereto, and which is responsive to acetoin. Consensus OR6B2 consists of the consensus amino acid sequence shown in SEQ ID NO: 8, derived by the above-mentioned method from an alignment of the amino acid sequence of OR6B2 shown in SEQ ID NO: 6 with the amino acid sequences of 181 olfactory receptors encoded by mammalian OR6B2 orthologs, and is responsive to acetoin. The ortholog is a gene highly homologous to OR6B2 among olfactory receptor genes possessed by mammalian species, and is a gene containing the same name as 6B2. Regarding mouse olfactory receptor genes and rat olfactory receptor genes, the most homologous species was selected as the ortholog. The amino acid sequence identity between consensus OR6B2 and OR6B2 is 92%.

[0048] In the method of the present invention, at least one olfactory receptor polypeptide selected from the above-mentioned olfactory receptor polypeptides may be used, or two or more of them may be used in combination. Preferably, one polypeptide selected from the group consisting of OR6Y1, OR6B2, consensus OR6Y1, and consensus OR6B2 is used, more preferably consensus OR6Y1 and / or consensus OR6B2 is used, and even more preferably consensus OR6Y1 is used.

[0049] In the method of the present invention, the olfactory receptor polypeptide can be used in any form as long as it does not lose its responsiveness to acetoin.For example, the olfactory receptor polypeptide can 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 their cultures; the membrane of olfactory cells carrying the olfactory receptor polypeptide; recombinant cells genetically engineered to express the olfactory receptor polypeptide or their cultures; the membrane of the recombinant cell that has the olfactory receptor polypeptide; artificial lipid bilayer membrane that has the olfactory receptor polypeptide, etc. All of these forms are included in the scope of the olfactory receptor polypeptide used in the present invention.

[0050] In a preferred embodiment, the olfactory receptor polypeptide is a cell that naturally expresses the olfactory receptor polypeptide, such as an olfactory cell, or a recombinant cell that has been genetically engineered to express the olfactory receptor polypeptide, or a culture thereof. The recombinant cell can be produced by transforming a cell with a vector incorporating a gene encoding the olfactory receptor polypeptide.

[0051] Preferably, to promote cell membrane expression of the olfactory receptor polypeptide, a gene encoding a receptor-transporting protein (RTP) is introduced into the cell together with the gene encoding the olfactory receptor polypeptide. Preferably, a gene encoding RTP1S is introduced into the cell 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:10, encoded by a gene having the nucleotide sequence of SEQ ID NO:9.

[0052] 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 culturing cells that express the olfactory receptor polypeptide, or directly dripping, sprinkling, or spraying the test substance onto the olfactory receptor polypeptide or cells or tissues containing it.

[0053] In the method of the present invention, the response of the olfactory receptor polypeptide is measured following the addition of a test substance to the olfactory receptor polypeptide. Measurement can be performed by any method known in the art for measuring olfactory receptor responses, such as measuring intracellular cAMP levels. For example, it is known that when olfactory receptors are activated by odorant molecules, they conjugate with intracellular G protein α subunits, which belong to the Gαs family, activating adenylate cyclase and thereby increasing intracellular cAMP levels ( Nat. Neurosci., 2004, 5:263-278 ). On the other hand, when olfactory receptors are activated by odorant molecules, they can also conjugate with proteins belonging to the Gq family, such as Gα15, and increase intracellular calcium ion levels. Therefore, the response of the olfactory receptor polypeptide can be measured by using the intracellular cAMP level or calcium ion level after the addition of odorant molecules, or the behavior of downstream molecules activated via these levels, as an indicator. Methods for measuring cAMP levels include ELISA and reporter gene assays. Other methods for measuring the response of olfactory receptor polypeptides include calcium ion concentration measurements, such as calcium imaging and TGFα shedding assays. Additionally, as an example of a method that uses the behavior of downstream molecules activated by cAMP levels as an indicator, the two-electrode voltage clamp method is effective in measuring the potential changes across the plasma membrane mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), which is activated by cAMP signals, in Xenopus oocytes.

[0054] In a first embodiment of the present invention, the method for evaluating and / or selecting an acetoin odor inhibitor 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 inhibitor of acetoin odor.

[0055] The test substance that enhances the response of the olfactory receptor polypeptide can first enhance the response of olfactory receptor, and then weaken the response of the olfactory receptor when exposed to acetoin.As a result, based on odor cross-adaptation, it can suppress the recognition of acetoin odor by individuals.Therefore, in the first embodiment, the acetoin odor suppressant is selected based on the odor cross-adaptation caused by olfactory receptor agonism.

[0056] The effect of a 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 in a control group. Examples of control groups include the olfactory receptor polypeptide to which no test substance has been added, the olfactory receptor polypeptide to which a control substance has been added, the olfactory receptor polypeptide to which a lower concentration of test substance has been added, the olfactory receptor polypeptide before the addition of the test substance, and cells in which the olfactory receptor polypeptide is not expressed. Preferably, the method of the present invention in the first embodiment comprises measuring the activity of the olfactory receptor polypeptide in the presence and absence of a test substance. Also preferably, the method of the present invention in the first embodiment comprises measuring the response to the agonist of cells in which the olfactory receptor polypeptide is expressed and cells in which the olfactory receptor polypeptide is not expressed in the presence of a test substance.

[0057] For example, in the first embodiment, if the response in test group is enhanced compared with that in control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.For example, if the response of the olfactory receptor polypeptide in test group is enhanced by preferably 120% or more, more preferably 150% or more, and even more preferably 200% compared with that in control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.Alternatively, if the response of the olfactory receptor polypeptide in test group is statistically significantly enhanced compared with that in control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.

[0058] One embodiment of the use of the acetoin odor suppressant selected according to the first embodiment is as follows: first, a subject who desires to suppress the acetoin odor is made to smell the suppressant before the subject is exposed to the odor. Alternatively, the suppressant is applied to the subject so that it has a stronger smell than the acetoin odor. As a result, even when the subject is exposed to the acetoin odor, the subject will perceive the odor as weak or not perceive it at all because their olfactory sensitivity to the odor has decreased.

[0059] In a second embodiment of the present invention, the method for evaluating and / or selecting an acetoin odor inhibitor according to the present invention comprises adding a test substance and an agonist of the olfactory receptor polypeptide to the above-mentioned olfactory receptor polypeptide; and measuring the response of the olfactory receptor polypeptide to the agonist. The agonist is preferably, but not limited to, acetoin. 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 acetoin odor.

[0060] In the second embodiment, the test substance that suppresses the response of the olfactory receptor polypeptide to the agonist of the olfactory receptor polypeptide is selected as an inhibitor of acetoin odor based on olfactory receptor antagonism.The effect of the test substance on the response of the olfactory receptor polypeptide to the agonist can be measured, for example, by comparing the response of the olfactory receptor polypeptide to the agonist (test group) to which the test substance has been added with the response of the agonist in the control group.Examples of the control group include those mentioned above.Preferably, the method of the present invention in the second embodiment comprises measuring the activity of the olfactory receptor polypeptide to the agonist in the presence and absence of a test substance.

[0061] For example, if the response in test group is more suppressed than that of control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.For example, if the response of the olfactory receptor polypeptide in test group is preferably suppressed by 60% or less, more preferably by 50% or less, and even more preferably by 25% or less compared with that of control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.Alternatively, if the response of the olfactory receptor polypeptide in test group is statistically significantly suppressed compared with that of control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to acetoin.

[0062] One embodiment of the use of the acetoin odor suppressant selected according to the second embodiment is as follows: first, a subject who desires to suppress the acetoin odor is made to smell the suppressant before or at the same time as the subject is exposed to the odor.As a result, even when the subject is exposed to the acetoin odor, the subject perceives the odor as weak or not perceive it because the olfactory sensitivity to the odor is reduced.

[0063] By the above procedure, an acetoin odor inhibitor can be obtained based on the response activity of olfactory receptor polypeptides that have acetoin odor responsiveness.If necessary, the acetoin odor inhibitory ability of the test substance selected above can be further evaluated by sensory testing.That is, in one embodiment of the method of the present invention, the test substance selected by the above procedure is obtained as a candidate substance for an acetoin odor inhibitor.Then, the acetoin odor inhibitory effect of the candidate substance is evaluated by sensory testing.The candidate substance that receives a better evaluation in sensory testing is selected as an acetoin odor inhibitor.

[0064] The sensory test of the candidate substance can be performed according to the deodorant evaluation procedure commonly performed in the art. If the candidate substance is an inducer of odor cross-adaptation, the order of application of the candidate substance and the target odor-causing substance to the evaluator can be adjusted. For example, when a test substance selected in the first embodiment described above is used as a candidate substance for a sensory test, the evaluator first smells the candidate substance and adapts to the odor. Then, the evaluator smells the target odor (preferably acetoin odor) and evaluates its intensity. The obtained evaluation result is compared with the intensity of the target odor when not adapted to the candidate substance. Also, for example, when a test substance selected in the second embodiment described above is used as a candidate substance for a sensory test, the evaluator smells the target odor (preferably acetoin odor) simultaneously with the candidate substance and evaluates the intensity of the target odor. The obtained evaluation result is compared with the intensity of the target odor alone. A candidate substance that is evaluated as reducing the intensity of the target odor as a result of the sensory test is selected as an acetoin odor suppressant.

[0065] The acetoin odor suppressant obtained by the present invention can be used as an active ingredient for suppressing acetoin odor. For example, the suppressant can be contained in a composition or article for suppressing acetoin odor as an active ingredient for suppressing acetoin odor. Alternatively, the suppressant can be used for the manufacture of a composition or article for suppressing acetoin odor. Examples of applications of the acetoin odor suppressant obtained by the present invention include, but are not limited to, sanitary products such as sanitary products and disposable diapers containing the agent; clothing, fabrics, or textiles such as underwear, undergarments, and linens containing the agent; laundry detergents or fabric softeners containing the agent; cosmetics, cleaning agents, topical preparations such as deodorants, medicines, and foods containing the agent; placing or spraying the agent in front of or in a toilet; having a person involved in excretion treatment in a hospital ward or nursing facility carry the agent or expose them to the agent before the treatment; and application to environments that generate acetoin odor.

[0066] The following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification as exemplary embodiments of the present invention, but the present invention is not limited to these embodiments.

[0067] [1] A method for evaluating and / or selecting an acetoin odor suppressant, which comprises measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR6Y1, OR6B2 and polypeptides having equivalent functions thereto after the addition of a test substance. [2] Preferably, the OR6Y1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and the OR6B2 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 6. The method described in [1]. [3] Preferably, the polypeptide having a function equivalent to that of OR6Y1 is a polypeptide having an amino acid sequence that is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more identical to the amino acid sequence shown in SEQ ID NO: 2, and is responsive to acetoin; Preferably, the polypeptide having a function equivalent to OR6B2 is a polypeptide having an amino acid sequence that is 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 identical to the amino acid sequence shown in SEQ ID NO: 6, and is responsive to acetoin, as described in [1] or [2]. [4] Preferably, the polypeptide having a function equivalent to OR6Y1 is composed of an amino acid sequence having an identity of 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 with the amino acid sequence shown in SEQ ID NO: 2, and has the amino acid residue of (2) at preferably at least one position, more preferably at least three positions, even more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions, corresponding to each amino acid position of (1) in Table 1 of the amino acid sequence shown in SEQ ID NO: 2, and is a polypeptide that is responsive to acetoin, Preferably, the polypeptide having a function equivalent to OR6B2 is a method described in any one of [1] to [3], which comprises an amino acid sequence that is 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 identical to the amino acid sequence shown in SEQ ID NO: 6, and has the amino acid residue (2) at preferably at least one, more preferably at least three, even more preferably at least five, even more preferably at least 10, and even more preferably all, of the positions corresponding to each amino acid position (1) in Table 2 of the amino acid sequence shown in SEQ ID NO: 6, and is a polypeptide that is responsive to acetoin. [5] Preferably, the polypeptide having a function equivalent to that of OR6Y1 is a polypeptide having an amino acid sequence having an identity of preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more with the amino acid sequence shown in SEQ ID NO: 4, and having acetoin responsiveness; Preferably, the polypeptide having a function equivalent to OR6B2 is a polypeptide 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 in SEQ ID NO: 8, and is responsive to acetoin, according to any one of [1] to [3]. [6] Preferably, the following: adding a test substance to the olfactory receptor polypeptide; and measuring the response of the olfactory receptor polypeptide to the test substance; The method according to any one of [1] to [5], comprising: [7] Preferably, the method described in [6] further comprises selecting a test substance that enhances the response of the olfactory receptor polypeptide to the test substance as an inhibitor of acetoin odor. [8] The method described in [6], preferably further comprising measuring the response of the olfactory receptor polypeptide in a control group. [9] Preferably, the method further comprises selecting a test substance that enhances the response of the olfactory receptor polypeptide to the test substance compared to the control group as an acetoin odor suppressant; More preferably, the method further comprises selecting 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 as an inhibitor of the acetoin odor, or selecting a test substance that statistically significantly enhances the response of the olfactory receptor polypeptide to the test substance compared to the control group as an inhibitor of the acetoin odor. [8] The method described in [8].

[10] Preferably, the following: Adding a test substance and an agonist of the olfactory receptor polypeptide to the olfactory receptor polypeptide; and measuring the response of the olfactory receptor polypeptide to the agonist; The method according to any one of [1] to [5], comprising:

[11] Preferably, the method described in

[10] further comprises selecting a test substance that inhibits the response of the olfactory receptor polypeptide to the agonist as an inhibitor of acetoin odor.

[12] The method described in

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

[13] Preferably, the method further comprises selecting a test substance that suppresses the response of the olfactory receptor polypeptide to the agonist compared to the control group as an acetoin odor suppressant; More preferably, the method further comprises selecting a test substance that suppresses the response of the olfactory receptor polypeptide to the agonist by 60% or less compared to the control group as an inhibitor of the acetoin odor, or selecting a test substance that statistically significantly suppresses the response of the olfactory receptor polypeptide to the agonist compared to the control group as an inhibitor of the acetoin odor.

[12] The method described in.

[14] The method according to any one of [1] to

[13] , wherein the olfactory receptor polypeptide is preferably expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide.

[15] Preferably, the response of the olfactory receptor polypeptide is measured by measuring the amount of intracellular cAMP using ELISA or a reporter gene assay, measuring the amount of calcium ions using calcium imaging or a TGFα shedding assay, or measuring the potential change inside and outside the cell membrane using a two-electrode voltage clamp method using Xenopus oocytes. The method according to any one of [1] to

[14] .

[16] The method according to any one of [1] to

[15] , preferably further comprising evaluating the test substance by a sensory test. [Example]

[0068] The present invention will be described in more detail below with reference to examples.

[0069] Example 1 Identification of olfactory receptors that respond to acetoin 1) Preparation of olfactory receptor-expressing cells To design consensus olfactory receptors, we used NCBI BLAST to search for candidate homologous genes of the target olfactory receptor gene. Orthologs were then identified from the resulting gene cluster. Specifically, genes with the same name as the target olfactory receptor were selected as mammalian orthologs from the top-ranked homologous genes found by BLAST. For example, in the case of human OR6Y1, 174 genes with names containing OR6Y1 were selected from the top 250 homologous genes found by BLAST, using the amino acid sequence of human OR6Y1 (NP_001005189.1) as the query sequence and the target organism name as mammal. Additionally, for genes from mice (Mus musculus) and rats (Rattus norvegicus), which use different olfactory receptor nomenclature systems, we selected one gene each with the highest homology from the top 250 genes in the search. These 176 genes were identified as mammalian orthologs. These 176 genes, plus human OR6Y1, yielded a total of 177 genes. The amino acid sequences were analyzed for alignment and consensus amino acids were identified as described below. For human OR6B2, BLAST search was performed using the amino acid sequence of human OR6B2 (NP_001005853.1) as the query sequence. The target organism was mammalian. From the top 250 homologous genes, 179 genes containing OR6B2 in their names were selected. Additionally, for genes from mice (Mus musculus) and rats (Rattus norvegicus), which use different olfactory receptor nomenclature systems, one gene each with the highest homology was selected from the top 250 genes. These 181 genes were identified as mammalian orthologs. The amino acid sequences of these 181 genes, plus human OR6B2, yielded a total of 182 genes. The amino acid sequences were analyzed for alignment and consensus amino acids were identified as described below.

[0070] Alignment analysis of the identified gene cluster was performed using ClustalW, and further adjustments were made to optimize the sequence based on amino acids or amino acid motifs highly conserved among olfactory receptors. Ballesteros-Weinstein residue numbering was assigned with reference to the results of an alignment of all mouse olfactory receptors shown in Non-Patent Document 3. Based on the alignment results, a consensus olfactory receptor was designed using Jalview. In the alignment, if there was one amino acid residue different from the amino acid residue in the reference amino acid sequence and with an occurrence frequency of 50% or more at a position corresponding to each amino acid position in the original reference human olfactory receptor amino acid sequence, the amino acid residue in the reference amino acid sequence was modified with that amino acid residue. Note that even if there was one amino acid residue different from the amino acid residue in the reference amino acid sequence and with an occurrence frequency of 50% at a position corresponding to each amino acid position in the original reference human olfactory receptor amino acid sequence, 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. The topology of the olfactory receptors was confirmed using the Transmembrane Hidden Markov Model (TMHMM). The DNA sequences encoding the various designed olfactory receptor polypeptides were synthesized by DNA synthesis, with the corresponding codons optimized for expression in human cultured cells. The codon-optimized sequence encoding the consensus OR6Y1 is shown in SEQ ID NO: 3, and the codon-optimized sequence encoding the consensus OR6B2 is shown in SEQ ID NO: 7. EcoRI and XhoI sites were added to both ends of this sequence, which was then recombined into the EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence in the pME18S vector. Additionally, the gene encoding human RTP1S, which translocates olfactory receptor proteins produced in cultured cells to the cell membrane, was inserted into the EcoRI and XhoI sites of another pME18S vector to create the pME18S-RTP1S vector.

[0071] A reaction solution with the composition shown in Table 3 was prepared, left to stand in a clean bench for 20 minutes, and then added to each well of a 96-well plate (BD). Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well at a concentration of 2 × 10 5 cells / cm 2 The cells were seeded at 100°C and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that did not express olfactory receptors (Mock) were prepared.

[0072] [Table 3]

[0073] 2) Luciferase assay Olfactory receptors expressed in HEK293 cells increase intracellular cAMP levels by coupling with endogenous GαS and activating adenylate cyclase. To measure odor responses, we used a luciferase reporter gene assay, which monitors the increase in intracellular cAMP levels as luminescence from the firefly luciferase gene (fluc2P-CRE-hygro). We also co-transfected the cells with the Renilla luciferase gene fused downstream of the CMV promoter (hRluc2P-CMV-hygro) and used it as an internal control to correct for errors in gene transfection efficiency and cell number.

[0074] The medium was removed from the culture prepared in 1) above, and 75 μL of test substance solution (final concentration: 1 mM acetoin (Tokyo Chemical Industry Co., Ltd.)) prepared in new medium was added. The cells were cultured in a CO2 incubator for 4 hours to allow sufficient expression of the luciferase gene in 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 intensity derived from firefly luciferase induced by stimulation with the test substance was divided by the luminescence intensity derived from Renilla luciferase to calculate the signal, which was used for analysis. The signal induced by acetoin stimulation was divided by the signal in cells without acetoin stimulation to calculate the fold increase, which was used as an index of response intensity.

[0075] 3) Results The results are shown in Figure 7. Of the consensus olfactory receptors tested, consensus OR6Y1 and consensus OR6B2 were found to respond to acetoin.

[0076] Example 2 Dose-dependent response of olfactory receptors to acetoin For consensus OR6Y1 and consensus OR6B2, which showed responsiveness to acetoin in Example 1, the dose dependence of the response to acetoin was analyzed in the same manner as in Example 1, except that the final concentration of acetoin was adjusted to 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM. The results are shown in Figure 8. Consensus OR6Y1 and consensus OR6B2 responded to acetoin in a dose-dependent manner.

[0077] The response of consensus olfactory receptors to odorants reflects the response of the original olfactory receptors in olfactory cells. Therefore, the results of Examples 1 and 2 identified the original OR6B2 and OR6Y1 as receptors for acetoin.

Claims

1. measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR6Y1, OR6B2, and polypeptides having equivalent functions thereto after the addition of a test substance; the OR6Y1 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, the OR6B2 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, The polypeptide having a function equivalent to that of OR6Y1 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 or 4, and is responsive to acetoin; The polypeptide having a function equivalent to OR6B2 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 or 8 and responsive to acetoin. Method for evaluating and / or selecting acetoin odor control agents.

2. The polypeptide having a function equivalent to that of OR6Y1 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, having an amino acid residue of (2) at at least one position corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 2 in Table 1 below, and having acetoin responsiveness, The polypeptide having a function equivalent to OR6B2 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6, having an amino acid residue of (2) at at least one position corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 6 in Table 2 below, and having acetoin responsiveness. The method of claim 1. 【Table 1】 【Table 2】

3. adding a test substance to the olfactory receptor polypeptide; and measuring the response of the olfactory receptor polypeptide to the test substance; 3. The method of claim 1 or 2, comprising:

4. The method of claim 3, further comprising selecting a test substance that enhances the response of the olfactory receptor polypeptide to the test substance as an inhibitor of acetoin odor.

5. The method of claim 3, further comprising measuring the response of the olfactory receptor polypeptide in a control group.

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

7. Adding a test substance and an agonist of the olfactory receptor polypeptide to the olfactory receptor polypeptide; and measuring the response of the olfactory receptor polypeptide to the agonist; 3. The method of claim 1 or 2, comprising:

8. The method of claim 7, further comprising selecting a test substance that inhibits the response of the olfactory receptor polypeptide to the agonist as an inhibitor of acetoin odor.

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

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

11. The method according to any one of claims 1 to 10, wherein the response of the olfactory receptor polypeptide is measured by measuring the amount of intracellular cAMP using ELISA or a reporter gene assay, measuring the amount of calcium ions using calcium imaging or a TGFα shedding assay, or measuring changes in the potential inside and outside the cell membrane using a two-electrode voltage clamp method using Xenopus oocytes.

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