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

Abstract

To search for substances capable of selectively inhibiting odors caused by heteroaromatic compounds.SOLUTION: A method for evaluating and / or selecting agents for inhibiting odors caused by heteroaromatic compounds includes measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and their functionally equivalent polypeptides after the addition of a test substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for searching for perfume materials that suppress odors caused by heterocyclic aromatic compounds. [Background technology]

[0002] In our living environment, there are many malodorous molecules with different polarities and molecular weights, and heterocyclic aromatic compounds are one of them. Heterocyclic aromatic compounds include compounds such as indole and skatole that are known to be the cause of fecal odor and bad breath (for example, Patent Documents 1 and 2). Technology that can suppress odors caused by heterocyclic aromatic compounds will lead to the solution of these malodor issues.

[0003] As a measure against odors caused by heterocyclic aromatic compounds, deodorizing technology has been proposed that adsorbs and decomposes the odor-causing substances to reduce their amount, but it takes time for the odor substances to be reduced, so it lacks immediate effectiveness. In addition, a technology has been proposed that masks the odors using fragrances that have a stronger smell than the heterocyclic aromatic compounds, but this technology does not deodorize the odors caused by heterocyclic aromatic compounds, and cannot fundamentally eliminate the odors caused by heterocyclic aromatic compounds. In addition, the odor of the fragrance may cause discomfort.

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

[0005] In the past, in the development of fragrance substances, the odor of candidate substances was evaluated by sensory testing by experts. However, sensory testing has problems such as the need to train experts who can evaluate odors and low throughput. In recent years, a method for searching for fragrance substances using the response of olfactory receptors to candidate substances as an index has been developed (Patent Document 3). Since the response of olfactory receptors is selective to odor substances, it is essential to first find an olfactory receptor that has selectivity for the target odor. If such an olfactory receptor is identified, for example, when the purpose is to deodorize a bad odor, an approach based on olfactory receptor suppression by an antagonist or cross-adaptation by an agonist becomes possible. In other words, it becomes possible to more effectively suppress odors caused by heterocyclic aromatic compounds, which are bad odors. In fact, several olfactory receptors that respond to heterocyclic aromatic compounds have been identified so far. For example, OR5P3, OR2W1, OR5K1 and OR8H1 (Patent Document 1) have been identified as olfactory receptors that respond to indole or skatole, OR4S2 and OR1A1 (Patent Document 2) have been identified as olfactory receptors that respond to indole, and OR52N2, OR11G2, OR5AC2, OR4C15, OR8S1, OR11H6 and OR11H4 (Patent Document 4) have been identified as olfactory receptors that respond to indole or skatole. However, existing receptor analysis methods have only succeeded in functional analysis of about 12% of all human receptors (Non-Patent Document 1). Thus, the olfactory system has not yet been elucidated, and therefore, there has been a problem in that it is not easy to find all olfactory receptors that are sensitive to a specific odor by molecular biological approaches.

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

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

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

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

[0010] [Patent Document 1] Patent No. 5593271 [Patent Document 2] Patent Publication No. 2021-69330 [Patent Document 3] JP 2018-74944 A [Patent Document 4] Special Publication No. 2016-523546 [Patent Document 5] JP 2023-2484 A [Non-patent literature]

[0011] [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) Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides a method for efficiently evaluating and / or selecting an agent for suppressing odor caused by a heterocyclic aromatic compound by using the response of an olfactory receptor as an indicator. [Means for solving the problem]

[0013] The present inventors have succeeded in identifying a new olfactory receptor that selectively responds to heterocyclic aromatic compounds by using the consensus method of Patent Document 5. The present inventors have also found that by using the response of the olfactory receptor or a polypeptide having a similar function as an index, it is possible to efficiently search for a substance that suppresses odors caused by heterocyclic aromatic compounds and to detect odors caused by heterocyclic aromatic compounds in a sample.

[0014] Therefore, the present invention provides the following 1) and 2). 1) A method for evaluating and / or selecting an inhibitor of odor caused by heterocyclic aromatic compounds, comprising measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12 and polypeptides having equivalent functions thereto after addition of a test substance. 2) A method for detecting an odor caused by a heterocyclic aromatic compound, comprising contacting a sample with at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR2AT4, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12 and polypeptides having equivalent functions thereto, and measuring the response of the olfactory receptor polypeptide to the sample. Effect of the Invention

[0015] According to the present invention, it is possible to efficiently evaluate or select a substance capable of selectively suppressing an odor caused by a heterocyclic aromatic compound, and to detect an odor caused by a heterocyclic aromatic compound in a sample. [Brief description of the drawings]

[0016] [Figure 1] Luciferase assay of olfactory receptors. Bar graphs show the response intensities for each consensus olfactory receptor when varying concentrations of skatole or indole were administered. The figure shows the EC50 obtained by curve fitting the data to a sigmoid. Error bars represent SEM (from three replicates in one experiment). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0018] As used herein, the term "agonist" refers to a substance that binds to and activates a receptor, whereas the term "antagonist" refers to a substance that binds to a receptor but does not activate the receptor or suppresses the response of the receptor to an agonist.

[0019] As used herein, "olfactory receptor agonism" refers to binding to a receptor and activating the receptor.

[0020] In this specification, "odor cross-adaptation (or olfactory cross-adaptation)" with respect to a target odor refers to a phenomenon in which olfactory sensitivity to a target odor causative substance is reduced or changed by receiving the odor of a substance other than the target odor causative substance in advance and becoming accustomed to the odor. The present inventors have previously revealed that "odor cross-adaptation" is a phenomenon based on olfactory receptor agonism (International Publication No. 2016 / 194788). That is, in "odor cross-adaptation", the olfactory receptor for the target odor causative substance responds to a different odor causative substance prior to responding to the target odor causative substance, and then desensitizes, so that even if it is later exposed to the target odor causative substance, it can only respond poorly, resulting in a reduction or alteration of the intensity of the target odor recognized by the individual. The mechanism of odor cross-adaptation caused by such behavior of olfactory receptors is also referred to as "odor cross-adaptation due to olfactory receptor agonism" in this specification.

[0021] As used herein, "suppression of a target odor by olfactory receptor antagonism" refers to suppressing the response of an olfactory receptor to a substance having a target odor by an antagonist, thereby suppressing the target odor recognized by an individual.

[0022] As used herein, the term "olfactory receptor polypeptide" refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and the term "polypeptide having a function equivalent to that of an olfactory receptor" refers to a polypeptide that can be expressed on the cell membrane, like an olfactory receptor, is activated by the binding of odor molecules, and, upon activation, has the function of increasing the amount of intracellular cAMP by coupling with intracellular Gαs to activate adenylate cyclase (Nat. Neurosci., 2004, 5:263-278).

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

[0024] As used herein, "at least 80% identity" with respect to a nucleotide sequence and an amino acid sequence means 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more identity. Also, "at least 90% identity" with respect to a nucleotide sequence and an amino acid sequence means 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity.

[0025] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0026] As used herein, amino acid modifications may be designated by the accepted IUPAC one-letter amino acid abbreviations: [original amino acid, position, modified amino acid].

[0027] In the present specification, the "corresponding position" on an amino acid sequence can be determined by aligning a target sequence with a reference sequence (the amino acid sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28 in the present invention) so as to give the maximum homology. Alignment of amino acid sequences can be performed using a known algorithm, and the procedure is known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin ([www.clustal.org]), the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0028] In this specification, the term "heterocyclic aromatic compound" refers to an aromatic compound that contains at least one type of heteroatom in the ring structure. Preferably, the heterocyclic aromatic compound is a nitrogen-containing aromatic compound that contains a nitrogen atom in the ring structure. More preferred examples of heterocyclic aromatic compounds include indole and skatole.

[0029] The "odor caused by a heterocyclic aromatic compound" suppressed by the present invention is an odor caused by the above-mentioned heterocyclic aromatic compound, preferably an odor caused by a nitrogen-containing aromatic compound, and more preferably an odor caused by at least one selected from the group consisting of indole and skatole. Both indole and skatole are known as causative substances of fecal odor, bad breath, etc. Therefore, the "odor caused by a heterocyclic aromatic compound" suppressed by the present invention may typically be fecal odor or bad breath.

[0030] The present inventors have previously discovered that by modifying the amino acid sequence of a target olfactory receptor based on a consensus amino acid sequence derived from an alignment of the amino acid sequence of the target olfactory receptor with the amino acid sequence of an olfactory receptor encoded by a specific ortholog or specific ortholog and paralog of the target olfactory receptor, according to criteria different from conventional standards, it is possible to improve the membrane expression of the modified olfactory receptor in cultured cells compared to the original olfactory receptor, improve the odor responsiveness of the modified olfactory receptor compared to the original olfactory receptor, and even if the membrane expression of the modified olfactory receptor in cultured cells is not improved compared to the original olfactory receptor, it is possible to improve the odor responsiveness of the modified olfactory receptor, furthermore, the modified olfactory receptor can well maintain the ligand selectivity of the original olfactory receptor before modification, and the analytical results obtained using the modified olfactory receptor well reflect the human sense of smell (Patent Document 5). In this specification, the olfactory receptor before modification is referred to as the "original olfactory receptor," modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence is referred to as "consensusization," and the consensus-based olfactory receptor is sometimes referred to as the "consensus olfactory receptor."

[0031] 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 an olfactory receptor encoded by a specific ortholog or a specific ortholog and paralog of the target olfactory receptor (e.g., at least 11 types of olfactory receptors selected from the group consisting of olfactory receptors encoded by orthologs of the target olfactory receptor in mammals) according to the following criteria (i) to (iii). (i) at each amino acid position of the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target olfactory receptor and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) when there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target olfactory receptor is identified as a consensus residue; (i-iii) if an amino acid residue is present in the olfactory receptor of interest and no amino acid residue is present with an occurrence frequency of 40% or more, it is identified as having no consensus residue; (i-iv) if no amino acid residue is present in the target olfactory receptor and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and if two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue; (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target olfactory receptor as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or the C-terminus of the olfactory receptor of interest and is not a methionine residue, the consensus residue N-terminal to the consensus residue consisting of the methionine residue at the position closest to the N-terminus is changed to no consensus residue; (iii) When a consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the olfactory receptor of interest and is not a methionine residue, the alignment is traced back one amino acid position at a time toward the N-terminus from the position of the consensus residue until a methionine residue appears, and the most frequently occurring amino acid residue is identified as the consensus residue. If there are two or more types of most frequently occurring amino acid residues, the amino acid residue with the smallest molecular weight among those amino acid residues is identified as the consensus residue. Additionally, herein, "modifying the amino acid sequence of an olfactory receptor based on a consensus amino acid sequence," i.e., "creating a consensus," refers to modifying at least one amino acid residue in the amino acid sequence of the olfactory receptor of interest that differs from the consensus amino acid sequence to an amino acid residue of the consensus amino acid sequence at the corresponding position.

[0032] The above-mentioned consensus olfactory receptor involves the introduction of amino acid mutations into the olfactory receptor, which may at first glance raise concerns about the possibility of providing measurement results that do not accurately reflect the original functions of the original olfactory receptor and human olfactory sensation. However, as shown in Patent Document 5, the consensus olfactory receptor maintains a high degree of ligand response selectivity of the original olfactory receptor and provides measurement results that best explain human olfactory sensation.

[0033] Therefore, although the amino acid sequence of the consensus olfactory receptor has been modified, its responsiveness reflects the responsiveness of the original olfactory receptor in olfactory cells, and its function as an olfactory receptor is equivalent to that of the original olfactory receptor.

[0034] The present inventor found that among the olfactory receptors to which the above-mentioned consensus was applied, consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3, consensus OR4B1, consensus OR5T3 and consensus OR8B12 respond to heterocyclic aromatic compounds. As described above, the consensus olfactory receptor maintains the ligand selectivity of the original olfactory receptor to a high degree. That is, the response of the consensus olfactory receptor to a certain odorant reflects the response of the original olfactory receptor to the odorant in the olfactory cell. Therefore, OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3 and OR8B12 can be identified as olfactory receptors that respond to heterocyclic aromatic compounds.

[0035] It has not previously been recognized that OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3 and OR8B12 are or may be responsive to heteroaromatic compounds.

[0036] As shown in Figure 1, consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3, consensus OR5T3 and consensus OR8B12 respond to indole, among which consensus OR2AT4 and consensus OR8I2 respond to indole in a concentration-dependent manner. Also, consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3 and consensus OR4B1 respond to skatole, among which consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1 and consensus OR8H2 respond to skatole in a concentration-dependent manner. Therefore, OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3 and OR8B12 are newly discovered heterocyclic aromatic compound receptors. Substances that suppress the response of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12 or polypeptides having equivalent functions thereto can cause changes in the recognition of odors caused by heterocyclic aromatic compounds in the central nervous system based on odor suppression by olfactory receptor antagonism, and as a result, can selectively suppress odors caused by heterocyclic aromatic compounds. On the other hand, substances that enhance the response of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, or polypeptides having similar functions, can cause changes in the central nervous system recognition of odors caused by heterocyclic aromatic compounds based on odor cross-adaptation due to olfactory receptor agonism, and as a result, can selectively suppress odors caused by heterocyclic aromatic compounds.Therefore, substances that suppress or enhance the response of these polypeptides can deodorize odors caused by heterocyclic aromatic compounds without causing problems such as discomfort due to the strong odor of the fragrance, which occurred in conventional deodorizing methods using deodorants or fragrances, or the problem of suppressing other odors.

[0037] Therefore, the present invention provides a method for evaluating and / or selecting an odor suppressant caused by a heterocyclic aromatic compound (hereinafter referred to as the odor suppressant evaluation and / or selection method of the present invention). The method includes measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto, after addition of a test substance. Based on the measured response, a test substance that enhances or suppresses the response of the olfactory receptor polypeptide is detected. The detected test substance is selected as an odor suppressant caused by a heterocyclic aromatic compound. That is, a test substance that enhances the response of the olfactory receptor polypeptide is selected as an inhibitor of the odor caused by a heterocyclic aromatic compound 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 inhibitor of the odor caused by a heterocyclic aromatic compound based on olfactory receptor antagonism.

[0038] The above-mentioned method for evaluating and / or selecting an odor suppressant of the present invention can be carried out in vitro or ex vivo.

[0039] The test substance used in the odor suppressant evaluation and / or selection method of the present invention is not particularly limited as long as it is a substance desired to be used as an odor suppressant caused by a heterocyclic aromatic compound. The test substance may be a naturally occurring substance, or a substance artificially synthesized by a chemical or biological method, or may be a compound, composition, or mixture.

[0040] The olfactory receptor polypeptide used in the odor suppressant evaluation and / or selection method of the present invention is at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto. OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, and OR8B12 are olfactory receptors expressed in human olfactory cells. The Accession No. and amino acid sequence of each olfactory receptor in the NCBI database are shown in Table 1.

[0041] [Table 1]

[0042] An example of a polypeptide having a function equivalent to OR2AT4 is a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:1 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR2AT4 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 2 below, and having responsiveness to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR2AT4 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 2 below, and having responsiveness to at least one selected from the group consisting of indole and skatole. Here, having L (2) at the amino acid position corresponding to the C-terminus of (1) in Table 2 below means that in a certain amino acid sequence, L is added to the C-terminus side of the position corresponding to the last amino acid position of the amino acid sequence shown in SEQ ID NO: 1. Note that in the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having amino acid residues (2) at all of the amino acid positions of (1) in Table 1 below is consensus OR2AT4 consisting of the amino acid sequence shown in SEQ ID NO: 3. In other words, Table 2 below shows the amino acid residues that differ between OR2AT4 and consensus OR2AT4.

[0043] Yet another example of a polypeptide having a function equivalent to OR2AT4 includes a consensus OR2AT4 consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. The consensus OR2AT4 consists of the consensus amino acid sequence shown in SEQ ID NO: 3, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR2AT4 shown in SEQ ID NO: 1 and the amino acid sequences of 197 types of olfactory receptors encoded by orthologues of OR2AT4 in mammals, and has responsiveness to indole and skatole. The orthologue is a gene that is highly homologous to OR2AT4 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that contains the same name as OR2AT4. The amino acid sequence identity between the consensus OR2AT4 and OR2AT4 is 94%.

[0044] An example of a polypeptide having a function equivalent to OR8I2 includes a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 4 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR8I2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, having an amino acid residue of (4) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (3) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 4, and having a responsiveness to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR8I2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, having an amino acid residue of (4) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (3) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 4, and having a responsiveness to at least one selected from the group consisting of indole and skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 4, a polypeptide having amino acid residues of (4) at all of the amino acid positions of (3) in Table 2 below is consensus OR8I2 consisting of the amino acid sequence shown in SEQ ID NO: 6. In other words, Table 2 below shows the amino acid residues that differ between OR8I2 and consensus OR8I2.

[0045] Yet another example of a polypeptide having a function equivalent to OR8I2 includes a polypeptide consisting of consensus OR8I2 consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. Consensus OR8I2 consists of the consensus amino acid sequence shown in SEQ ID NO: 6, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR8I2 shown in SEQ ID NO: 4 and the amino acid sequences of 118 olfactory receptors encoded by mammalian orthologues of OR8I2, and is responsive to indole and skatole. The orthologue is a gene that is highly homologous to OR8I2 among olfactory receptor genes possessed by mammalian species and contains the same name as OR8I2. The amino acid sequence identity between consensus OR8I2 and OR8I2 is 95%.

[0046] An example of a polypeptide having a function equivalent to OR3A3 includes a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR3A3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, at least one, preferably at least three, more preferably at least five, even more preferably at least 10, and even more preferably all of the amino acid residues of (6) at positions corresponding to each amino acid position of (5) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 7, and responsive to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR3A3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all of the amino acid residues of (6) at positions corresponding to each amino acid position of (5) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 7, and responsive to at least one selected from the group consisting of indole and skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 7, a polypeptide having amino acid residues (6) at all of the amino acid positions (5) in Table 2 below is consensus OR3A3 consisting of the amino acid sequence shown in SEQ ID NO: 9. In other words, Table 2 below shows the amino acid residues that differ between OR3A3 and consensus OR3A3.

[0047] Another example of a polypeptide having a function equivalent to OR3A3 includes a polypeptide consisting of the consensus OR3A3 consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. The consensus OR3A3 is composed of the amino acid sequence of OR3A3 shown in SEQ ID NO: 7, the amino acid sequences of seven olfactory receptors encoded by the orthologues of OR3A3 in mammals, the amino acid sequence of OR3A2 shown in SEQ ID NO: 31, which is a paralog with high homology to OR3A3, and the consensus amino acid sequence shown in SEQ ID NO: 9, which is derived by the above-mentioned method from the alignment of the amino acid sequences of 144 olfactory receptors encoded by the orthologues of OR3A2 in mammals, and has responsiveness to indole and skatole. The orthologue of OR3A3 is a gene that is highly homologous to OR3A3 among olfactory receptor genes possessed by biological species belonging to mammals, and has the same name as OR3A3. The orthologue of OR3A2 is a gene that is highly homologous to OR3A2 among olfactory receptor genes possessed by biological species belonging to mammals, and has the same name as OR3A2. The amino acid sequence identity between the consensus OR3A3 and OR3A3 is 89%.

[0048] An example of a polypeptide having a function equivalent to OR3A1 includes a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 10 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR3A1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 10, at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably all positions corresponding to each amino acid position (7) in the amino acid sequence shown in SEQ ID NO: 10 in Table 2 below, and having an amino acid residue of (8) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions, and having a responsiveness to at least one species selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR3A1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 10, at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position (7) in the amino acid sequence shown in SEQ ID NO: 10 in Table 2 below, and having an amino acid residue of (8) at at least one position selected from the group consisting of indole and skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 10, a polypeptide having amino acid residues (8) at all of the amino acid positions (7) in Table 2 below is consensus OR3A1 consisting 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 OR3A1 and consensus OR3A1.

[0049] Yet another example of a polypeptide having a function equivalent to OR3A1 includes a polypeptide consisting of consensus OR3A1, which consists of the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 90% identity thereto, and which is responsive to at least one member selected from the group consisting of indole and skatole. The consensus OR3A1 is composed of the consensus amino acid sequence shown in SEQ ID NO: 12, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR3A1 shown in SEQ ID NO: 10 and the amino acid sequences of 194 olfactory receptors encoded by orthologues of OR3A1 in mammals, and is responsive to indole and skatole. The orthologue is a gene that is highly homologous to OR3A1 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as OR3A1. The amino acid sequence identity between the consensus OR3A1 and OR3A1 is 93%.

[0050] An example of a polypeptide having a function equivalent to OR8H2 includes a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR8H2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13, at least one, preferably at least three, more preferably at least five, even more preferably at least 10, and even more preferably all of the positions corresponding to the amino acid positions (9) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 13, and having an amino acid residue (10) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions, and having a responsiveness to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR8H2 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13, at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to the amino acid positions (9) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 13, and having an amino acid residue (10) at at least one position selected from the group consisting of indole and skatole. Here, having TK (10) at the amino acid position corresponding to the 90th position of (9) in Table 2 below means that in a certain amino acid sequence, the amino acid residue at the position corresponding to N at the 90th position of the amino acid sequence shown in SEQ ID NO: 13 is replaced with T, and K is inserted immediately thereafter. Also, having LK (10) at the amino acid position corresponding to the C-terminus of (9) in Table 2 below means that in a certain amino acid sequence, LK is added to the C-terminus side of the position corresponding to the last amino acid position of the amino acid sequence shown in SEQ ID NO: 13. In addition, a polypeptide having amino acid residues (10) at all positions of each amino acid position of (9) in Table 2 below in the amino acid sequence shown in SEQ ID NO: 13 is consensus OR8H2 consisting 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 OR8H2 and consensus OR8H2.

[0051] Yet another example of a polypeptide having a function equivalent to OR8H2 includes a polypeptide consisting of consensus OR8H2 consisting of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. Consensus OR8H2 is composed of the amino acid sequence of OR8H2 shown in SEQ ID NO: 13, the amino acid sequences of six olfactory receptors encoded by the orthologues of OR8H2 in mammals, the amino acid sequence of OR8H1 shown in SEQ ID NO: 32, which is a paralog with high homology to OR8H2, and the consensus amino acid sequence shown in SEQ ID NO: 15 derived by the above-mentioned method from the alignment of the amino acid sequences of 225 olfactory receptors encoded by the orthologues of OR8H1 in mammals, and has responsiveness to indole and skatole. The orthologue of OR8H2 is a gene that is highly homologous to OR8H2 among olfactory receptor genes possessed by mammalian species and has the same name as OR8H2. The orthologue of OR8H1 is a gene that is highly homologous to OR8H1 among olfactory receptor genes possessed by mammalian species and has the same name as OR8H1. The amino acid sequence identity between consensus OR8H2 and OR8H2 is 87%.

[0052] An example of a polypeptide having a function equivalent to OR8J1 includes a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 16 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR8J1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16, having an amino acid residue of (2) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (1) in Table 3 of the amino acid sequence shown in SEQ ID NO: 16 below, and having responsiveness to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR8J1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16, having an amino acid residue of (2) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (1) in Table 3 of the amino acid sequence shown in SEQ ID NO: 16 below, and having responsiveness to at least one selected from the group consisting of indole and skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 16, a polypeptide having amino acid residues (2) at all of the amino acid positions (1) in Table 3 below is consensus OR8J1 consisting 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 OR8J1 and consensus OR8J1.

[0053] Yet another example of a polypeptide having a function equivalent to OR8J1 includes a polypeptide consisting of consensus OR8J1 consisting of the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. Consensus OR8J1 consists of the consensus amino acid sequence shown in SEQ ID NO: 18, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR8J1 shown in SEQ ID NO: 16 and the amino acid sequences of 54 olfactory receptors encoded by orthologues of OR8J1 in mammals, and has responsiveness to indole and skatole. The orthologue is a gene that is highly homologous to OR8J1 among olfactory receptor genes possessed by organisms belonging to mammals and includes the same name as OR8J1. The amino acid sequence identity between consensus OR8J1 and OR8J1 is 93%.

[0054] An example of a polypeptide having a function equivalent to OR8J3 includes a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 19 and having responsiveness to at least one member selected from the group consisting of indole and skatole. Another example of a polypeptide having a function equivalent to OR8J3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, having an amino acid residue of (4) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position of (3) in Table 3 below in the amino acid sequence shown in SEQ ID NO: 19, and having responsiveness to at least one selected from the group consisting of indole and skatole. Yet another example of a polypeptide having a function equivalent to OR8J3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, having an amino acid residue of (4) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position of (3) in Table 3 below in the amino acid sequence shown in SEQ ID NO: 19, and having responsiveness to at least one selected from the group consisting of indole and skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 19, a polypeptide having amino acid residues (4) at all of the amino acid positions (3) in Table 3 below is consensus OR8J3 consisting 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 OR8J3 and consensus OR8J3.

[0055] Another example of a polypeptide having a function equivalent to OR8J3 includes a polypeptide consisting of consensus OR8J3 consisting of the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to at least one member selected from the group consisting of indole and skatole. The consensus OR8J3 consists of the consensus amino acid sequence shown in SEQ ID NO: 21, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR8J3 shown in SEQ ID NO: 19 and the amino acid sequences of 147 olfactory receptors encoded by orthologues of OR8J3 in mammals, and has responsiveness to indole and skatole. The orthologue is a gene that is highly homologous to OR8J3 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as OR8J3. The amino acid sequence identity between the consensus OR8J3 and OR8J3 is 92%.

[0056] An example of a polypeptide having a function equivalent to OR4B1 includes a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 22 and that is responsive to skatole. Another example of a polypeptide having a function equivalent to OR4B1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 22, having the amino acid residue (6) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position (5) in the amino acid sequence shown in SEQ ID NO: 22 in Table 3 below, and having responsiveness to skatole. Yet another example of a polypeptide having a function equivalent to OR4B1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 22, having the amino acid residue (6) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position (5) in the amino acid sequence shown in SEQ ID NO: 22 in Table 3 below, and having responsiveness to skatole. In addition, in the amino acid sequence shown in SEQ ID NO: 22, a polypeptide having amino acid residues (6) at all of the amino acid positions (5) in Table 3 below is consensus OR4B1 consisting of the amino acid sequence shown in SEQ ID NO: 24. In other words, Table 3 below shows the amino acid residues that differ between OR4B1 and consensus OR4B1.

[0057] Another example of a polypeptide having a function equivalent to OR4B1 includes a polypeptide consisting of consensus OR4B1 having the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to skatole. Consensus OR4B1 consists of the consensus amino acid sequence shown in SEQ ID NO: 24, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR4B1 shown in SEQ ID NO: 22 and the amino acid sequences of 220 olfactory receptors encoded by orthologues of OR4B1 in mammals, and has responsiveness to skatole. The orthologue is a gene that is highly homologous to OR4B1 among olfactory receptor genes possessed by organisms belonging to mammals and contains the same name as OR4B1. The amino acid sequence identity between consensus OR4B1 and OR4B1 is 93%.

[0058] An example of a polypeptide having a function equivalent to OR5T3 includes a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 25 and having responsiveness to indole. Another example of a polypeptide having a function equivalent to OR5T3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 25, at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably all positions corresponding to each amino acid position (7) in the amino acid sequence shown in SEQ ID NO: 25 in Table 3 below, and having an amino acid residue (8) at each position, and having a responsiveness to indole. Yet another example of a polypeptide having a function equivalent to OR5T3 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 25, at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position (7) in the amino acid sequence shown in SEQ ID NO: 25 in Table 3 below, and having an amino acid residue (8) at each position, and having a responsiveness to indole. In addition, in the amino acid sequence shown in SEQ ID NO: 25, a polypeptide having amino acid residues (8) at all of the amino acid positions (7) in Table 3 below is consensus OR5T3 consisting of the amino acid sequence shown in SEQ ID NO: 27. In other words, Table 3 below shows the amino acid residues that differ between OR5T3 and consensus OR5T3.

[0059] Another example of a polypeptide having a function equivalent to OR5T3 includes a consensus OR5T3 consisting of the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to indole. The consensus OR5T3 is composed of the consensus amino acid sequence shown in SEQ ID NO: 27, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR5T3 shown in SEQ ID NO: 25 and the amino acid sequences of 48 olfactory receptors encoded by orthologues of OR5T3 in mammals, and is responsive to indole. The orthologue is a gene that is highly homologous to OR5T3 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as OR5T3. The amino acid sequence identity between the consensus OR5T3 and OR5T3 is 94%.

[0060] An example of a polypeptide having a function equivalent to OR8B12 includes a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28 and having responsiveness to indole. Another example of a polypeptide having a function equivalent to OR8B12 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 28, having the amino acid residue (10) at at least one position, preferably at least three positions, more preferably at least five positions, even more preferably at least 10 positions, and even more preferably at all positions corresponding to each amino acid position (9) in the amino acid sequence shown in SEQ ID NO: 28 in Table 3 below, and having responsiveness to indole. Yet another example of a polypeptide having a function equivalent to OR8B12 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 28, having the amino acid residue (10) at preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably at all positions corresponding to each amino acid position (9) in the amino acid sequence shown in SEQ ID NO: 28 in Table 3 below, and having responsiveness to indole. In addition, in the amino acid sequence shown in SEQ ID NO: 28, a polypeptide having amino acid residues (10) at all of the amino acid positions (9) in Table 3 below is consensus OR8B12 consisting of the amino acid sequence shown in SEQ ID NO: 30. In other words, Table 3 below shows the amino acid residues that differ between OR8B12 and consensus OR8B12.

[0061] Another example of a polypeptide having a function equivalent to OR8B12 includes a polypeptide consisting of consensus OR8B12 consisting of the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% identity thereto, and having responsiveness to indole. Consensus OR8B12 consists of the consensus amino acid sequence shown in SEQ ID NO: 30, which is derived by the above-mentioned method from the alignment of the amino acid sequence of OR8B12 shown in SEQ ID NO: 28 and the amino acid sequences of 144 olfactory receptors encoded by orthologues of OR8B12 in mammals, and has responsiveness to indole. The orthologue is a gene that is highly homologous to OR8B12 among olfactory receptor genes possessed by organisms belonging to mammals, and is a gene that includes the same name as OR8B12. The amino acid sequence identity between consensus OR8B12 and OR8B12 is 91%.

[0062] [Table 2]

[0063] [Table 3]

[0064] In the method for evaluating and / or selecting odor suppressants of the present invention, at least one selected from OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto may be used, but any two or more of them may also be used in combination. Preferably, at least one selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3, consensus OR4B1, consensus OR5T3 and consensus OR8B12 is used, and more preferably, at least one selected from the group consisting of consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3, consensus OR4B1, consensus OR5T3 and consensus OR8B12 is used.

[0065] In one example, when the heterocyclic aromatic compound is indole, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR5T3, OR8B12, and polypeptides having equivalent functions thereto, more preferably at least one selected from the group consisting of OR2AT4, OR8I2, and polypeptides having equivalent functions thereto, even more preferably at least one selected from the group consisting of OR2AT4, OR8I2, consensus OR2AT4, and consensus OR8I2, and even more preferably at least one selected from the group consisting of consensus OR2AT4 and consensus OR8I2.

[0066] In another example, when the heterocyclic aromatic compound is skatole, the olfactory receptor polypeptide is preferably at least one selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, and polypeptides having equivalent functions thereto, and more preferably at least one selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, and polypeptides having equivalent functions thereto. More preferably, at least one selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1 and consensus OR8H2 is used, and even more preferably, at least one selected from the group consisting of consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1 and consensus OR8H2 is used.

[0067] In the odor suppressant evaluation and / or selection 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 heterocyclic aromatic compounds.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 carrying the olfactory receptor polypeptide; artificial lipid bilayer membrane carrying the olfactory receptor polypeptide, etc.All of these forms are included in the scope of the olfactory receptor polypeptide used in the present invention.

[0068] 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 genetically engineered to express the olfactory receptor polypeptide, or a culture thereof. The recombinant cell can be prepared by transforming a cell with a vector incorporating a gene encoding the olfactory receptor polypeptide.

[0069] Preferably, in order to promote the cell membrane expression of the olfactory receptor polypeptide, a gene encoding an RTP (receptor-transporting protein) 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:34, encoded by a gene having the nucleotide sequence of SEQ ID NO:33.

[0070] 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 in which cells expressing the olfactory receptor polypeptide are cultured, and directly adding, dripping, sprinkling or spraying the test substance onto the olfactory receptor polypeptide or cells or tissues containing it.

[0071] In the method for evaluating and / or selecting an odor suppressant 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. The measurement may be performed by any method known in the art as a method for measuring the response of an olfactory receptor, such as measuring the amount of intracellular cAMP. For example, it is known that when an olfactory receptor is activated by an odor molecule, it conjugates with a G protein α subunit classified as the Gαs family in the cell to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP (Nat. Neurosci., 2004, 5:263-278). On the other hand, when an olfactory receptor is activated by an odor molecule, it can also conjugate with a protein belonging to the Gq family, such as Gα15, in the cell, and increase the amount of calcium ions in the cell. Therefore, the response of the olfactory receptor polypeptide can be measured by using the amount of intracellular cAMP or calcium ion after the addition of an odor molecule, or the behavior of downstream molecules activated via them, as an index. Methods for measuring the amount of cAMP include ELISA, reporter gene assay, and a method using a cAMP-responsive reporter protein. In a method using a cAMP-responsive reporter protein, for example, a firefly luciferase genetically modified to contain a portion of a cAMP-binding protein is used, and the amount of cAMP is measured by monitoring the luminescence value of the luciferase corresponding to the amount of cAMP (GloSensor (trademark) cAMP Assay (Promega)). As another method for measuring the response of an olfactory receptor polypeptide, a method for measuring calcium ion concentration includes a calcium imaging method and a TGFα shedding assay. In addition, as an example of a method using the behavior of downstream molecules activated via the amount of cAMP as an index, a two-electrode voltage clamp method is also effective for measuring the potential change inside and outside the cell membrane via the cystic fibrosis transmembrane conductance regulator CFTR, which is activated by a cAMP signal, in Xenopus oocytes.

[0072] In a first embodiment of the present invention, the method for evaluating and / or selecting an inhibitor of an odor caused by a heterocyclic aromatic compound according to the present invention comprises adding a test substance to the above-mentioned olfactory receptor polypeptide; 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 an odor caused by a heterocyclic aromatic compound.

[0073] The 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 heterocyclic aromatic compound by enhancing the response of the olfactory receptor in advance.As a result, it is possible to suppress the recognition of the odor caused by the heterocyclic aromatic compound by an individual based on odor cross-adaptation.Therefore, in the first embodiment, a suppressant of the odor caused by the heterocyclic aromatic compound is selected based on the odor cross-adaptation by olfactory receptor agonism.

[0074] 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 (test group) to which a test substance has been added with the response in a control group. Examples of the control group 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 a test substance has been added, the olfactory receptor polypeptide before the addition of a test substance, and cells in which the olfactory receptor polypeptide is not expressed. Preferably, the odor suppressant evaluation and / or selection method of the present invention in the first embodiment includes measuring the activity of the olfactory receptor polypeptide in the presence and absence of a test substance. Also preferably, the odor suppressant evaluation and / or selection method of the present invention in the first embodiment includes measuring the response of cells in which the olfactory receptor polypeptide is expressed and cells in which the olfactory receptor polypeptide is not expressed to the test substance in the presence of a test substance.

[0075] For example, if the response in the test group is enhanced compared to the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to heterocyclic aromatic compounds.For example, if the response of the olfactory receptor polypeptide in the test group is enhanced preferably by 120% or more, more preferably by 150% or more, and even more preferably by 200% compared to the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to heterocyclic aromatic compounds.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 heterocyclic aromatic compounds.

[0076] Examples of odors caused by heterocyclic aromatic compounds that are suppressed by the inhibitor of odors caused by heterocyclic aromatic compounds selected according to the first embodiment include fecal odor, halitosis, indole odor, and skatole odor. Preferably, fecal odor, halitosis, indole odor, and skatole odor are used when OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, or a polypeptide having a function equivalent thereto, are used as the olfactory receptor; fecal odor, halitosis, and skatole odor are used when OR4B1 or a polypeptide having a function equivalent thereto are used; and fecal odor, halitosis, and indole odor are used when OR5T3, OR8B12, or a polypeptide having a function equivalent thereto are used. More preferably, it is fecal odor or halitosis.

[0077] One embodiment of the use of the suppressant of the odor caused by the heterocyclic aromatic compound selected according to the first embodiment is as follows: first, the subject who desires to suppress the odor caused by the heterocyclic aromatic compound smells the suppressant before the subject is exposed to the odor.Alternatively, the suppressant is applied to the subject so that the odor is stronger than the odor caused by the heterocyclic aromatic compound.As a result, even if the subject is exposed to the odor caused by the heterocyclic aromatic compound, the subject feels the odor weak or does not feel it because the olfactory sensitivity to the odor is reduced.

[0078] In a second embodiment of the present invention, the method for evaluating and / or selecting an inhibitor of an odor caused by a heterocyclic aromatic compound 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 not limited to, but is preferably a heterocyclic aromatic compound, more preferably a nitrogen-containing heterocyclic aromatic compound, and even more preferably at least one selected from the group consisting of indole and skatole. Even more preferably, when OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3 or a polypeptide having an equivalent function thereto is used as the olfactory receptor, at least one selected from the group consisting of indole and skatole is used, when OR4B1 or a polypeptide having an equivalent function thereto is used, skatole is used, and when OR5T3, OR8B12 or a polypeptide having an equivalent function thereto is used, indole 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 substances are selected as suppressants of odors caused by heterocyclic aromatic compounds.

[0079] In the second embodiment, a test substance that inhibits the response of the olfactory receptor polypeptide to an agonist of the olfactory receptor polypeptide is selected as an inhibitor of odor caused by heterocyclic aromatic compounds based on olfactory receptor antagonism.

[0080] 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 which the test substance has been added (test group) to the agonist with the response of the control group to the agonist. Examples of the control group include those described above. Preferably, the method for evaluating and / or selecting an odor suppressant of the present invention in the second embodiment includes measuring the activity of the olfactory receptor polypeptide against the agonist in the presence and absence of a test substance.

[0081] For example, if the response in the test group is more suppressed than that in the control group, the test substance can be selected as a substance that suppresses the response of the olfactory receptor polypeptide to heterocyclic aromatic compounds.For example, if the response of the olfactory receptor polypeptide in the test group is preferably suppressed to 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 heterocyclic aromatic compounds.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 heterocyclic aromatic compounds.

[0082] Examples of odors caused by heterocyclic aromatic compounds that are suppressed by the suppressor of odors caused by heterocyclic aromatic compounds selected according to the second embodiment include odors caused by heterocyclic aromatic compounds that are suppressed by the suppressor of odors caused by heterocyclic aromatic compounds selected according to the first embodiment described above.

[0083] One embodiment of the use of the suppressant of the odor caused by the heterocyclic aromatic compound selected according to the second embodiment is as follows: first, the subject who desires to suppress the odor caused by the heterocyclic aromatic compound smells the suppressant before or at the same time that the subject is exposed to the odor.As a result, even if the subject is exposed to the odor caused by the heterocyclic aromatic compound, because the olfactory sensitivity to the odor is reduced, the subject feels the odor weakly or does not feel it.

[0084] By the above procedure, an inhibitor of an odor caused by a heterocyclic aromatic compound can be obtained based on the response activity of an olfactory receptor polypeptide responsive to an odor caused by a heterocyclic aromatic compound. If necessary, the ability of the test substance selected above to inhibit an odor caused by a heterocyclic aromatic compound may be further evaluated by a sensory test. That is, in one embodiment of the odor inhibitor evaluation and / or selection method of the present invention, the test substance selected by the above procedure is obtained as a candidate substance for an inhibitor of an odor caused by a heterocyclic aromatic compound. Then, the inhibitory effect of the candidate substance on an odor caused by a heterocyclic aromatic compound is evaluated by a sensory test. The candidate substance that is evaluated better in the sensory test is selected as an inhibitor of an odor caused by a heterocyclic aromatic compound.

[0085] The sensory test of the candidate substance can be performed according to the evaluation procedure of deodorants that is usually performed in the field. If the candidate substance is an inducer of cross-adaptation of odor, the order of application of the candidate substance and the substance causing the target odor to the evaluator can be adjusted. For example, when the test substance selected in the first embodiment described above is used as a candidate substance for sensory test, the evaluator first smells the candidate substance and adapts to the odor. Then, the evaluator smells the target odor (preferably the odor caused by a heterocyclic aromatic compound) and evaluates its intensity. The obtained evaluation result is compared with the intensity of the target odor when the candidate substance is not adapted. Also, for example, when the test substance selected in the second embodiment described above is used as a candidate substance for sensory test, the evaluator smells the target odor (preferably the odor caused by a heterocyclic aromatic compound) at the same time as the candidate substance, and evaluates the intensity of the target odor. The obtained evaluation result is compared with the intensity of the target odor alone. Candidate substances that are evaluated as reducing the intensity of the target odor as a result of the sensory test are selected as inhibitors of odors caused by heterocyclic aromatic compounds.

[0086] The odor suppressant of heterocyclic aromatic compounds obtained by the odor suppressant evaluation and / or selection method of the present invention can be used as an active ingredient for suppressing odor caused by heterocyclic aromatic compounds. For example, the suppressant can be contained in a composition or article for suppressing odor caused by heterocyclic aromatic compounds as an active ingredient for suppressing odor caused by heterocyclic aromatic compounds. Alternatively, the suppressant can be used for the manufacture of a composition or article for suppressing odor caused by heterocyclic aromatic compounds. Examples of applications of the inhibitor for odors caused by heterocyclic aromatic compounds obtained by the odor inhibitor evaluation and / or selection method of the present invention include, but are not limited to, sanitary products such as sanitary products and disposable diapers containing the agent; clothing accessories such as underwear, undergarments, and linens, cloth products, or textiles containing the agent; laundry detergents or fabric softeners containing the agent; cosmetics, cleaning agents, external preparations such as deodorants, medicines, foods, etc. containing the agent; placing or spraying the agent in front of or in toilets; placing or spraying the agent in hospital wards or nursing facilities, etc.; methods in which a person involved in excretion treatment in a hospital ward or nursing facility, etc., carries the agent or is exposed to the agent before the treatment; and application to environments in which odors caused by heterocyclic aromatic compounds are generated.

[0087] In addition, as described above, OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3 and OR8B12 are newly discovered heterocyclic aromatic compound receptors, so that OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12 or polypeptides having equivalent functions thereto can be used to detect odors caused by heterocyclic aromatic compounds. Therefore, the present invention provides a method for detecting odors caused by heterocyclic aromatic compounds (hereinafter referred to as the detection method of the present invention). The method includes contacting a sample with at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto; and measuring the response of the olfactory receptor polypeptide to the sample. Based on the measured response, the odor caused by the heterocyclic aromatic compound contained in the sample is detected.

[0088] Here, the term "detection" can be replaced with "evaluation" or "measurement", and "detection of an odor caused by a heterocyclic aromatic compound" includes proving the presence or absence of an odor caused by a heterocyclic aromatic compound and measuring the level of an odor caused by a heterocyclic aromatic compound.

[0089] The sample used in the detection method of the present invention is not particularly limited as long as it is a sample for which confirmation of the presence or absence or the level of odor caused by a heterocyclic aromatic compound is desired.The sample may be a compound, a composition or a mixture, and may be a gas, a liquid or a solid.The sample is preferably a real sample, and examples of the real sample include samples derived from body fluids (e.g., saliva, sweat, urine), wastewater, food, fragrances, cosmetics, medicines, detergents, daily necessities and laundry.

[0090] The olfactory receptor polypeptide and its form used in the detection method of the present invention are the same as the olfactory receptor polypeptide and its form used in the odor suppressant evaluation and / or selection method of the present invention.

[0091] Methods for contacting a sample with the olfactory receptor polypeptide include, but are not limited to, adding the sample to a culture medium in which cells expressing the olfactory receptor polypeptide are cultured, and directly adding, dripping, scattering or spraying the sample onto the olfactory receptor polypeptide or onto cells or tissues containing the same.

[0092] The response of the olfactory receptor polypeptide can be measured using a method similar to that used in the evaluation and / or selection method of the odor suppressant of the present invention.

[0093] Based on the measured response, the odor caused by the heterocyclic aromatic compound in the sample is detected.The sample that causes the response of the olfactory receptor polypeptide can be evaluated as the sample that exhibits the odor caused by the heterocyclic aromatic compound, in other words, the sample that contains the heterocyclic aromatic compound.The stronger the response intensity caused by the sample, the stronger the odor caused by the heterocyclic aromatic compound exhibited by the sample, in other words, the more the sample contains the heterocyclic aromatic compound.

[0094] Preferably, the response of the olfactory receptor polypeptide to a sample can be evaluated by comparing the response of the olfactory receptor polypeptide (test group) contacted with a sample with the response of the olfactory receptor polypeptide in a control group.The control group can include the olfactory receptor polypeptide without the addition of a sample, the olfactory receptor polypeptide with the addition of a control substance, the olfactory receptor polypeptide contacted with a lower concentration of a sample, the olfactory receptor polypeptide before contacting with a sample, etc.If the response in the test group is higher than that in the control group, the sample is evaluated as a sample that exhibits an odor caused by a heterocyclic aromatic compound.

[0095] Therefore, in one embodiment of the detection method of the present invention, the response of the olfactory receptor polypeptide in the presence and absence of a sample is measured, and then it is determined whether the response in the presence of a sample is higher than the response in the absence of a sample.If the response in the presence of a sample is higher, the sample is evaluated as a sample that exhibits an odor caused by a heterocyclic aromatic compound.In a preferred embodiment, if the response intensity of the olfactory receptor polypeptide in the presence of a sample is preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more compared to the absence of a sample, the sample is evaluated as a sample that exhibits an odor caused by a heterocyclic aromatic compound.In another preferred embodiment, if the response intensity of the olfactory receptor polypeptide in the presence of a sample is statistically significantly increased compared to the absence of a sample, the sample is evaluated as a sample that exhibits an odor caused by a heterocyclic aromatic compound.

[0096] Alternatively, the response of the olfactory receptor polypeptide to a sample can be evaluated by comparing the response of the olfactory receptor polypeptide (test group) contacted with a sample with a calibration curve prepared in advance from the response when a standard sample containing a heterocyclic aromatic compound of known concentration is used as the sample. If the response in the test group is within the quantitative range of the calibration curve, the sample is evaluated as a sample that exhibits an odor caused by a heterocyclic aromatic compound, and the level of the odor can be calculated based on the calibration curve.

[0097] Examples of odors caused by heterocyclic aromatic compounds detected by the detection method of the present invention include fecal odor, halitosis, indole odor, and skatole odor. Preferably, fecal odor, halitosis, indole odor, and skatole odor are detected when OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, or a polypeptide having a function equivalent thereto, are used as the olfactory receptor. Fecal odor, halitosis, and skatole odor are detected when OR4B1 or a polypeptide having a function equivalent thereto is used. Fecal odor, halitosis, and skatole odor are detected when OR5T3, OR8B12, or a polypeptide having a function equivalent thereto are used. Fecal odor or halitosis are more preferably detected. EXAMPLES

[0098] The present invention will be described more specifically below with reference to examples.

[0099] Example 1 Identification of olfactory receptors that respond to heterocyclic aromatic compounds 1) Creation of olfactory receptor expressing cells A consensus olfactory receptor was designed in the same manner as described in the Examples of Patent Document 5. In designing the consensus olfactory receptor, a search for homologous gene candidates of the target olfactory receptor gene was performed using NCBI BLAST. Orthologs were identified for the obtained gene group. Specifically, from the top homologous genes searched by BLAST, genes with the same name as the target olfactory receptor were selected as orthologs. For example, in the case of human OR2AT4, the amino acid sequence of human OR2AT4 (NP_001005285.1) was used as the query sequence, and 197 genes with OR2AT4 in their names were selected from the top 250 homologous genes searched by BLAST. These 197 genes were identified as orthologs. For the amino acid sequences of these 197 genes and human OR2AT4, a total of 198 genes were subjected to alignment analysis and consensus amino acid identification as described below. Consensus amino acids were also identified for other human olfactory receptors, OR8I2, OR3A1, OR8J1, OR8J3, OR4B1, OR5T3, and OR8B12. In the case of human OR3A3, we tried to identify the ortholog group in the same manner as above, except that the amino acid sequence of human OR3A3 (NP_001373027.1) was used as the query sequence, but only seven genes were selected as orthologs. Therefore, from the top 250 homologous genes searched using the amino acid sequence of human OR3A3 as the query sequence, we focused on human OR3A2 (NP_002542.3), which is a paralog with high homology to human OR3A3, and identified the ortholog group and selected 144 genes as orthologs. We performed alignment analysis and consensus amino acid identification for the amino acid sequences of a total of 153 genes, including human OR3A3, orthologs of human OR3A3, and human OR3A2, as described below. In the case of human OR8H2, we attempted to identify the ortholog group in the same manner as above, except that the amino acid sequence of human OR8H2 (NP_001005200.1) was used as the query sequence, but only six genes were selected as orthologs. Therefore, from the top 250 homologous genes searched using the amino acid sequence of human OR8H2 as the query sequence, we focused on human OR8H1 (NP_001005199.1), a paralog with high homology to human OR8H2, to identify the ortholog group and selected 225 genes as orthologs. We performed alignment analysis and consensus amino acid identification for the amino acid sequences of a total of 233 genes, including human OR8H2, orthologs of human OR8H2, and human OR8H1, as described below.

[0100] The alignment analysis of the identified gene group was performed using ClustalW, and further adjustments were made to optimize the olfactory receptors based on amino acids or amino acid motifs highly conserved among olfactory receptors. Based on the alignment results, a consensus olfactory receptor was designed using Jalview. In the alignment, when there is one amino acid residue different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% or more at a position corresponding to each amino acid position of the original human olfactory receptor amino acid sequence serving as a reference, the amino acid residue of the reference amino acid sequence was modified to the amino acid residue. Even if there is one amino acid residue different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% at a position corresponding to each amino acid position of the original human olfactory receptor amino acid sequence serving as a reference, if the occurrence frequency of the amino acid residue of the reference amino acid sequence is also 50%, the amino acid residue of the reference amino acid sequence was not modified. In the alignment, when there is a deletion with an occurrence frequency of 40% or more at a position corresponding to each amino acid position of the original human olfactory receptor amino acid sequence serving as a reference, the amino acid residue of the reference amino acid sequence was modified to a deletion. On the other hand, when an amino acid with an occurrence frequency of 60% or more exists at a position corresponding to the deletion position of the original reference human olfactory receptor amino acid sequence, the reference amino acid sequence was modified to insert the most conserved amino acid at the deletion position. When there are two or more types of the most conserved amino acid, the amino acid with the smallest molecular weight was modified to insert. The topology of the olfactory receptor in the design was confirmed using TMHMM (Transmembrane Hidden Markov Model). The DNA sequences encoding the various designed olfactory receptor polypeptides were obtained by DNA synthesis after optimizing the base sequence codons corresponding to the amino acid sequences for expression in human cultured cells. The base sequences with optimized codons encoding consensus OR2AT4, consensus OR8I2, consensus OR3A3, consensus OR3A1, consensus OR8H2, consensus OR8J1, consensus OR8J3, consensus OR4B1, consensus OR5T3, and consensus OR8B12 consisting of the amino acid sequences shown in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 are shown in SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, and 29, respectively. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined into the EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector. In addition, a gene encoding human RTP1S, which transports olfactory receptor proteins produced in cultured cells onto the cell membrane, was inserted into the EcoRI and XhoI sites of another pME18S vector to create the pME18S-RTP1S vector.

[0101] A reaction solution with the composition shown in Table 4 was prepared, left to stand in a clean bench for 20 minutes, and then added to each well of a 96-well plate (BD). For the olfactory receptors OR8I2, OR5T3, and OR8B12, the compositions shown in Table 5 were used. Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well at 2 × 10 5 cells / cm 2 The cells were seeded at 100°C and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that do not express olfactory receptors (Mock) were prepared.

[0102] [Table 4]

[0103] [Table 5]

[0104] 2) Luciferase assay Olfactory receptors expressed in HEK293 cells increase the amount of intracellular cAMP by coupling with endogenous GαS and activating adenylate cyclase. To measure odor responses, a luciferase reporter gene assay was used to monitor the increase in intracellular cAMP as luminescence values ​​derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a Renilla luciferase gene fused downstream of the CMV promoter (hRluc2P-CMV-hygro) was simultaneously transfected and used as an internal standard to correct errors in gene transfection efficiency and cell number.

[0105] The medium was removed from the culture prepared in 1) above, and 75 μL of test substance solution (indole (CAS: 120-72-9, Tokyo Chemical Industry) or skatole (CAS: 83-34-1, Kanto Chemical), concentration range 0 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM) prepared in new medium was added. The cells were cultured in a CO2 incubator for 4 hours to allow the luciferase gene to be fully expressed in the cells. Luciferase activity was measured using Dual-Glo TM The measurements were performed using a luciferase assay system (Promega) according to the product's operating manual. In each well of a 96-well plate, the luminescence value derived from firefly luciferase induced by stimulation with the test substance was divided by the luminescence value derived from Renilla luciferase to calculate the signal (fluc / hRluc) and used for analysis. For the signals under each transfection condition, the signal value (fluc / hRluc) under the condition without stimulation with odorant was set to 0%, and the signal value (fluc / hRluc) under stimulation with 10 μM forskolin was set to 100%, and the signal was used for analysis as the response (%).

[0106] 3) Results Among the consensus olfactory receptors tested, those that responded to indole or skatole are shown in Figure 1. Figure 1 also shows the response values ​​of each olfactory receptor to each concentration of indole or skatole. A statistically significant difference was observed between the response value (Response (%)) of each olfactory receptor to the highest concentration tested, 300 μM, and the response value (Response (%)) of cells under mock conditions in which the receptor is not expressed (Student's t-test, P<0.05). For each olfactory receptor, a significant difference was also observed using the same statistical method when comparing the signal value (fluc / hRluc) under conditions in which no stimulation with odorants was performed with the signal value (fluc / hRluc) under conditions in which stimulation with the highest concentration of 300 μM was performed. The values ​​in Tables 6 and 7 are EC values ​​calculated by regressing the Response (%) values ​​onto a sigmoid curve. 50 (μM). The EC 50 Olfactory receptors without a description of (μM) are receptors that can respond to indole or skatole, but there was insufficient data to regress to a sigmoid curve within the concentration range tested in this study, and the EC 50This means that the β-amyloid ... Among them, the consensus OR2AT4, the consensus OR8I2, the consensus OR3A3, the consensus OR3A1 and the consensus OR8H2 were also confirmed to respond to skatole in a dose-dependent manner, and thus the consensus OR2AT4, the consensus OR8I2, the consensus OR3A3, the consensus OR3A1, the consensus OR8H2, the consensus OR8J1, the consensus OR8J3, the consensus OR4B1, the consensus OR5T3 and the consensus OR8B12 were found to respond to at least one heterocyclic aromatic compound selected from the group consisting of indole and skatole.

[0107] [Table 6]

[0108] [Table 7]

[0109] The responses of the consensus olfactory receptors to odorants reflect those of the original olfactory receptors in olfactory cells. Therefore, the above results identify the original OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3 and OR8B12 as receptors for heterocyclic aromatic compounds.

Claims

1. A method for evaluating and / or selecting an agent for suppressing odor caused by heterocyclic aromatic compounds, comprising measuring the response of at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto, after addition of a test substance.

2. 2. The method of claim 1, wherein the odor caused by the heterocyclic aromatic compound is fecal odor or breath odor.

3. 3. The method according to claim 1 or 2, wherein the heterocyclic aromatic compound is at least one selected from the group consisting of indole and skatole.

4. The method according to any one of claims 1 to 3, wherein the OR2AT4 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, the OR8I2 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, the OR3A3 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7, the OR3A1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10, the OR8H2 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13, the OR8J1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, the OR8J3 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 19, the OR4B1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, the OR5T3 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 25, and the OR8B12 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

28.

5. the polypeptide having a function equivalent to OR2AT4 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1 and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR8I2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 10, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8H2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR4B1 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 22 and having responsiveness to skatole; the polypeptide having a function equivalent to OR5T3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25 and having responsiveness to indole; The polypeptide having a function equivalent to OR8B12 is a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28 and having responsiveness to indole. The method according to any one of claims 1 to 4.

6. The polypeptide having a function equivalent to OR2AT4 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, having an amino acid residue of (2) at at least one position corresponding to each amino acid position of (1) in the amino acid sequence shown in SEQ ID NO: 1 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole, the polypeptide having a function equivalent to OR8I2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4, having an amino acid residue of (4) at at least one position corresponding to each amino acid position of (3) in the amino acid sequence shown in SEQ ID NO: 4 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7, having an amino acid residue of (6) at at least one position corresponding to each amino acid position of (5) in the amino acid sequence shown in SEQ ID NO: 7 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 10, having an amino acid residue of (8) at at least one position corresponding to each amino acid position of (7) in the amino acid sequence shown in SEQ ID NO: 10 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR8H2 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13, having an amino acid residue of (10) at at least one position corresponding to each amino acid position of (9) in the amino acid sequence shown in SEQ ID NO: 13 in Table 1 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16, 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: 16 in Table 2 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 19, having an amino acid residue of (4) at at least one position corresponding to each amino acid position of (3) in the amino acid sequence shown in SEQ ID NO: 19 in Table 2 below, and having responsiveness to at least one member selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR4B1 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:22, having an amino acid residue of (6) at at least one position corresponding to each amino acid position of (5) in the amino acid sequence shown in SEQ ID NO:22 in Table 2 below, and having responsiveness to skatole; the polypeptide having a function equivalent to OR5T3 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25, having an amino acid residue of (8) at at least one position corresponding to each amino acid position of (7) in the amino acid sequence shown in SEQ ID NO:25 in Table 2 below, and having responsiveness to indole; The polypeptide having a function equivalent to OR8B12 is a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, having an amino acid residue of (10) at at least one position corresponding to each amino acid position of (9) in the amino acid sequence shown in SEQ ID NO:28 in Table 2 below, and having responsiveness to indole. The method according to any one of claims 1 to 5. 【Table 1】 【Table 2】

7. the polypeptide having a function equivalent to OR2AT4 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 3 and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR8I2 is a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:6, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A3 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:9, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR3A1 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 12, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8H2 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 15, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J1 is a polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 18, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to that of OR8J3 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:21, and having responsiveness to at least one selected from the group consisting of indole and skatole; the polypeptide having a function equivalent to OR4B1 is a polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:24 and having responsiveness to skatole; the polypeptide having a function equivalent to OR5T3 is a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:27 and having responsiveness to indole; The polypeptide having a function equivalent to OR8B12 is a polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 30 and having responsiveness to indole. The method according to any one of claims 1 to 4.

8. 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 claims 1 to 7, comprising:

9. The method of claim 8, further comprising selecting a test substance that enhances the response of the olfactory receptor polypeptide to the test substance as an inhibitor of odor caused by a heterocyclic aromatic compound.

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

11. The method of claim 10, 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 odor caused by heterocyclic aromatic compounds.

12. 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 claims 1 to 7, comprising:

13. The method of claim 12, further comprising selecting a test substance that inhibits the response of the olfactory receptor polypeptide to the agonist as an inhibitor of odor caused by a heterocyclic aromatic compound.

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

15. The method of claim 14, further comprising selecting a test substance that inhibits the response of the olfactory receptor polypeptide to the agonist compared to the control group as an inhibitor of odor caused by a heterocyclic aromatic compound.

16. Contacting a sample with at least one olfactory receptor polypeptide selected from the group consisting of OR2AT4, OR8I2, OR2AT4, OR3A3, OR3A1, OR8H2, OR8J1, OR8J3, OR4B1, OR5T3, OR8B12, and polypeptides having equivalent functions thereto; and measuring the response of the olfactory receptor polypeptide to the sample; A method for detecting an odor caused by a heterocyclic aromatic compound, comprising:

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

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